FOOD AND DRUG ADMINISTRATION

	CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

	TWENTY-SEVENTH MEETING OF THE

	BIOLOGICAL RESPONSE MODIFIERS ADVISORY COMMITTEE

	8:40 a.m.

	Thursday, July 13, 2000

	Hilton Hotel
	620 Perry Parkway
	Gaithersburg, Maryland  20877

	ATTENDEES

COMMITTEE MEMBERS:

DANIEL R. SALOMON, M.D., Chairman
Associate Professor
Department of Molecular and Experimental Medicine
The Scripps Research Institute
10550 N. Torrey Pines Road, MEM 55
La Jolla, California  92037

GAIL DAPOLITO, Executive Secretary
Scientific Advisors & Consultants Staff
Center for Biologics Evaluation and Research
Food and Drug Administration
1401 Rockville Pike
Rockville, Maryland  20852-1448

RICHARD E. CHAMPLIN, M.D.
Professor of Medicine
Department of Blood and Marrow Transplantation
University of Texas M.D. Anderson Cancer Center
1515 Holcombe Boulevard, Box 24
Houston, Texas  77030

JOANNE KURTZBERG, M.D.
Professor of Pediatrics
Department of Pediatrics
Duke University Medical Center
Box 3350, Suite 1400 North Pavilion
Durham, North Carolina  27710

W. MICHAEL O'FALLON, PH.D.
Professor of Biostatistics
Department of Health Sciences Research
Mayo Clinic
Harwick Building, Room 766-A
Rochester, Minnesota  55905

EDWARD A. SAUSVILLE, M.D., PH.D.
Associate Director
Developmental Therapeutics Program
National Cancer Institute
National Institutes of Health
6116 Executive Boulevard, Suite 500
Rockville, Maryland  20892

	ATTENDEES  (Continued)

COMMITTEE MEMBERS:  (Continued)

ALICE WOLFSON, J.D.
Bourhis, Wolfson and Schlichtmann
1050 Battery Street
San Francisco, California  94111

TEMPORARY VOTING MEMBER:

HUGH AUCHINCLOSS, JR., M.D.
Professor of Surgery
Department of Surgery
Harvard Medical School
Massachusetts General Hospital
GRB504, 55 Fruit Street
Boston, Massachusetts   02144-2696

CONSULTANTS:

DAVID A. DRACHMAN, M.D.
Professor and Chair
Department of Neurology
University of Massachusetts Medical School
55 Lake Avenue N., Room S5-753
Worcester, Massachusetts  01655

THOMAS B. FREEMAN, M.D.
Professor
Department of Neurosurgery
University of South Florida College of Medicine
#4 Columbia Drive, Suite 730
Tampa, Florida  33606

STEVEN A. GOLDMAN, M.D., PH.D.
Professor of Neurology and Neuroscience
Department of Neurology and Neuroscience
Cornell University Medical College
1300 York Avenue
New York, New York  10021

	ATTENDEES  (Continued)

CONSULTANTS:  (Continued)

JEFFREY H. KORDOWER, PH.D.
Professor of Neurological Sciences
Department of Neurological Sciences
Rush Presbyterian-St. Luke's Medical Center
2242 W. Harrison Street
Chicago, Illinois  60612

JOHN W. McDONALD, III, M.D., PH.D.
Assistant Professor of Neurology and
  Neurological Surgery 
Department of Neurology
Washington University School of Medicine
Campus Box 8111
660 South Euclid Avenue
St. Louis, Missouri  63110-1093

VASSILIS E. KOLIATSOS, M.D.
Associate Professor
Department of Pathology
The Johns Hopkins University School of Medicine
Ross 558 Research Building
720 Rutland Avenue
Baltimore, Maryland  21205-2196

JEFFREY D. MACKLIS, M.D.
Associate Professor of Neurology
Department of Neurology
Harvard Medical School
Children's Hospital
354 Enders Building
320 Longwood Avenue
Boston, Massachusetts  02115

MAHENDRA S. RAO, M.D., PH.D.
Associate Professor
Department of Neurobiology and Anatomy
University of Utah Medical School
531 Wintrobe Building
50 North Medical Drive
Salt Lake City, Utah  84132

	ATTENDEES  (Continued)

CONSULTANTS:  (Continued)

EVAN Y. SNYDER, M.D., PH.D.
Assistant Professor in Neurology
Department of Pediatrics
Harvard Medical School
248 Enders Building
300 Longwood Avenue
Boston, Massachusetts  02115

JEREMY SUGARMAN, M.D., M.P.H., M.A.
Associate Professor of Medicine
Center for Study of Medical Ethics and Humanities
Duke University Medical Center
Box 3040, Trent Drive
Durham, North Carolina  27710

CATHERINE M. VERFAILLIE, M.D.
Professor of Medicine
Department of Medicine
University of Minnesota Medical Center
Cancer Center Research Building, Room 660F
Box 806 Mayo
420 Delaware Street, S.E.
Minneapolis, Minnesota  55455

MICHAEL D. WALKER, M.D.
National Institute of Neurological Disorders and Stroke
National Institutes of Health
Neuroscience Center, Room 2223
6001 Executive Boulevard
Bethesda, Maryland  208892-2581

GUESTS:

FRED H. GAGE, PH.D.
Professor
Laboratory of Genetics
The Salk Institute
10010 N. Torrey Pines Road
La Jolla, California  92037

	ATTENDEES  (Continued)

GUESTS:  (Continued)

JOHN D. GEARHART, PH.D.
Professor and Director of Developmental Genetics
Department of Obstetrics and Gynecology
The Johns Hopkins University School of Medicine
600 N. Wolfe Street, Park Building, B2-202
Baltimore, Maryland  21287-2501

RICHARD C. MULLIGAN, M.D.
Mallinckrodt Professor of Genetics
Department of Genetics
Harvard Medical School
Children's Hospital
861 Enders Building
320 Longwood Avenue
Boston, Massachusetts  02115

MARK NOBLE, PH.D.
Professor
Department of Oncological Sciences
Huntsman Cancer Institute
University of Utah
2000 Circle of Hope, Room 4280
Salt Lake City, Utah  84112

JOHN Q. TROJANOWSKI, M.D., PH.D.
Professor
Department of Pathology and Laboratory Medicine
University of Pennsylvania School of Medicine
3rd Floor Maloney Building
3600 Spruce Street
Philadelphia, Pennsylvania  19104-4283

NIH PARTICIPANT:

ARLENE CHIU, PH.D.
Project Director
National Institute of Neurological Disorders and Stroke
National Institutes of Health
6001 Executive Boulevard, Room 2205
Bethesda, Maryland  20892

	ATTENDEES  (Continued)

FOOD AND DRUG ADMINISTRATION PARTICIPANTS:

DONALD W. FINK, JR., PH.D.
Biologist
Division of Cellular and Gene Therapies
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-527)
Rockville, Maryland  20852

MALCOLM MOOS, JR., M.D., PH.D.
Medical Officer
Division of Cellular and Gene Therapies
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-524)
Rockville, Maryland  20852

PHILLIP D. NOGUCHI, M.D.
Director
Division of Cellular and Gene Therapies
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-515)
Rockville, Maryland  20852

MERCEDES SERABIAN, M.S.
Division of Cellular and Gene Therapies
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-579)
Rockville, Maryland  20852

JAY P. SIEGEL, M.D.
Director
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-500)
Rockville, Maryland  20852

BARBARA WILCOX, PH.D
Biologist
Division of Therapeutic Proteins
Office of Therapeutics Research and Review
Center for Biologics Evaluation and Research
1401 Rockville Pike (HFM-524)
Rockville, Maryland  20852

	ATTENDEES  (Continued)

ALSO PRESENT:

AUSIM AZIZI, M.D., PH.D.
MCP Hahnemann University 

L. TONY BECK, PH.D.
Tissue Engineering Sciences

JONATHAN DINSMORE, PH.D.
Diacrin

CURT FREED, M.D.
University of Colorado

DARWIN PROCKOP, M.D., PH.D.
MCP Hahnemann University 

LOLA M. REID, PH.D.
Renaissance Cell Technologies

JORDANA SONTAG

	C O N T E N T S

AGENDA ITEM	PAGE

CONFLICT OF INTEREST STATEMENT
	by Ms. Gail Dapolito	15

FDA INTRODUCTION
	by Dr. Malcolm Moos	19

OVERVIEW:  STEM CELL BIOLOGY 
CURRENT KNOWLEDGE AND UNANSWERED QUESTIONS
	by Dr. John Gearhart	24

ISOLATION, IDENTIFICATION, AND CHARACTERIZATION
OF BONE MARROW-DERIVED NON-HEMATOPOIETIC STEM
CELLS CAPABLE OF ASSUMING NEURONAL PHENOTYPES
	by Dr. Catherine Verfaillie	61

CHARACTERIZATION OF LINEAGE-RESTRICTED,
SELF-RENEWING PRECURSORS FROM
NEUROEPITHELIAL STEM CELLS
	by Dr. Mahendra Rao	101

ISOLATION, IDENTIFICATION, AND CHARACTERIZATION
OF ADULT HUMAN NEURAL PROGENITOR CELLS
	by Dr. Steven Goldman	128

ANTICIPATING ETHICAL ISSUES IN CLINICAL EXPERIMENTS
USING STEM CELLS FOR NEUROLOGICAL DISORDERS
	by Dr. Jeremy Sugarman	173

OVERVIEW:  ANIMAL MODELS FOR PROGNOSTIC SAFETY
ASSESSMENT OF NEURAL STEM CELL TRANSPLANTATION
IN HUMANS
	by Dr. Fred Gage	224

ANIMAL MODELS FOR EVALUATING CELL THERAPY OF
NEURODEGENERATIVE DISEASES
	by Dr. John Trojanowski	258

MIGRATION AND INTEGRATION OF TRANSPLANTED STEM
CELLS WITHIN THE RECIPIENT NERVOUS SYSTEM
	by Dr. Evan Snyder	298

ANIMAL MODELS OF SPINAL CORD INJURY AS 
PARADIGMS FOR STEM CELL THERAPY
	by Dr. John McDonald	326

	P R O C E E D I N G S
	(8:40 a.m.)
		DR. SALOMON:  Good morning, everybody.  I'd like to welcome you to the latest meeting of the FDA's Biological Response Modifiers Advisory Committee.  My name is Dan Salomon.  I've got the pleasure of chairing this session today and tomorrow.  Again, I'd like to welcome all of you to the Hilton Hotel.
		What I always try and do at the beginning of these sessions just so that we get to know each other because a lot of people here don't know each other -- I certainly don't know everybody on the committee.  There are some experts here from the field of neural stem cell transplantation and neural science that I'd like to have introduce themselves.  So, what we've usually done is just gone around the table so everybody gets a quick idea of who's sitting here.  So, I'd like to do that.  It's a little more difficult in this setting because of the way we've sort of staggered these chairs.  It's usually easier because it's a circle, but if we could start maybe at the last table in the back right and sort of go through there.  Can you just tell us briefly who you are and your area of interest?
		DR. CHIU:  Arlene Chiu and I'm from NINDS, the Neurology Institute.
		DR. MULLIGAN:  I'm Rich Mulligan from Harvard Medical School and Children's Hospital and involved in stem cell research and gene transfer research.
		DR. NOBLE:  Mark Noble, University of Rochester.  I'm a precursor cell biologist working in both general principles in precursor cell biology and in studying oligodendrocyte biology and repair of demyelinating damage.
		DR. GEARHART:  John Gearhart, Johns Hopkins Medicine, interested in human embryonic stem cells.
		DR. GAGE:  Fred Gage from the Salk Institute.  I'm a neurobiologist.
		DR. GOLDMAN:  (Inaudible.)
		DR. SALOMON:  I should point out to everyone that you press the button.  This little red light turns on for all us who forget to turn it back off again after you've spoken. 
		DR. RAO:  Mehandra Rao from the University of Utah.  I'm a neurobiologist interested in stem cells in the nervous system.
		DR. SNYDER:  Evan Snyder, Children's Hospital, Boston.  I'm a neurobiologist and also a pediatrician, and I study stem cell biology.
		DR. VERFAILLIE:  Catherine Verfaillie at the University of Minnesota.  I am a hematologist.  I'm interested in stem cells from bone marrow.
		DR. O'FALLON:  Michael O'Fallon, Mayo Clinic, a member of the committee.  I'm a biostatistician.  I'm not quite sure what I'm going to make of all this basic science here in the next two days, but I'll be very interested.
		(Laughter.)
		MS. WOLFSON:  Alice Wolfson.  I'm an attorney from San Francisco and I'm the consumer representative.
		MS. DAPOLITO:  Gail Dapolito, Executive Secretary for the committee.
		DR. SALOMON:  Dan Salomon.  I'm from the Scripps Research Institute in La Jolla, California.  My interests are in gene therapy, hematopoietic stem cell transplantation, and islet cell transplantation and organ transplantation.
		DR. SAUSVILLE:  My name is Ed Sausville.  I'm a medical oncologist from the National Cancer Institute in the Developmental Therapeutics Program, and my interest is in the development of novel small molecules and biologicals for the treatment of cancer.
		DR. AUCHINCLOSS:  My name is Hugh Auchincloss, and I'm a transplant surgeon at Harvard Medical School and a very recent member of the Biological Response Modifiers Committee.
		DR. CHAMPLIN:  Richard Champlin.  I'm with the blood and marrow transplant program at the M.D. Anderson Cancer Center.  I'm a hematologist.
		DR. DRACHMAN:  David Drachman, U Mass Medical Center.  I'm a neurologist with an interest in Alzheimer's and other degenerative disorders.
		DR. KOLIATSOS:  Vassilis Koliatsos from Johns Hopkins.  I'm a neurobiologist and clinician interested in neuroplasticity and the mechanisms of regeneration in CNS in the context of neurodegenerative disorders. 
		DR. KORDOWER:  Jeff Kordower from Rush Presbyterian Medical Center in Chicago.  I'm interested in gene therapy and cell transplantation.
		DR. MACKLIS:  Jeffrey Macklis from Children's Hospital and Harvard Medical School.  My lab studies the repair of circuitry in the cerebral cortex by neural transplantation or activation of endogenous precursors.
		DR. WALKER:  Michael Walker, neurosurgeon, Neurology Institute, NIH.
		DR. WILCOX:  I'm Barbara Wilcox.  I'm a neurobiologist with CBER.
		MS. SERABIAN:  I'm Mercedes Serabian with the Office of Therapeutics Research and Review of the Division of Clinical Trials.  I'm a toxicology reviewer.
		DR. FINK:  I'm Donald Fink with the Division of Cell and Gene Therapy.  I'm interested in neurotrophic factors.
		DR. MOOS:  Malcolm Moos, also from Cellular and Gene Therapy.  My research interests are in pattern formation and cell and tissue fate specification.
		DR. NOGUCHI:  I'm Phil Noguchi, Director of Cell and Gene Therapy in the Office of Therapeutics.
		DR. SIEGEL:  Jay Siegel, Director of the Office of Therapeutics.
		DR. SALOMON:  Thank you all very much.  Again, welcome.
		I have some administrative things to quickly go over.  
		First, I'd like to welcome two new members of the Biological Response Modifiers Advisory Committee:  Dr. Joanne Kurtzberg and Ms. Alice Wolfson, our new consumer representative.
		There are also five panelists today who have been introduced but who are participating as guests, and I'd like to read their names just into the record.  It's Dr. Fred Gage, Dr. John Gearhart, Dr. Richard Mulligan, Dr. Mark Noble, Dr. John Trojanowski, and Dr. Arlene Chiu.
		I've also been asked to inform you that there are some revised questions in the blue folders.  This continues to be an iterative process.  Who knows.  There may be revised questions tomorrow too.
		What we're going to do is have presentations today with some discussion certainly.  However, when discussions begin to veer toward things that are very specific questions for tomorrow, we'll kind of get into them a little bit and then decide to put them off or amplify them or return to them tomorrow.  We'll just have to see what the chemistry is for that. 
		Finally, as Chair, I feel like the most important thing that happens in the next two days is that everybody sitting at the table today, as well as those of you in the public, feel like you have had access to make your points clear and add to the conversation.  I think that's what we're here to do.  Certainly on my part, there's no specific agenda except to address the questions, as best we can, that the FDA has put to us.  So, if at any time, as things progress two days, somebody feels like I didn't get my point across or something, I really would rather have you come to me at that point and I will do everything possible to bring the point back around to discussion and have everything included.  I say that also for the people in the audience.
		So, with that, Gail Dapolito will read us the conflict of interest statement. 
		MS. DAPOLITO:  Yes.  Good morning, Dr. Salomon.
		I'd like to also take this opportunity to introduce Ms. Rosanna Harvey, the committee management specialist.  She and Ms. Denise Royster will be at the registration table to help out with any questions or assistance you might need for the committee.
		I'd like to read the conflict of interest statement.  This announcement is made part of the public record at this meeting of the Biological Response Modifiers Advisory Committee on July 13 and 14, 2000.  
		Pursuant to the authority granted under the committee charter, the Director of FDA's Center for Biologics Evaluation and Research has appointed Dr. Hugh Auchincloss as a temporary voting member.  
		Based on the agenda made available, it has been determined that the agenda addresses general matters only.  For this meeting, general matters waivers have been approved by the agency for all special government employees who are participants.  The general nature of the matters to be discussed by the committee will not have a unique and distinct effect on any of the participants' personal or imputed financial interests.  
		In regards to FDA's invited guests, the agency has determined that the services of these guests are essential.  The following interests are being made public to allow meeting participants to objectively evaluate any presentation and/or comments made by the guests.
		Dr. Arlene Chiu is employed by the National Institute of Neurological Disorders and Strokes, NIH.  Dr. Fred Gage is employed by the Salk Institute.  He is a scientific advisor for Cell Genysis, Signal Therapeutics, and Stem Cell, Inc. and has financial interests in several firms that could be affected by the committee discussions. 
		Dr. John Gearhart is employed by Johns Hopkins University.  He receives financial support for his laboratory from Geron.  
		Dr. Richard Mulligan is employed by Harvard University. 
		Dr. Mark Noble is employed by the University of Utah.  He's also a founding scientist of Acorda Therapeutics.  He consults with Acorda and has a financial interest in a firm that could be affected by the committee discussions. 
		Dr. John Trojanowski is employed by the University of Pennsylvania School of Medicine.  He's the co-founder of Layton BioScience, serves as the principal investigator on several federally supported grants, and has a financial interest in a firm that could be affected by the committee discussions. 
		In the event that the discussions involve other products or firms not already on the agenda for which FDA's participants have a financial interest, the participants are aware of the need to exclude themselves from such involvement and their exclusion will be noted for the public record.
		With respect to all other meeting participants, we ask in the interest of fairness that you state your name, affiliation, and address any current or previous financial involvement with any firm whose product you wish to comment upon. 
		Copies of the waivers addressed in this announcement are available by written request under the Freedom of Information Act. 
		At this time we would also like to request that as a courtesy to the participants and to your neighbors in the audience that cellular phones be turned off.  Please step outside in the foyer if you wish to use your cell phone.  We also ask that pagers be set on the silent mode.
		Thank you very much.  Dr. Salomon, I'll turn it over to you.
		DR. SALOMON:  Basically the only other thing I'll say -- you guys got the drill as we went around.  One of the things that's very important is that we create a written record.  Just to make it easier for the transcriber, try and speak every time into the microphone, and when you're done speaking, turn the thing off, otherwise it picks up the background and she won't be able to get a clear recording.  
		Then let's start.  We begin with the FDA introduction by Malcolm Moos.
		DR. MOOS:  Good morning, everyone.  I'd like to make a few general remarks aimed largely at the audience because they're not quite as familiar with the process of these advisory committee meetings as some of those of us sitting around the table. 
		One of the more commonly held models of the nature of the interactions between the Food and Drug Administration and its constituent body as the public/patient advocacy groups and so forth is depicted schematically on the first slide.  
		Now, although I'd like to point out that if circumstances warrant, we do have the statutory authorities for things to degenerate to this level.
		(Laughter.)
		DR. MOOS:  By and large, that's not how it works.
		In fact, if we go back to civics 101, it's important to remember that the public, through the President and cabinet officials and through Congress, delegates to us the job of riding herd on the development of promising new therapies.  In fact, one thing that we are empowered specifically to do, in order to help us do our jobs right, is to go directly to experts in the public in order to gain the expertise that we need to approach difficult, new issues. 
		Now, certainly the FDA has to be sensitive to emerging technologies.  For those of you in the back who can't read the caption, it says, "Look what they're doing," they being the folks who've gotten the clue that flying is faster than walking.  
		So, it's natural for us to start looking for new technologies with enthusiasm, but at the same time, it's important to understand that -- the caption reads, "early experiments in transportation" -- there are certain pitfalls that can be anticipated.  And perhaps more dangerous than difficulties that are obvious are cases that arise when we are confident in our abilities, we think that we are cruising at altitude and that everything is going to be a smooth flight, when in fact our judgments have blinded us to the obvious potential for great misfortune.  
		Although I think Mr. Larsen's cartoons make some of these points very elegantly, I don't want anyone to get the idea that we think of this as a big joke.  There is great excitement in the issue of stem cell biology, but at the same time, there are various types of hazards, some of which we can foresee, some of which we can't.  It is important to let everyone know that we have to be very sensitive to the fact that the most fundamental tenet of clinical medicine is:  "First do no harm."
		The task and advisory committee meeting I think is summed well by what a Hewlett Packard executive formulated as the quadrants of confidence.  He started with conscious confidence.  You know something and you know you know it.  I think this quadrant right now is perhaps the smallest of the ones that we have.  It will be very useful for us to establish as a group what sorts of things about the biology, the manufacture, the testing of stem cells we are fairly confident in that we think we know.  
		It will be also useful to address this quadrant, the quadrant of conscious incompetence, things that we know we don't know that we have to find out.  In fact, I look at this quadrant as the quadrant of the professional.  The professional establishes the things that really need to be taken care of and addressed carefully and one by one systematically pursues this.  We want to figure out how we can take precautions about this quadrant, how to be the competent copilot in the previous cartoon, to take things from the arena of where we don't know that we don't know it and place it first into this quadrant and then into this quadrant. 
		I'll leave this quadrant until later.  This deals with issues that we don't worry about until licensure, and we can talk more about that in a subsequent workshop.
		So, the other thing I'd like to establish is certain things that we're not going to talk about.  We have a limited amount of time and a lot to get accomplished in that time, and there are certain things that are going to be off the table. 
		The first thing we're not going to talk about is whether the FDA is going to be regulating these things.  There has been a court decision that reinforces the 1993 stem cell policy that we promulgated, and in fact, the entire convening of this workshop is predicated on the fact that we will have jurisdiction over stem cell based products. 
		Similarly we're not going to talk about xenotransplantation because we've already done that.  We are quite sensitive to the fact that many types of manufacture of these products involve things like mouse feeder cells and perhaps other types of technologies that will involve animal tissue, but those issues are not specific to stem cell or neural stem cell biology and so they will not be addressed today.
		Similarly, there are some issues that would be important to address in trials involving diseases of the nervous system which are not specific to therapies using stem cells, and we won't talk about them. 
		Finally, we are quite sensitive to the fact that certain sources of stem cells are very controversial and certain sources are not.  The FDA does not have a position on whether embryonic or fetal tissue is or isn't appropriate.  We don't have the expertise to make that call.  We don't have the authority.  Finally, whether stem cells are coming from fetal or embryonic tissues or from adult tissues or other types of sources or not, the way we look at testing, control of the manufacture, the right types of animal experiments is pretty much the same.
		So, to place this in a pithy nutshell, what we want to do in the next day and a half or so is to figure out what we know, if there are areas of consensus.  If there's not consensus, are there a few sharply divided viewpoints?  What are they?  And finally, what must we learn both now -- by that I mean before any human trials can start -- and later, which means before marketing approval.  The reason that this last thing is on this slide at this early stage is that there may be some kinds of technical issues that need to be addressed that are going to take quite a while, and we want the whole community to be aware that this may be the case so that they won't be on the critical path to licensure.
		With that, I will close and turn the discussion over to Dr. John Gearhart.  I think I'd like to say in closing that we're very excited to have the panel of experts that we have managed to convene and have enjoyed very much working closely with our colleagues from NIH in establishing this.  Dr. Gearhart.
		DR. GEARHART:  Good morning.  I'm obviously delighted that there are certain topics that are off the table and that we can talk about some of the biology. 
		Now, even following an hour conversation with Malcolm the other day, I'm still not sure what I'm supposed to talk about.  Now, this may sound strange to a certain extent.  There is a lot, as far as the experimental side of things with human embryonic stem cells, going on.  So, I think what I'll try to do is to summarize where we are in this field, giving you some examples but, because of restrictions in time, not dealing with a lot of examples.
		One of the lessons that I have learned this past year was nicely summed up actually in Sherwin Neuland's admonition to the FDA in Monday's New York Times op-ed piece on a different topic, but dealing with the fact that we should really make a habit of two things when we're dealing with human disease.  One is to help, or at least to do no harm. 
		Our thoughts this past year have really turned to the latter.  We are thrilled by what the cells are doing in culture and in some of our initial experiments with respect to animal studies, but our attention is now more focused on how to demonstrate that these cells will do no more as far as harm is concerned.
		In the first part of this talk, I'm going to talk a little bit about stem cells.  Now, we have obviously a distinguished group of investigators on stem cells, and anymore I'm almost afraid to define a stem cell.  Things have changed so drastically over the past year, but nonetheless, it won't stop me from attempting to do it.  So, if I could have the first slide, please.
		Well, stem cells have two important properties, the first of which is a capacity for self-renewal, which means that they have the ability to produce more cells like themselves.  The second property that is important is that a stem cell is capable of differentiation.  It's these two properties and certain degrees of it, which we can talk about, that define stem cells.  It's really a definition that's experimental in nature.
		Now, in this definition we say nothing about the extent of proliferation.  Some stem cells we know can divide indefinitely; others have a very much more short span, or the cell cycling rate of those divisions and also the developmental plasticity of a stem cell.  What can it form?  As we're learning now, where we thought there was less plasticity in stem cells, we're now seeing a great deal of plasticity. 
		In the classic picture of stem cells -- and this really derived from the work of Till, McCollough, Seminovich in the '70s and was borne out by types of experiments, which we need not go into at the moment -- we had a picture that in very early development, we had cells that were capable of forming a large number of lineages.  As development progressed, these cells became much more restricted in their lineages, but still had capacities to divide.  And finally, we would get into this area we call lineage-restricted stem cells.  They would be for neural components or hematopoietic components, et cetera. 
		Now, obviously, although this type of chart is still illustrative of what we think is going on, maybe the issues of where in this context these cells are are going to vary, and you'll hear some examples of that today.
		So, where do we get stem cells?  In this drawing, what is depicted is in human terms.  We don't know all of this as far as humans are concerned.  Most of it is animal work, but we know that we can get stem cells from pre-implantation staged embryos, these structures here, just before implantation into the uterine wall, and we'll talk about that in a few minutes.  
		We know that the fetus has been a good source of various kinds of stem cells, and I'll talk about one specific kind this morning.  And Evan and others will talk bout other sources here. 
		Finally, over this past year and a half to 2 years, the realization that the adult is also a very good source of a number of stem cells.  The interest of this conference is with respect to the central nervous system, but we know bone marrow, we know muscle and others are now viewed as having stem cell populations with a great deal of developmental plasticity.
		Well, let's concentrate on embryonic sources. These structures here represent embryos that are within the first week following fertilization.  They are about 100 microns in diameter, just visible to the human eye.  These structures here are what you would find in the oviducts on the way to the uterus in human terms.  
		They consist of a couple cell populations, depicted in this cartoon, in which we have an outside layer of cells here which is responsible for the implantation process, and these are the cells that will give rise to placental tissues.  In an embryo of 200 or 300 cells, about 85 percent of the cells are in this layer here. 
		There is also another group of cells present, referred to as the inner cell mass.  These cells are the direct precursors of the embryo proper.  
		So, we have embryonic structures formed out of these, and extra-embryonic, placental structures out of this outside tissue.
		If you remove the outside tissue -- and you do this through immunosurgery -- place this group of cells in culture under specific conditions, you can derive embryonic stem cells.  This was initially done in the early '80s in the mouse, and with respect to the human, Jamie Thomson's group in Wisconsin reported this in 1978.  Since then, four or five other groups have now obtained human embryonic stem cells using this structure here called the blastocyst, which is derived from in vitro fertilization techniques.
		A second procedure that has been found in mammals to work is depicted in this slide with respect to a human embryo.  Investigators in the early 1990's were interested in obtaining cultures of primordial germ cells, and their interest here was really to study germ cells.  The primordial germ cell represents the lineage that's set aside very early in embryogenesis that gives rise to eggs and sperm.  They are diploid.  This is before the meiotic events. 
		It was found in the rodents and subsequently by us in humans that we can identify the stage in which these primordial germ cells can be recovered, placed in culture, and embryonic stem cell lines obtained from them.
		I'm going to now talk about our lines in some general terms, to give you a feeling of where they come from and what we've done with them.  The end product here, though, is very similar to the type of cell that Jamie Thomson and others have isolated from blastocysts, and I'll tell you about differences as we go along. 
		This is a 3-week human embryo.  It's a drawing from Emil Vecci's classic work illustrating the embryonic axis here with the developing CNS, heart, caudal region, the yolk sac, and this is going to develop into the amniotic cavity.  Early, one sees a group of cells, 50 to 100 in number, that's located extra-embryonically.  These cells arose embryonically, have been translocated to an extra-embryonic site.  And over the next few weeks, these cells divide and then eventually -- this is a sagittal section through a 4-to 5-week human embryo.  You see these black dots here that represent the migratory path that these cells have taken from an extra-embryonic site coming through the gut epithelium, the dorsal mesentery, into this large structure here, which will condense down to form either an ovary or a testis. 
		These cells now number in the thousands.  We estimate in humans that there are about 20,000 cells when they come to this ridge or this developing gonad.  It's during this period of time that we isolate these cells.
		In actual tissue or what one is looking at here is this bundle of tissue that comprises the developing gonad, along with rudiments of the kidney.  It's this tissue here that we recover from an embryo, the crown-rump length of about 1 centimeter.
		We place these cells in culture.  They are individual cells to begin with, these large cells.  They are extremely mobile in a dish.  This is the mouse equivalence to the human.  And over a period of several weeks, under appropriate culture conditions, they sit down and form colonies.  And we'll talk about these colonies. 
		Now, what's important here?  Well, there are several issues that are important here.  
		The culture conditions for these cells we find have a requirement for a specific group of growth factors.  We grow them in tested fetal calf serum, and we grow them on feeder layers.  All of these, of course, raise issues as relates to how reproducible the results are, and the source, for example, of fetal calf serum and of the feeder layer requirement that Malcolm had alluded to. 
		These cells have an absolute requirement for the feeder layer, and we use a mouse feeder layer.  We can also use human, certain tissues from the human, but this has been a problem.  We are trying to grow these cells off of the feeder layer.  It has proved to be quite difficult up to this point, but it is certainly a target of our work.
		That calf serum which has been found to be ideal for mouse embryonic stem cells is not good for human embryonic stem cells.  So, we now get into this issue of how do you test and what is it you're looking for here without really looking on the human cells.  We're trying to find surrogate cells to test this on to begin with, but that's the nature of the beast.
		There are some laboratories that are making progress in using serum-free media with mouse embryonic stem cells, but the rate of growth, the rate of differentiation, and the variety at this point of differentiative products is very minimal, as one may expect, because of the source of growth factors and whatnot in the calf serum.
		Not all cells placed in culture grow, although it is a very high rate of growth.  Not all grow and some grow to limited degrees as well.  Again, we don't know the basis of this at all. 
		This illustrates the colony morphology of these stem cells.  They have a cobblestone appearance and this appears a few weeks after being placed in culture.  In the background here, one can see the feeder layers.
		Well, how do we define a stem cell?  Now, this gets into another area, and that is that we have a series of biochemical markers, of cell epitope markers, molecular markers, the sum total of which we say you have a stem cell.  No one marker is indicative of the stem cell.  And this is another issue.  Some of these markers are stage-specific embryonic antigens, and we find, for example, that they vary among species, so that what is appropriate for the mouse may not be appropriate for the macaque or the human.  So, we look at the consistency of this panel rather than any as an absolute marker.  
		And this is another issue.  We are trying to define these cells from the molecular standpoint, their gene expression profiles.  But we're a long way from solving this issue at the moment.  So, it's another area that we have to really explore.
		Now, having said all this, I would say that we will not use embryonic stem cells as the source of tissues for direct transplantation into anyone.  These are pluripotential cells that can form many different structures, and it's really the derivatives of these cells that we are after.  So, this is a starting material.  This isn't what's going to be a licensed product I believe that's going to go into a patient.  The concern here is that if you transplant these cells, without question you're going to get teratocarcinomas.  And we've done this extensively in mouse many years ago.  So, this is starting material.
		It's important that these cells to me are karyotypically normal and that over many passages they remain so.  You can have male or female cells.  That's an important issue. 
		Now, another aspect of this is that we have embryonic stem cells, which is a term used very generally, but it refers specifically to those cells that are derived from the inner cell mass, those cell lines.  The type of cell that we've derived from primordial germ cells has another name, embryonic germ cell, and it was given that name to distinguish it from the blastocyst-derived ES cell.
		The issue here is how similar or dissimilar are these two cell populations.  I would tell you that where both of these in the mouse would go germ line, if you want them, you can get in vitro differentiation, and you can get tumors if you place them in different sites within an animal.  They share these features in common.  But the cells are not identical.  We know of a number of differences between ES and EG-derived cells from mouse studies.  No lab has yet had both human ES and EG in them to study together, but they are different.  There's no question that they are different.  They're derived from different sources, as you can see.  I want to emphasize this.  So, what maybe come down for ES cells as a uniform kind of thing will be different from what we would expect from EG.  But we do know that they are capable of all of these functions down below here. 
		Now, some of you may remember this from last fall.  In Germany investigators are not permitted to use human ES cells, but they're permitted to use human EG cells.  And there are reports that the imprinting within these cells, which is a molecular -- what it is, in alleles that you may inherit from your mom and dad may have a different molecular structure about them that comes on to it subsequently from your inheritance that are then expressed in a specific pattern during development.  This phenomenon is allele-specific.  It's called imprinting and it's very important.  If that imprint is not there, it can lead to birth defects or it could lead to death. 
		What we have found with the human lines we're working with in looking at several loci, looking polymorphisms of expression at these loci, and the methylation patterns, is that these cells are normally imprinted.  But it is an issue. 
		But I should now back up and tell you that even among mouse ES cells and among mouse ES/EG lines, that there's a variability in a number of parameters.  There's no uniformity there either.  So, it may not be expected in the human side of things, but it's just something to be concerned about. 
		What's in a word?  Just to remind you that we refer to these cells as being pluripotent because they cannot form the extra-embryonic structures of the placenta.  If they could, we would probably refer to them as totipotent.  That means they could form all cell types, but totipotent carries with it the issue of being able to form an embryo.  But these cells are pluripotent.  Another term that's used is multipotent here. 
		We've talked about embryonic germ cells, EG, ES.  Another one to alert you to are embryonal carcinoma cells.  These are all very closely related.  The top cell type here is the stem cell of teratocarcinomas, of these special tumors that arise from germ cells.  
		As many of you are aware, cell lines derived from human EC cells are now used in a clinical trial in stroke at the University of Pittsburgh.  Whether there is concern about this with respect to the origin of the cells, the fact that they're hyperdiploid, and whatnot, is something that I am concerned about.  We can talk about this later, but they are related in origin.
		So, now we have these stem cells in culture, these pluripotent stem cells.  How do we get them into these different derivatives that we'd like to use in basic science studies or in therapies?  So far to date, the procedure involves really one of affecting their environment.  We know that in mammalian development that the fate of cells in early embryogenesis is really determined by the environment that these cells see.  So, by controlling that environment, you can then determine the fate, or at least enhance or affect the fate, of a cell.
		How do you do this?  Well, different combinations and permutations of growth factors and cytokines.  This is what has proved to be most effective.
		With embryonic stem cells, the state of the art is that you first work through a structure called an embryoid body.  There is an "i" and a "d" on this, just to alert you.  It's a pathologic structure.  It's not an embryo.  What these are are aggregates of these cells that you permit them to aggregate in culture.  When you get these little balls of cells, they are depicted like so.  You can have hundreds of cells or thousands of cells, and then by treating these embryoid bodies with cytokines or dissociating them and treating them with cytokines, one can then isolate from them different cell types.  This is a histologic picture staining for some neuroendocrine cells within an embryoid body.  What you then do is go in and isolate and enhance the growth of these cell types. 
		So, this is done -- for example, you'll hear from John McDonald of taking embryoid bodies, treating them with retinoic acid and this enhances to some degree the formation of neural structures.  About 10 percent of your wells will have some neurons in them under these conditions, and this is an illustration of some of those.  That's unimportant.  It's been well-documented and published.
		Ron McKay came up with a very nice procedure that we use routinely now, of taking embryoid bodies, treating them with a series of cytokines, forming both neurons and glial populations of cells.  This procedure takes a few months in culture.  And then out of these pots at the bottom, one can identify specific types of neurons or glia.  This is the pots that we use for transplantation, and it works very well.  But again, the important point here is that this is all being done from the outside of a cell by treating them with different cytokines. 
		These illustrate then the staining properties that one would like to see in some of these neuronal precursors and finally into some specific types of neuron. 
		This is not 100 percent.  In fact, you're lucky if it's 25 percent.  There has been a recent publication from Ron McKay's lab where he's now getting upwards of, I think it is, maybe in the 40 to 50 percent range of dopaminergic neurons by specific growth factors downstream. But again, this is not a process which is 100 percent.
		Now, I want to give an illustration here.  It's not on your mind, being mostly neural inclined here, but I want to make a point with this.  We have been trying to isolate hematopoietic stem cells from our populations of cells.  One can do this using techniques that Michael Wiles and Gordon Keller have used.  We're again starting with embryoid bodies and treating these with cytokines that we know are important in hematopoiesis.  We can get at pots of cells that are enriched for different lineages within hematopoiesis, be they going through the erythropoiesis with macrophages or whatnot.
		Now, the important thing here is this.  We've been taking these cells that have been isolated and we've been cell sorting at this level here for specific antigens, whatever the latest is for defining a hematopoietic stem cell, and then transplanting these into lethally irradiated animals.  So, we've done this with a number of antigens.  It's unimportant here.  
		The important point is this, that we are getting long-term grafts, but along with that, out of over 150 animals that we've transplanted, 2 of these animals have developed teratocarcinomas.  And we're concerned about this.  What have we done here?  Have we carried along a stem cell after literally weeks in culture and then cell-sorting based on antigens that we know embryonic stem cells don't have?  What has happened here?  Have we had some kind of reversion or whatnot?  Don't know, but it is of concern to us. 
		Now, let me tell you where our work is currently and where we think that most of the cells that we're going to use in the human story are going to come from.  Slightly different than what I just told you about, about taking embryoid bodies, treating them with cytokines, and trying to enhance or direct differentiation into specific lineages.
		We know that embryoid bodies can have a variety of cell types within them.  So, our current experiments deal with taking an embryoid body, dissociating it, playing it out in conditions that are ideal, or have been reported to be ideal, for specific cell lineages, be they neural, hematopoietic, endothelial, endodermal, whatnot, and then cloning the populations out of this, and using these cells then as the source of material for transplantation studies.  So, part of this paradigm is you're growing up these embryoid bodies that come in different varieties.  They can be cystic.  They can be solid groups of cells.  Over time you can enhance within those or select within those populations of cells. 
		We can use markers.  This is an example of different lineages, just PCR-based markers as endpoints to say that we're into a specific lineage. 
		This is a very busy slide, but what it shows is some of the markers for different lineages and whether we've used a PCR-based analysis or antibody to detect on the surface of cells or in cells the specific markers.
		This illustrates our endodermal markers that we're now using for pancreatic and liver development, some of the markers we use there, an antibody to verify that we have expression.
		This is an important slide.  What we have done is to take these clonal lines that -- we initially were looking at just different groups of markers to say we have neural, endothelial, whatnot.  The cell lines grow robustly, on and on.  We were concerned about the following thing, that when we went back and took our neural cells and looked at other markers, hematopoietic markers or anything else, we would find those markers also on many of the cells.  So, initially we were just focused on specific subsets of genes, but when we back and tested all of them, we would find they have a lot in common.  So, this may say something about using molecular markers for defining a cell population.
		But we were concerned that we had a heterogeneous population, but when we did subcloning from these, we found that, indeed, there were characteristics of all the cells within these populations.  I think it's an important lesson here both in the biology maybe of stem cells and in any type of marker system we would like to use to define a given population of cells for product. 
		Now, we are just in the initial phases of going into culture modeling, into animal models.  We're going this with Jeff Rothstein and Tony Ho at Hopkins and a number of other groups.  So, we don't have a lot of data other than some preliminary things to say, that when you take some of our human embryoid body derived stem cells, that we can, indeed, form some motor neurons or oligodendrocytes and whatnot under appropriate conditions.  We don't have a lot of data on this.  We're moving into stroke models shortly with Dick Traytsman, Parkinson's, others.  So, this is where our work is at this point in time, defining some of these stem cells and moving into mouse modeling.
		These are examples of forming oligos and whatnot and motor neurons. 
		Now, we're not going to get into these kinds of things.  I didn't know what to be prepared for.  I want to skip ahead, though, to just a few items. 
		One is that this field is evolving.  There are a large number of investigators working on mouse embryonic stem cells, wanting to work on human, working up conditions, as I mentioned, of trying to get feeder layer independent cells, of trying to define the growth paradigms to get into different lineages, to use also genetics to try to create transgenic lines, if you will, that would only then give rise to specific lineages but using genes that are early on in pathways.  This is sort of where we are from that standpoint.
		Now, a couple of things that we're looking at downstream.  One is we've already shown that we can get a number of derivatives out of this.  Whether or not they're lineage-restricted, we are now giving our hematopoietic cells to the neural people and vice versa to see if they will give rise to appropriate structures.  
		We're interested down here in the issue of transplantation and what we can do genetically within these cells to try to make, for example, a universal donor.  But this involves transfections, gene modifications.  That again is an issue to be addressed.  It's not unique to the stem cell story, but at least we're using that approach.  It has to be considered.
		Another issue that many labs are working on is trying to match host to a stem cell, the issue of nuclear cloning or nuclear transfer in which we take a nucleus of an adult, place it into an egg cytoplasm and generate a stem cell out of this, so it would match that patient. 
		We're also, interestingly, moving on to reprogramming adult cells.  Now, this is taking cytoplasm from EG cells, combining it with nuclei of differentiated cells, and beginning to show that you can reprogram these nuclei.  Now, what impact will this have if you come up with a population of cells here that are stem cells in nature and you can differentiate them down different pathways?  This will happen. 
		We're concerned here with respect to the origin of tissues, of infectious agents.  The interesting thing at Hopkins, we're having some difficulty in being able to gain information in this area from any patients.  We are not permitted in the fetal tissue area to take patient records and whatnot.  This is completely anonymous in a sense.  Although identifiers are kept, we can't go back and get that information.  So, we have to do testing on all of our tissue, and even that is controversial.  The issue through the feeder layers and the issue through, for example, the serum that we're using.
		We're concerned about stable properties with continuous culture.  Is the differentiative capacity going to remain unchanged?  How do we determine this?  Do we have stable gene profiles?  And do they remain karyotypically stable over a long period of time?  These are things that we're looking at.  Can we really regulate or control the differentiation of these cells?  
		Non-tumorigenic issues here.  We have a number of fail-safe systems that we're now considering that if a cell turns tumorigenic, that we can bump it off internally.
		The issue of graft rejection and the fact that we may have to be using genetic manipulation.  Of course, that brings with it a number of factors as well.
		Now, one last comment.  As you know, there are Rhesus ES cells.  There has been a push by the NIH to use the Rhesus and Rhesus ES cells as a test before we get into human clinical trials.  That would mean that we would take these cells and do the same thing with them that we've done with the human cells or with the mouse cells.  That's an issue which I think should be discussed.  I'm not in particular favor of it at this point. 
		There was also the issue of the Rhesus studies themselves of the time frame of taking human cells and putting it into a Rhesus.  How long would you want to do this to look for tumors, for example?  And on and on.  So, there are some other issues that I think we can chat about at this meeting.
		Well, I hope in this few minutes I've given you a flavor as to where we are in these studies, where some of the points are that we should be talking about.  I still feel that the embryonic source of cells will prove to be a reliable source and a good source for finally getting into human therapies with transplantation studies. 
		Thank you very much.
		(Applause.)
		DR. REID:  Lola Reid from the University of North Carolina.  I have a couple questions.
		One is that you had defined all stem cells as being capable not only of differentiating but also of self-replicating.  That's an area where I think there's getting to be increasing controversy over whether that is always true.  Certainly for the totipotent stem cells and the embryonic stem cells that is true, that they self-replicate, but for the determined stem cell or, as you're calling them, lineage-restricted stem cells, those are ones in which, at least by assays such as the telomerase assay, they can be restricted in terms of their true self-replicative ability.  So, one issue is what is the evidence now that determines stem cells truly self-replicate.
		The second issue is that you are going through this elegant procedures from germ cells or from embryonic stem cells into lineage-restricted cells and then getting some evidence that they can be tumorigenic when you inject them back in vivo.  But if you were to isolate out determined stem cells from the normal tissue and compare those back with what you think are those lineage-restricted cells, you should be able to get a better idea of what might be distinctions in them and get better markers for being able to identify the determined or lineage-restricted stem cells from those that are in fact still totipotent.
		DR. GEARHART:  Let's take your second question first.  I would agree we would love to learn from our colleagues here what features we should be looking at at these stem cells downstream that would define a population of, let's say, lineage-restricted cells.  We have to wait for that as we're learning.  So, that will come I'm sure. 
		We are not doing that within our own laboratory.  We're relying obviously on our colleagues.
		The first issue as to whether all stem cells can proliferate where they have a self-renewal capability, I agree with you we're getting into an area now of again trying to define a stem cell.  But does it, in a way, really matter?  What you can demonstrate or what we would like to demonstrate in the use of a stem cell population is you have to have that capability, if you're going to grow large numbers of cells, to be able to effect any kind of transplantation therapies.  A cell has to have the ability to replicate.  One isn't going to just remove a few cells and transplant them.  You need really thousands, millions.  And if you go into any FDA-approved kind of thing, you're going to have to have a bank that's going to be stable and it's going to be proliferative.
		DR. REID:  The question of extensive growth potential is separate from self-replication.  Obviously, stem cells in general -- I don't know of any stem cells that don't have actually quite extensive growth potential, but the issue of self-replication is that they can form a daughter cell that is absolutely identical to the parent cell.  That has certainly been, I think, proven for totipotent stem cells, but I think the evidence is waning on even the most well studied of the determined stem cells, that of the hematopoietic stem cells.  So, a "hematopoietic stem cell" isolated from an older animal will have less self-replicative ability than one that is isolated from an embryo. 
		So, I think that that's an issue that gets muddled, particularly with reviewers or discussions.  They keep demanding that that be part of the criteria when, in fact, what we're now seeing is that it's not applicable to lineage-restricted stem cells, or determined stem cells.
		DR. SALOMON:  Yes.  Picking up to try and pick one point that I think we should try and return to tomorrow is that these questions identify an issue for how the FDA is going to look at all of this. 
		So, one idea that you floated, John, is, well, we'll have a master cell bank, which the FDA would love.  I'm not certain that the biology is going to allow that for many of the types of stem cells that we're going to bring forward into clinical trials in the near future for the reasons that the speaker pointed out, that you may be able to get so many replications of so long a time, and then you're going to have to go back to your source.  So, I think that's going to be a very important question to think about because if you regulate from a master cell bank point of view, that's a very, very different prospect than regulating from something in which, let's say, every 2 months, every 10 patients or something, you have to go back to the source.  I think we should be careful not to close that off unless the experts in the group say, no, we can really do this master cell bank thing.
		DR. NOBLE:  Mark Noble, University of Utah at the moment, University of Rochester in real life. 
		One of the assumptions, John, that's made in a lot of this work is that once you have identified a lineage-restricted precursor cell, you have essentially identified that lineage.  Now, we know from current studies in the oligodendrocyte lineage that this just isn't true, that we have thus far a minimum of three -- possibly four, but certainly three -- oligodendrocyte precursor cells with very, very different biological properties, particularly in respect to their self-renewal properties which may have tremendous implications for the utility in tissue repair.  So, as we go on to consider how we're going to develop cell banks, this question of how we're going to prospectively recognize those cells that actually do have the extended capacity for division seems to be part of the discussion we need to have.
		DR. SAUSVILLE:  Ed Sausville from NCI.
		Could you clarify what the HLA restrictions on the use of these types of cells might be and the extent to which immunologic barriers might either require diversity of sources or are not an issue?
		DR. GEARHART:  Well, it's obviously a major issue here.  We would like to think that we in the future can provide cells to patients with a minimum of immunosuppressive therapy out of this technology.  We've had discussions with a number of transplantation immunologists as to, if we failed at this, how many different cells we would have to generate from different HLA types and whatnot, that we would have a bank that could service a large population of individuals.  And there doesn't seem to be a consensus on this in any fashion of whether we should have 20, 40, 80, 100, or hundreds.  So, I can't give you an answer.  It has been a topic of debate from that standpoint.  That's about as far as we've gone. 
		We are now looking at gene alterations within class 1 and class 2 molecules to see how far afield we can go with respect to the mouse work in getting a good degree of transplantation success with a minimum amount of rejection.  So, this is where we are in mouse work, not human.
		DR. CHAMPLIN:  To state the obvious, in bone marrow transplants we use stem cells, even highly enriched stem cells, and HLA restriction there is very important, and even one HLA/allele mismatch leads to a markedly increased risk of rejection and graft versus host disease.  So, whether or not that will be true of other stem cell populations is unclear.
		One other comment in terms of assaying cells.  The phenotype of the cell depends on the culture conditions that they're being prepared and that the growth factors in the milieu will lead to differentiation in one direction or another.  So, it's not just the cell itself, but it's the conditions in which they're assayed.  So, it is obviously a highly complex question.
		DR. GEARHART:  I should have pointed out that ES cells and EG cells do not exist in situ.  These are artifacts of culture, and that's another issue here.  These aren't a naturally occurring stem cell within an embryo or a fetus.
		DR. PROCKOP:  I'm Darwin Prockop from Tulane now.  I'm here kind of as an advocate of the FDA considering the possibility of using cells from the same patient, that is, stem-like cells maybe lineage-restricted.  I think that's a real possibility.  It's a possibility that our laboratory and Berber Laboratories are pursuing.  So, you'll be hearing the cells that Catherine Verfaillie will be describing quite soon, I believe, in the next talk. 
		Under conditions we've recently developed, we seem to have no limit on number of cells we can produce.  So, from small bone marrow aspirate, we can produce 10 to the 13th cells in 6 to 8 weeks.  We have fully characterized those cells, but we don't think we've reached the limit of expansion, and they still stay multipotential.  So, I would very much hope the FDA still considers that possibility of cells coming from the patient who's going to be treated. 
		DR. SALOMON:  One thing that you brought up I wanted to get your comment on -- and I'm sure we're not going to settle it here, but I think one of the fascinating things is the observation that if you put these into an adult animal, I'm assuming, that you're getting a teratocarcinoma.  As a result, what you're doing is culturing for several months in growth factors prior to transplantation.
		One of the things that simplistically has been said is an advantage of stem cells in many different reviews of the subject has been that the signals for stem cell differentiation exists in a local site.  There's nothing simple in stem cell biology.  Forgive me for simplicity.  But the simplistic idea would be then if you put these in a site, that they shouldn't develop as a teratocarcinoma.  In other words, where are these microenvironmental signals?  Because I think thinking about these microenvironmental signals is going to be very critical, for example, in neural cell transplantation, right, in order to guide these down the right developmental paths?
		Do you have a comment?
		DR. GEARHART:  My comment on this would be a factor of cell number.  We've known for many years working with mouse ES, mouse EC cells that it's an issue -- for example, when you return cells to what's called a blastocyst, if you're making a chimera, you can override that system very easily by placing too many cells within that environment.  It may become an issue of how many cells or what the draw-down is with respect to factors within an environment based on the number of cell types that you have there.  That's an easy explanation. 
		The hematopoietic story I gave you is a little bit more complicated I think because we've had these cells in culture for months, essentially, and then FACS sorted them and then came out at the other end with these two tumors.  Now, this was in an initial experiment of which we did 70 animals.  We found them there.  We've subsequently repeated and haven't found any more tumors. 
		But is it an issue that we've taken a cell, it's somehow differentiated to a certain degree, and then has dedifferentiated, for a lack of a better term?  Or have we carried always along this stem cell -- I mean, this one that's an ES cell rather than a more differentiated type?  Don't know.
		DR. GAGE:  I think the issue of local environment is really critical in the process of differentiation of the cells.  But I also really support John's statement that the ideal cell for transplantation wouldn't be the purified, most primitive stem cell necessarily because that cell may be less able to read that local environment.  Particularly when going back into the adult host or the damaged host in some way, while there is a local environment that contains cues that can lead that cell down to perhaps appropriate lineage, it has to be mature enough to be able to read those signals.  So, once again, we come back to this idea as to whether or not the most primitive cell is actually mature enough to be read by the local environment that can drive the cell down that lineage, and understanding how to get from the primitive cell to a cell that can read those environmental cues that remain I think is really a very important challenge for everybody.
		DR. SALOMON:  Excellent.  I think that then should be considered another underpinning of the discussion in the next two days.
		DR. MULLIGAN:  In the case of the concept of lineage-restricted stem cells from your EG cells, what specific cases do you have where you can actually get from your bulk culture an amplifiable lineage-restricted stem cell population?  I'm thinking about cell banking.  I think what you were saying was it would be nice if you have something that was focused on a lineage, but I'm not clear whether you've actually shown that that is possible.
		DR. GEARHART:  Well, our lab has been looking in three areas.  One is the neural cells; the second, hematopoietic; the third is endodermal.  
		What we have been able to demonstrate is we have a number of clonal lines with the neural phenotype that have been working very effectively, long-term culture robustly growing that we've been using in a number of co-culture experiments.  They're now into the transplantation experiments.  They've been in continuous culture since last August approximately with the same markers and empirically giving us the same results.  So, we do have I think a more general neural line from that standpoint.
		The endodermal lines apparently are working the same way.
		Is that the question?
		DR. MULLIGAN:  That's the question.
		I guess then the other point is the definition of lineage-restriction, of course, depends on what it's put in and where it sits.  So, what's the likelihood that, although in vitro it may look like a lineage-restricted stem cell, again depending on where you put it, it may have great plasticity.  So, is it necessarily possible that there is such a thing as a lineage-restricted stem cell?
		DR. GEARHART:  I worry about this all time, obviously.  We are getting examples of this cross-talk of taking a neural cell, giving it to our hematopoietic collaborators, and showing that it will do something different in another environment.  So, we are seeing examples of this.  But we don't have a lot of numbers to show you, but we are learning that we have some examples of that.
		DR. MULLIGAN:  On Rusty's point, maybe it's possible that exactly the opposite might be the case, that is, having a more early cell may actually make it more capable of sensing injury and doing the right thing than a more differentiated.  So, I was curious, Rusty, what case in point makes you think that having a more differentiated stem cell would allow it to better sense local environment?
		DR. GAGE:  Well, a very specific example would be in certain areas of the brain where neurogenesis continues.  If you implant into those areas neural cells -- let's say, fetal-derived propagated cells -- that they migrate just to this region, they can differentiate into neurons as evidenced by morphological criteria.  If you take a more primitive cell like a hematopoietic cell and put them into that same area, they don't.  They get to the area and they don't read, as one example. 
		So, I'm not saying that there couldn't be other environments where primitive cells could do it, but I submit that in some cases the environment may be quite restricted as to what signals it can demonstrate.  It doesn't mean we can't change the environment, but there may be some restriction in those environments.
		DR. MULLIGAN:  I guess I would caution us that we think that this lineage restriction issue may be very, very complicated and by, for instance, taking the hematopoietic stem cells in vitro and putting them in some other broth or cocktail of factors, they may then behave like a neural-restricted stem cell.
		DR. MACKLIS:  If I could make a couple of comments on these last two points.  I think another issue we need to raise that comes directly from what Rusty Gage said is how sharply do we define the pie of what we call lineage-restricted.  There are estimates of hundreds of types of neurons in the central nervous system, maybe a hundred types in the cerebral cortex itself.  Is neural-restricted or neuron-restricted enough? 
		A second point is, regarding Rich Mulligan's point, there is evidence from our lab and many other labs that later and later stage, partially differentiated neuroblasts or neurons can at least repair certain circuitry with much higher efficiency.  I think it would argue that we really need to take those many, many steps from the immature cells up through maturity correctly.
		DR. MULLIGAN:  But, again, I think the issue that I'm raising is, is that a truly fixed property of those cells?  Although they behave in that way, do we know enough about what their state is to make sure that that state is a truly fixed state as opposed to if those cells or another cell type was, again, cultured or manipulated in a different fashion, they would behave in that --
		DR. MACKLIS:  Right.  Well, I assume that's the lower right, knowing incompetence or something.  I think we know that we don't know enough yet. 
		DR. CHAMPLIN:  Clearly at some point, cells become irreversibly differentiated.  You can't make a granulocyte into a neural cell, although that may be possible at a more primitive point in hematopoiesis.  
		At least my view of this is this is a spectrum of differentiation where there's not discrete cells as much as a continuum of cells with different proliferative and differentiative potentials that slowly diminish as the cells mature along a given lineage.  So, trying to define each point on this continuum is certainly complex. 
		DR. MACKLIS:  If I could make a quick point on that, there is some new evidence, some of it still controversial, that even what we thought were continuously differentiating glial lineage, away from the neuronal lineage, some of those cells may actually dedifferentiate into precursor cells and then back into neurons, and that's just emerging now.  And I think we all have to deal with that.
		DR. REID:  I wanted to raise the issue on this microenvironment.  One of the areas of microenvironment, that at least in the past was not discussed as much, was the matrix chemistry.  I listened to some groups from Johnson and Johnson where they were discussing issues of fetal brain transplant, and I was stunned to learn that when they're doing this for Parkinson's patients, they had to use as many as eight fetal brains in order to try to get some measure of efficacy on some of the patients. 
		And there have been preliminary studies from Titan in which, when they take particular neuronal cells ‑‑ this was not human.  I think it was in pig cells -- and bind them in any kind of adhesion state, that they got tremendous increase in their efficacy.  
		So, I think one of the whole areas of microenvironment that may help in directing toward lineages is that the adherent cell populations, lung, liver, brain, are not going to be ones where we can just simply inject them.  We may try that but I think it will be tremendously improved if the injection procedures will involve actually embedding them in some form of matrix before we inject them.  It will dramatically improve efficiency and the survivability of the cells.
		DR. SNYDER:  I think another thing that at least we in the nervous system have to realize is that we don't know exactly what it's going to take to restore function in most diseases, and even though we tend to think of diseases as being one cell type that needs to be replaced, if we simply replaced the neurons, we're home free, the reality is we may need to recreate the whole milieu, which means more than one cell type, which means the neurons, but maybe also the glia that create homeostasis, that nurture, that support, the myelinate.  It may be that we need to do co-grafts.  We may need to implant very restricted cells at the exact same time as more plastic cells that play off of these cells, play off of the environment to really reconstruct the milieu.  So, it may be more complicated than simply saying let's just replace a dopaminergic neuron or a GABAergic neuron or a motor neuron.
		DR. RAO:  I just wanted to reiterate what all of this tells us is how little we do know about a lot of the things that we want to regulate.  We don't know what's true about the matrix.  We don't know what's true about definitions of the cells that we want to use, and we don't know about the environment in which we want to put the cells back in.  We should keep all of that in mind.
		DR. SALOMON:  I think that's probably a good place to stop for the moment.  We've got two days to discover what we don't know.  I suppose these things are sort of humbling experiences for all of us. 
		I certainly think that the idea of defining what is the state of knowledge and where the gray areas are is exactly what the FDA wants from us.  I don't think anybody here should be concerned about stopping and saying, hey, really I don't have the foggiest idea.  I think that's okay. 
		I'd like to introduce the next speaker.  I'm trying to remember my French.  Is it Catherine Verfaillie or Verfaillie?
		DR. VERFAILLIE:  Verfaillie.
		DR. SIEGEL:  Could I just add while she's getting hooked up?  There may have been some misperceptions from one of the questions from the floor.  We're certainly not here to decide which is the right cell source or which is the right matrix or whatever.  We're trying to get information about what are the appropriate controls, testing, and whatever to set the ground for safety for human research.
		DR. VERFAILLIE:  I will also be talking about cells that actually are not embryonal in origin but actually can be derived from some adult tissues which may have potential of differentiating in a number of different lineages, potentially also in neuronal or neuroectodermal cell types.
		This is sort of the same that you just saw from Dr. Gearhart.  There are totipotent stem cells in an embryo obviously, and then there are embryonic stem cells.  Then you have the mature lineage-specific stem cells which are present both in embryos, fetuses, and in adults.  There's a lot know, for instance, about the hematopoietic stem cells, and there's a very quickly growing body of evidence or studies that actually address stem cells for other organs both endodermal, neuroectodermal, and then mesodermal.
		My lab has for a long period of time worked on hematopoietic stem cells.  A few years ago, we started to also think about a different cell type which is called a mesenchymal stem cell, or a cell that was known to be in the bone marrow that can differentiate to certain forms of mesoderm, cartilage, bone, fat, fibroblasts.  So, that's really where the studies I will present to you today and I actually put it in perspective of the field today is starting with cells that we thought initially were mesenchymal cells.  But it seemed to have a subgroup of cells that have much broader potential than pure mesenchymal.  I will come back to this also in the latter part of my talk.  
		There is from adult sources of tissues now growing evidence that certain of these cells have plasticity and can maybe become a cell that we never thought they could become.  The question ultimately is going to be, is that really a lineage-committed cell or is it a subpopulation of the lineage-committed cells that have more immature features and actually could remain multipotent and actually aren't truly lineage-committed yet?  And I don't think there's really any answer in this area at this point in time. 
		So, when we started this, the area of mesenchymal cells has been longstanding.  Actually the first person to identify the cell is Fridenshtein back in the '70s who isolated from bone marrow a cell he called colony-forming unit-fibroblast which was a cell that, when cultured in the presence of fetal calf serum, adheres to plastic and it makes small colonies.  He initially described it in mouse.  Lots of people who work in the hematopoietic field have used this because these cells actually support hemopoiesis.
		A big group who has done a lot of work on this is the group from Arnie Caplan in Cleveland and some of that technology has actually moved to Osiris where they also used a similar approach to purify mesenchymal cells and then identified a number of antibodies that if used in combination, identifies cells that have the potential to become bone, cartilage, fat, skeletal muscle, and possibly heart muscle, although that's not 100 percent proven at this point in time.
		Paul Simmons identified an antibody that he called Stro-1 which was made against what he thought initially a CD34 positive blood cell in the bone marrow, but it ultimately turned out to be something that recognized a stromal precursor.  Therefore, Stro-1.  And if he uses this antibody together with VCAM, he can isolate, to almost homogeneity, cells that have the ability to make bone, cartilage, and fat.  And he calls these cells osteoprogenitor cells.
		Darwin Prockop, who is here, has done a lot of work on these cells too.  He also uses the plastic adhesion method.  He has been able to show that these cells can differentiate again in bone, cartilage, fibroblasts, fat, and muscle cells and possibly -- and I will get back to that too -- cells with neuroectodermal characteristics, cells that at least express proteins consistent with astrocytes and possibly even other neuronal cell types.
		When we started work, we actually thought that we should take bone marrow cells, and since we wanted non-hematopoietic cells, we depleted blood from the bone marrow by depleting 45 and then cultured these cells in relatively defined culture conditions.  The initial idea was that we were going to use these cells pretty quickly as stromal support for transplantation of hematopoietic cells or as gene vehicles for certain genetic disorders.  So, we wanted to come up with a "defined culture condition" and try to get away from fetal calf serum as much as we could to try to be able to move into the clinic quickly.  
		So, we used low glucose DMEM, PDGF, beta-beta, EGF, and then a number of nutrients in the culture system.  And 7 to 21 days after we started depleting these cells in culture, we see small clusters, some of them spindle-like and some of them almost star-like cells that grow out of these cultures.
		The frequency in an average college student at the University of Minnesota is about 1 in 2,000 to 1 in 5,000 of the 45 negative glyco-4 neo-negative cells.  If you take small children, the frequency is actually higher.  If you take people that are 50 years old, the frequency is lower.  So, it's anywhere between 1 in 1,000 to 1 in probably 8,000 to 10,000.
		We now know that what we grow out of these initial cultures is a very much of a mixture of cells of a large population of cells that are truly mesenchymal stem cells, which are cells that can make cartilage, bone, and fibroblasts and adipocytes, and then we believe a very small subpopulation of these cells which has much more multipotent capabilities.  So, we actually called this cell a multipotent stem cell, or actually being a hematologist, usually we call it a multipotent progenitor cell because stem cell has a bad connotation to it. 
		We don't know the phenotype of the cells up front.  We've done extensive phenotypic characterization of the cells once we have sufficient cells to actually find out what they are.  The multipotent progenitor cell phenotype -- again, it's not a homogeneous population, but if we have these cells in the population, the cells usually don't express CD44.  They don't express HLA-DR type 2.  They express little or no HLA-ABC, the beta-2-microglobulin negative.  They express a number of characteristics down here.  They obviously respond to PDGF, and so they have the PDGF receptor.  They have an FGF receptor, EGF receptor.  Interestingly, a small subpopulation of the cells expresses the LIF receptor, and there's gp130 present but not any other members of the gp130 family, such as IL-6 or 11 or CNTF receptor.  A small subpopulation of the cells is stained with SSEA4, which is an antibody that recognizes ES cells and EG cells. 
		Once the cells are grown to confluency, they actually only have mesenchymal cell capacity anymore.  And at that point in time, the phenotype switches.  They lose this SSEA4 expression and they actually become 44 positive.  They remain HLA-DR negative but become HLA-ABC positive and beta-2-microglobulin positive.  They also lose expression of the LIF receptor.
		If we take these cells and culture them, we can generate up to -- we have now made 70, 80 cell doublings, and that's not shown here. 
		Initially if we grow the cells and we don't subclone cells, we have a cell doubling of the majority of the cells every 40 to 48 hours.  If we go beyond here, and actually over time, we actually grow out a cell population that grows much, much slower, and the cell doubling looks almost like 96 to 100 hours.  At this point in time the cells are actually much more homogeneous.  Actually the markers that I showed you on the previous slide that would recognize cells with more multipotent potential become expressed significantly more.
		What I didn't bring, we actually have molecular markers in cells that are grown out here.  They're very low expressed here, but are expressed forms cells that are maintained for very long periods of time in culture.  There are markers that would be found in germ cells just like OCT4, SOC-2, and then obviously the LIF receptor.  So, you actually can enrich for these markers if we culture the cells for very long periods of time.  We actually get a much more homogeneous cell population.
		One of the things we've started to look at is whether these cells have extensive self-renewal potential or extensive growth potential.  So, the cells express telomerase.  If we culture the cells under the right conditions, which means very low density, and split them on a very regular basis, you can see here that we don't use telomere length after 35 cell doublings.  We haven't really gone any further than that yet.  
		This is a 52-year-old donor.  We compared telomere lengths at 10 cell doublings and 35 cell doublings compared to telomere lengths that we found in lymphocytes in the same donor.  You can see that the telomere length is much longer in these multipotent-containing cell populations compared to lymphocytes.  These is just a cell line with short telomeres and long telomeres as a control.
		Now, how do we need to do this to get these multipotent cells?  
		First of all, we can't grow them at very high cell density.  So, they have to remain under 4 to 5 times 10 to the third cells per square centimeter if we grow them.
		We have tried to clone them singly, and we have had a very, very difficult time.  So, we actually have been able to get down to 10 cells per row and 1 or 2 cells per row, but we can't do this from fresh bone marrow.  So, we actually have to grow the initial cell population and then subclone it at that point in time.  I think we can now do it at 2 cells per row, and we're trying to get down to 1 cell per row. 
		If they become confluent they actually lose this multipotentiality and they lose the longevity.  They start losing telomere length.  They do no longer express telomerase.  As I will show you, they actually don't have the ability anymore to differentiate into even endothelium, which is also a mesodermal cell type.
		We tried to repeat this in mouse.  Actually in mouse it's even more interesting because we cannot even start with CD45 negative cells.  There is something in the rest of the bone marrow that supports the initial growth of the cells.  But after you start using full bone marrow, you can actually come up with the same cells that actually have the same cell surface markers and obviously have significant cell expansion.  We actually haven't been able to show all the differentiations quite yet.  
		So, it looks like these cells are present in bone marrow from humans anywhere from the age of 2 to 55, as well as in mouse.  In mouse, there is suggestive evidence that they may actually be present in other organs as well and that they may be present in organs outside of the bone marrow. 
		As I mentioned, if we take these cells, 1 and 2 to up to 10,000 cells, depending on the age of the cells, will be mesenchymal cells mainly and a small population of multipotent cells.  If we subclone and actually try to come up with the frequency of multipotent cells, we think 1 in the initial 1,000 cells is a multipotent cell.  And by doing sequential subcloning, you can actually increase the frequency.  So, we're really working hard to come up with populations that are much purer so we can come up with positive selectable markers, whether it's cell surface or whether is by genetic markers.
		The other interesting thing that we found is that we found that these cells express the LIF receptor.  If we now sort up front from fresh bone marrow, based on the LIF receptor, we actually enrich significantly for cells with this characteristic.  However, LIF doesn't seem to be required in the culture of the human cells even though it is required in the culture of mouse cells.
		I'm not going to go through all the different lineages.  I just listed them here.  The mesoderm we can divide into splanchnic mesoderm and visceral mesoderm.  And mesenchymal cells have been defined as cells that can differentiate in most cell types of the splanchnic mesoderm, so fibroblasts, adipocytes, osteoblasts, cartilage, and skeletal muscle.
		The cell populations that we have, whether they are containing the multipotent cells or not, can be induced to differentiate along these lineages.  We can get homogeneous cultures of bone, and we can get homogeneous cultures of cartilage, which is articular cartilage, but there is also hypertrophic cartilage in there.  So, we're actually, aside from looking at some other cell types, trying to understand why we differentiate the cells too far and actually get hypertrophic cartilage rather than regular cartilage.
		If you try to induce muscle differentiation, this does not go spontaneous.  So, none of these differentiations do happen spontaneously, except that if you let the cultures become confluent, you get fat and fibroblasts.  So, in contrast to embryoid bodies where you get spontaneous differentiation with removal of leukemia inhibitory factor of certain cell types, we do not see that.  So, to induce skeletal muscle differentiation, we can either actually use 5-azacytidine or induce the cells short term with retinoic acid and maintain them in the same culture, and we get cells that are myoblastic in characteristics.  They form myotubes, so they actually have some of the contraction characteristics of skeletal muscle cells.
		Cardiomyocytes.  We also don't see spontaneous differentiation into this direction.  If we treat the cells with retinoic acid and then FGF and BMP-4, we do get cells that express markers of cardiomyocytes.  We have spontaneous areas of contraction in the dish, but significantly less so than what you see when you have embryoid bodies that differentiate.  And we actually do not have full proof that we get cardiomyocytes at the end.
		If we treat the cells -- and I didn't point this out -- the undifferentiated cells express one of the receptors for VEGF, the Flk1 positive, and so if we treat the cells simply with vascular endothelial growth factor, we can make it differentiate into cells that express von Willebrand factor and a number of other markers consistent with endothelial cells.  If we grow them onto collagen gels, they actually make vascular tubes, and so it looks like we can make these cells differentiate into endothelial cells.
		For the lineages listed here, we have data, using retroviral marking, that a single cell can give rise to all of the cells with protein characteristics consistent with the different cell types using clonal insertion site analysis of cells that were cultured initially transduced, and then we can find the same insertion location in all the differentiated progeny.
		So, compared to mesenchymal cells that have been described, which make usually these types of cells and possibly cardiomyocytes and smooth muscle cells, if we have more undifferentiated cells present, we definitely can make endothelial cells.  And I will get back to that too.  There is quite a bit of evidence in the literature that there's, indeed, bone marrow-derived cells that can make endothelium, that can make skeletal muscle, and it can make all of these cell types.
		Being a hematologist, if we think about mesoderm, the other mesodermal cell type is obviously blood.  So, based on the data from a number of investigators showing the possible existence of hemangioblasts, cells that are initially Flk1 positive but don't express any markers of endothelium or blood, but depending on how you culture them, you can make them become endothelial cells, meaning CD34 positive that then express von Willebrand factor or hematopoietic CD34 positive cells that do not express von Willebrand factor, we thought that we should at least give it a try and see if we could take the same cultures and induce them to differentiate into something that would have hematopoietic characteristics.
		To try to do that, we used a couple fetal feeder layers that were from fetal liver and tried a number of different cytokine combinations.  Initially we just analyzed the cultures by molecular markers and looked for things that were present in early hematopoietic cells like GATA-1, cKit, and a number of other things.  And we were all excited when we saw that these markers came up, but we haven't really made hematopoietic cells out of these cells in any way, shape, or form.
		But when we tried to do that, we kept seeing cells that had morphology that didn't look at all like hematopoietic cells to me.  I'm not a neuroscientist but even from back in medical school, some of these cells looked like they had at least the morphology of neural type cells.
		So, in this particular culture, we took these mesodermal progenitor cells, which again weren't homogeneous at all -- this was a mixture of cell types -- and cultured them with a number of hematopoietic cytokines, stem cell factor, vascular endothelial growth factor on this fetal liver feeder that supports murine and human hematopoietic stem cells, AFT024.  And these cultures also had EGF.  So, that's what we kept coming up with. 
		We had to go to the literature because, as I said, my lab is not at all neuroscience oriented.  So, we actually looked in the literature and found out that EGF and basic FGF are two growth factors that are important in neurogenesis.  We know that this feeder makes a large amount of basic FGF.
		So, we've repeated some of these studies in more purified conditions and actually used basic FGF together with EGF and then looked over time whether we would again be able to induce differentiation to cells with morphology and markers that would be consistent with neuroectodermal cells.  If you do this under defined conditions, 80 to 90 percent of the cells probably died during the initial phases of the culture, and then we get cells to differentiate into cells that express markers that are consistent with neuroectodermal cells, like tubulin-beta-3, neurofilaments, NSE, and MAP-2.  So, the vast majority of the cells under these conditions have neuronal markers, although there's a small subpopulation of cells, usually about 10 to 20 percent of the cells that survive, that have markers consistent with astrocytes and oligodendrocytes.  We don't have any functional data on the cells at this point in time. 
		We confirmed this by PCR and Western Blot and showed again that you find myelin basic protein, GFAP, and neurofilament-200 in the cells that are induced to differentiate, again here with EGF and basic FGF.  And we can find at the protein level the same markers to be expressed.
		In collaboration with a person at the Neurosurgery Department at our institution, we have started to take some of these cells and implant them into the brain of immunosuppressed rats.  We've implanted them mainly as undifferentiated cells.  We've taken some of the basic FGF induced cells and implanted them, and they do survive in vivo, but we don't really have much more data than that.
		We've put them in undifferentiated.  We're not sure what we actually see.  We see that cells survive, and these were GFAP-marked.  Unfortunately, the way they treated the brain, we've had a hard time getting around the problems with autofluorescence, and so we actually haven't really been able to use the green fluorescent protein and have had to come in with a secondary antibody.  
		So, you can see here, these are the cells that were implanted 2 weeks before.  These undifferentiated multipotent cells are very large and the cells that you see here are very large too.  Interestingly, some of these cells express markers consistent with neural differentiation even though at 2 weeks of time in the brain they're very large still, and we're actually not sure whether this is differentiation or whether this is nonspecific staining at this point in time.
		This is 2 weeks after transplantation into the rat brain, nestin staining, and this is 6 weeks after.  This is the area where we put the graft.  You can see cells with a much more elongated morphology than the cells that you saw on the previous slide.  They're nestin positive, but we couldn't prove at this point that these are not rat in origin and cells that were recruited from the normal rat brain into the area where we put the cells in.
		Interestingly, though, if you looked at an animal in whom we had caused a stroke and actually asked the question whether implantation of these undifferentiated cells in the brain would have a functional effect, this is shown here.  So, this is a sham animal, and this is just one test that was done 6 weeks after implantation of the multipotent cells.  This is limb placement in a sham animal.  This is an animal in whom we caused a parietal infarct 2 weeks prior to implantation of the cells infused with saline.  This is the medium that comes from the cultured cells.  So, there's just a medium from the multipotent cell culture.  And this is where we implant the multipotent cells in the brain.
		Again, this shows that there is improvement functionally, but this doesn't prove in any way, shape, or form that the improvement is due to neural connections or actually recruitment of cells present in the rat brain to the area because of the implantation of multipotent cells here.
		Now, do we have proof that any of these cells are stem cells?  In the previous discussion, there were a lot of questions about how long self-renewal has to be.  Does it have to be unlimited?  Does it have to be for a certain period of time?  We have evidence that in the most undifferentiated cells telomerase is present.  We don't see shortening of telomeres, so we've only looked really at 35 to 40 cell doublings, but we can grow the cells to 70, 80, or more cell doublings if we grow them under the right conditions, low density and with subcloning.
		We have evidence that these cells remain cytogenetically stable at least in 3 of the donors that we've tested currently.
		We have shown multi-lineage differentiation, but to prove that it was a stem cell, we would have to really be able to show this at the clonal level.  We haven't been able to sort them singly, but using retroviral markers, we've at least shown that the mesodermal differentiation is single cell-derived.  And I can't prove to you today that actually we can get some of these neuroectodermal marking cells also from the same cell derived.  
		We have no data currently on if we implant these systemically in an animal what will happen to this.  We know that they survive and you can find them in different tissues, but we do not know currently whether they will differentiate into the right phenotype.  Obviously, that's why we're working on the mouse model, to try to be able to do this in a more rigorous fashion than trying to do human into an immune deficient animal.
		So, how does this fit with what we all thought?  We all have thought about tissue-specific stem cells present in postnatal individuals, whether it's humans or nonhumans.  We've always thought that hematopoietic stem cells were hematopoietic stem cells and mesenchymal stem cells were mesenchymal stem cells and neural stem cells were neural stem cells, and that there wasn't really this ability within this population to take on other fates. 
		But over the last two or three years, there is a very significant number of papers that have come out suggesting that there is more plasticity in stem cells than we thought to be present beforehand.  What I don't know is whether these cells are committed, dedifferentiate, and redifferentiate, or if there is really a subpopulation of cells that will have this more multipotent potential that would be more similar to that of the multipotent ES cell or embryonic germ cell.
		There is the paper that came out two or three years ago now by Ferrari, showing that at least in a model where they cause damage in the muscle, bone marrow cells could contribute to muscle regeneration in the animal.
		Richard Mulligan's group has shown that a population of cells that is very quiescent in the bone marrow that contains hematopoietic stem cells, which he calls side population cells, if that is infused in animals that have muscular dystrophy, that these very rare side population cells from the bone marrow, which have hematopoietic potential, can contribute to muscle formation in these animals.  It can actually improve the muscular dystrophy in these animals.
		The Pittsburgh group a couple of years ago has shown that in a mouse transplant model, that cells that were transplanted with bone marrow into an animal can contribute to liver regeneration.  This has been confirmed by a group from Yale and New York both in mouse and possibly in the humans.
		Irv Weissman and Marcus Grompe have the same data.  If they use a, quote, hematopoietic stem cell population for mouse that is fairly well phenotypically defined, that these cells not only can reconstitute the hematopoietic system, but may also be able to reconstitute the liver.
		There's a number of groups that have shown that bone marrow can contribute to endothelium.  As I mentioned in the beginning, Dr. Prockop's group, Dr. Kopen and his group have shown that if you implant mesenchymal cells in the brain of a rat, that you have cells with astrocytic characteristics.  
		And my lab has shown that, again, the cells at least have the phenotype of neuroectodermal cells.  Whether they function as neuroectodermal cells is still a question.
		The other cell source that has been looked at is Peggy Goodell who has shown that SP cells from muscle can actually differentiate in blood.
		Then there is a number of groups who have suggestive evidence that neural stem cells might actually have the ability to differentiate into hematopoietic cells.
		So, the question really will be is there plasticity, and can bone marrow become endothelium?  And is it actually a cell that switches its genetic makeup and becomes another cell, or is there a small subpopulation of cells that can be identified phenotypically and by genetic markers that is a multipotent cell that is a descendent somehow from ES cells or embryonic germ cells that is present in multiple different organs, and if you are able to select the mouse and put them back into the right environment, can take up the fate of that environment?  So, it goes it round and round that it is indeed a subpopulation of cells and most organs that have this pluripotent potential is still a question.
		So, since this is a neural stem cell oriented meeting, what is the evidence that marrow cells can become neural cells?  There is, I would say, currently soft evidence that that might be possible.  As I mentioned, Dr. Prockop's group has shown cells with astrocyte morphology and astrocyte markers and possibly even neural markers that can be derived from mesenchymal cells derived from humans.  And our lab has the same evidence possibly in vitro as well as in vivo. 
		But I don't think there is any data currently to indicate that we go through the regular neural cell development.  We have cells that stain positive with nestin, but we don't know whether there is a neural stem cell-like cell derived from multipotent cells or whether the differentiation is actually correct.
		We obviously have no data whatsoever at this point in time that these neuroectodermal-like cells have functioned as such cells.
		So, I think in this area the biggest question really is going to be to try to, first of all, come up with a much more better defined cell population.  Currently a lot of laboratories are using plastic adhesion, which is a fairly crude method to purify the cells.  What we did initially was CD45 depletion, and that is a very crude method too.  The frequency of the cell that has this multipotentiality is extremely low in there. 
		So, a major effort in multiple labs is ongoing to try to come with positive selectable markers, because they're cell surface markers or gene trap methods to try to come up with a more purified cell population such that we can actually evaluate the cells much better and actually define exactly whether a single cell has this differentiation potential. 
		So, there is really very little known about cell surface markers.  There's very little known about the expressed gene profile.  Currently we really are depending on functional definitions, which means that you have to grow the cells for 6 months in the laboratory and be able to show that you get lineage differentiation from a single cell.  But actually coming up with these markers will be of extreme importance to try to really nail down what we have.
		There is some suggestive evidence that cells might be able to differentiate into neuroectodermal cells, but that will need to be nailed down much more extensively before these cells could be used in a clinical setting.
		The studies that I showed you on the functional recovery of some of these animals are consistent with some of the studies that Dr. Prockop has where he also shows functional improvement after implantation of these cells in the brain, but it could essentially be that you implant the cell that produces certain cytokines that recruit local stem cells or progenitor cells in the brain itself that then ultimately make functional improvements.
		Once we have nailed down the cell, then the same questions will come up as what came up with the embryonic stem cells and embryonic germ cells:  At which point do you then use these cells?  
		We have given mixtures of cells to now SCID mice and looked at them 6 to 8 weeks later and haven't seen teratomas.  The cells we injected, though, knowing what we know, back then had very few of these multipotent cells present.  So, I think if we get better at purifying the multipotent cells, we may see incorrect differentiation as has been shown for embryonic stem cells, although currently we don't really have any data to suggest that.  We haven't really seen any teratomas being formed. 
		But if you think about a cell that has the potential to make bone and cartilage and to use that cell and take it undifferentiated and implant it in the brain or any other organ, it may not be something that you want to do.  There may be some problems with even more primitive cells like ES cells and embryonic germ cells to do that.
		So, I think these questions will only be able to be answered once we have a better handle on what these multipotent cells that are present in individuals or animals have as characteristics.
		I think that's it.  I'll stop there.
		(Applause.)
		DR. SALOMON:  Yes, please.
		DR. KOLIATSOS:  Thank you.
		Dr. Verfaillie, your approach raises acutely the issue of who is what and who becomes what in this field.  In particular, the first point I would like to raise is that I would be very careful, very cautious before I characterize any type of immunoreactivity in the brain, be it GFAP or nestin or what have you, as originating from the cells you put in the brain, unless if you use dual labeling procedures or have wonderful cytological and morphological evidence.  I don't know if these are the cells you put or the reactions of the brain to the cells you put or, more importantly and perhaps more interestingly, induction of indigenous neurogenetic gradients and other processes by the cells you put in the brain.  We have a tendency to focus too much on what our cells do and we don't forget that we may change fundamentally neurobiology by putting these cells, be they hematopoietic or neural stem cells, in the brain.
		DR. VERFAILLIE:  Yes, I fully agree with that.  I think I was trying to be very cautious in trying to say what we have at this point in time.  We have cells that have these markers, but we don't have dual labeling.  The main reason is we're actually doing everything over with cells that haven't been GFAP-labeled because that seems to be a major problem in us trying to find out.  So, we're planning to either use BrdU-labeled cells or specific human antibodies to try to double label and prove that the transplanted cells are indeed the cells that have these markers.
		The cells I showed you were 2 weeks after transplant.
		DR. KOLIATSOS:  Which is not trivial to do, by the way.
		DR. VERFAILLIE:  Right, I know.  So, that's one of the reasons why we don't have it yet.
		(Laughter.)
		DR. KOLIATSOS:  The second point I'd like to say is that here we tend to be very particular about what constitutes a germinal cell or an embryonic stem cell.  Having a neuroscience background, I would like not to be less strict in how I define a neuron.  Neurons are cells that make all these wonderful phenotypic markers and at the same time make connections in the afferent and efferent sense and also generate, propagate potentials and transduce electrical into chemical in the synapse.  And for our patients in particular, I would like nothing less but our cells, whatever we put into brain, to eventually become, if they are going to be used as neurons, to become neurons with all the features of neurons.
		DR. VERFAILLIE:  Correct.  That's right.  I fully agree there too.  I think we have data to suggest that proteins get turned on that are consistent with T-cells, but we have no functional data.  Even though we saw functional improvement, I would be the last one to say that this is due to a nexus between the new cells and the host brain cells.  It might well be cytokines being produced by the undifferentiated cells you place into the brain.
		DR. SAUSVILLE:  So, to pursue that thought from our questioner, do you therefore recommend or do you think we should consider actual assays of function for particular uses?  Because if you pursue your argument, a substantia nigra neuron is not the same as a caudate neuron is not the same as a spinal cord neuron.  So, how would you address the uses, or do you think we should be addressing potential uses with respect to the functions we would assay?
		DR. KOLIATSOS:  The answer is in some form, yes.  It depends on the clinical question.  It depends on the context.  But you need certainly more information, and certainly you need to have an idea what generates your functional advantage or benefit.  Is it the cells you put or what you've done to the host tissue?  You have to have some measures to figure those things out because I believe they have clear implications in terms of longevity of the treatment approach, complications, et cetera, et cetera.
		The argument here again is that I believe I have the sense that here we're facing a dark area, a new biology which needs to be defined in a very fundamental, basic way, not what the cells are, but what also they do to the entire circuitry and whatever is going on in the brain.  It's a totally new area and needs to be treated as such.
		DR. MACKLIS:  If I could simply reinforce that comment, I would argue not just in some cases yes, but I'd say absolutely yes, that we have to figure out the disease process we'd like to reverse and whether that can be reversed simply by a neurotransmitter production locally as a mini pump versus rebuilding circuitry, and that we need to look at that anatomically and functionally.  Also, individual markers may not at all define a neuron.  In fact, a cell may express a neuronal marker and might express a glial marker at the same time.
		DR. GAGE:  Did you say that your cells make FGF?
		DR. VERFAILLIE:  No, they do not make FGF.  They're responsive to FGF, but they do not make FGF.  Actually we looked at it.  When they are treated with FGF on the date and start making it, I don't really know that.
		DR. GAGE:  Do you know what other cytokines are made by --
		DR. VERFAILLIE:  In the undifferentiated cells, we find KGF, VEGF.
		DR. GAGE:  Is VEGF secreted?  Do you know if any of these are secreted from the cell?
		DR. VERFAILLIE:  We don't know if it's secreted, but they do make it.  They make keratocyte growth factor.  They vascular endothelial growth factor, and I'm blanking on the third one that we've actually found to made at the RNA level and protein level.  We don't know the amount that is there.  We haven't looked whether they make BDNF or GDNF or any of the other neuronal growth factors and cytokines.  So, we haven't really looked at that.
		DR. SALOMON:  Have you looked at angiopoietin, by the way?  That came up in a gene screen in Science a couple months ago in CD34 stem cells.
		DR. VERFAILLIE:  We've done several arrays and I can't remember whether angiopoietin is on there.  I would have to look that up.  I don't know.  But they definitely do make VEGF.
		DR. GAGE:  So, in a general sense, ideally in neurobiology we'd like the cells to differentiate into competent cells for the local environment and reconstitute the area.  But maybe another goal we should be at least aware of or attuned to is that some of these cells might in and of themselves provide local factors which could have some reparative properties locally.  We're sort of casting this off as an artifact of the cell and, in fact, maybe in some cases that's exactly what we'd like the cells to do.  If we can learn more about what the cells do and differentiate them down lineages where they persist, perhaps in an undifferentiated state, but secret factors which can induce repair, that should be or could be a target for utilization as much as a completely differentiated cell which may be even a more difficult task to achieve.
		DR. MACKLIS:  I didn't mean in any way to cast that aside.  It was more that I think we should have that as the goal.  Oh, in this circumstance, we'd like these cells to make X and Y factor and prove that they do that.  Or maybe it will be in the upper left.  It will be -- whatever it was -- unknowing competence, that maybe we'll just find it out serendipitously and then we'll figure that out. 
		DR. SALOMON:  Dr. Prockop?
		DR. PROCKOP:  I'd like to congratulate Catherine on summarizing a huge amount of data very beautifully for us here.   This is a new area.  There are many very big questions that we really can't speak to.  
		But on one point, I think the data are quite good that at least some of these cells take on the characteristics of astrocytes in the central nervous system.  Don Phinney and Ausim Azizi have been able to do experiments with double labeling with BrdU and markers.  Ausim Azizi, who is here, has actually been able to grow out astrocytes and infusing human astrocytes in rat brain and grow out human cells that have the morphology of astrocytes and stained for GFAP.  
		That doesn't prove functionality, but coming back to Fred's point there, our thinking about these cells is, yes, maybe as wild type cells in the brain, they may do nice things.  They may repair the brain.  We don't know that.  There are many big questions to be answered.  But I would submit that they really last a long time and they migrate and integrate in the brain.  
		So, one appeal to us is just vectors for the kinds of growth factors that might be helpful in one condition or another.  They do last long periods of time.  Ausim Azizi has been able to recover them after 6 months, even human cells in rat brain, but we of course prefer to stay with rat cells in rats and so on. 
		But I think the potential is very broad with a lot of big questions still unanswered.
		DR. DRACHMAN:  The other side of that is where we put those cells.  Why did the, say, hippocampal neurons die to begin with?  We are hoping or making the assumption that all we need to do is put in at least pluripotential cells.  The circuitry will be regenerated, but we're putting it into a microenvironment that no longer supported the cells that were there to begin with.  What is our belief or what is our faith that whatever is needed to redirected even wonderfully defined, really pluripotential cells or cells that have gone down a further pathway that whatever microenvironment it is needed in order to reestablish circuitry exists in the host?
		DR. VERFAILLIE:  Well, I would like to answer.  I don't know anything about circuitry in the host.  I think in other organs, for instance, the data on bone marrow cells repopulating endothelial cells happens in areas where there is damage to the endothelial cells.  So, there is obviously the cues locally to make primitive cells from bone marrow become endothelial cells.  The data that is out there on bone marrow cells differentiating into skeletal muscle would suggest the same thing.  
		So, there's something about a damaged microenvironment, at least inflammatory damage, that causes that.  Well, muscular dystrophy that might be degenerative -- you can maybe that under the same category.  But that wasn't in a transplant setting.  So, there was, again, probably inflammatory damage done first for the cells to get there.
		DR. DRACHMAN:  Yes.  That makes perfect sense, but we're talking about things like Alzheimer's disease where something has gone wrong that has enabled cells to die.  So, that's a little bit different.
		DR. CHAMPLIN:  There's precedence for other organ systems in hematopoietic transplants where we transplant stem cells to treat hematopoietic diseases.  Aplastic anemia is the classic example of a disease we can treat by just giving more stem cells, at least in some patients.  Some patients have stem cell injury from a virus or something, and if that virus is then gone, you can then reconstitute hematopoiesis with stem cells.  On the other hand, if they have an autoimmune disease that's ongoing, just giving stem cells will not work. 
		So, in the analogous situation, if you have a neural disease where there is an active disease process that will affect the new stem cells, of course they wouldn't work unless you could overcome that underlying process.  If it is a degenerative disease or if it is a toxic injury that is no longer present, then perhaps stem cells would be successful in and of themselves.
		DR. RAO:  I think there are two reasons to be hopeful.  One is that there's been a lot of recent data which suggests that there's ongoing neurogenesis.  We also know from a lot of data in the past, there's ongoing synaptogenesis.  So, we know that there is remodeling of circuits and connections which are being made and that these are being made all stages of development.  So, cues exist.
		The other, I think, positive note to remember is that a lot of the neurodegenerative diseases are very slow and ongoing processes which take decades to appear.  So, if you could provide a reasonable number of cells, even if they responded the same in cells, the likelihood of seeing symptoms would be long enough that there would be therapeutic value.  Does that make --
		DR. DRACHMAN:  Not really. 
		(Laughter.)
		DR. DRACHMAN:  That's a little bit different.  That's not quite the point.  The point isn't whether we supply viable cells, but whether the microenvironment to redirect them exists at that point.  That's different.  Those are host factors that are really important where circuitry is key.  That may not be so with Parkinson's disease where really you're looking for a dopamine pump, to be crude.
		DR. RAO:  No.  What I was pointing out is that there is ongoing synaptogenesis and ongoing circuitry involvement or control.  So, in that normal environment, irrespective of whether you put in cells or not, there are signals which are directing synaptogenesis and growth of cells, and that's been clearly evident.
		DR. DRACHMAN:  Synapse is really one of the keys of things like Alzheimer's disease.
		DR. RAO:  And that's a slow, ongoing process which takes over 25 years perhaps to happen.  Therefore, even if you transplanted cells and they integrated, it might take 25 years before you get Alzheimer's again.
		DR. DRACHMAN:  It's worth a try.
		DR. KOLIATSOS:  With all due respect, I disagree.  I really disagree.  I think this point actually is probably irrelevant because at the time you're called to do the treatment, the patient already has reached a threshold.  Unless if you have biological markers to predict when somebody starts having Alzheimer's or ALS 10 or 20 years ahead of time, this point is irrelevant.  Unfortunately, when we're going to be called upon to put the cells, the patient is already symptomatic.  So, there are plaques forming.  There is cell death going on.  The whole environment is totally different and has been different for decades.
		DR. MULLIGAN:  I just wanted to get back to the question of mechanism.  It was reminiscent of a lot of the gene transfer work in the past where there has been, I think, the continued argument about how much do you need to know about mechanism.  If it works, go for it.  I think that has led to some difficulties. 
		Here I think the FDA has asked us to think about the question of how much preclinical work is necessary, and I think we're going to get right back to that very issue of when you implant neural stem cells, is it injury, is it really cytokines, is it forming the right connections?  I think that we're going to have to think very, very carefully about how much we do want to know.  
		A good case in point is, I'm reminded by, the TK suicide gene therapy that people worked on where there is very clear mechanism thought to be accounting for the preclinical success, which I think in time proved clearly not to be the mechanism.  And this led to a very large, costly pursuit that is of questionable ultimate value.
		In this case here, I think you really have to ask the question how useful would it be to know that your neural stem cells are releasing several growth factors, and that's the only thing that accounts for the success because then you might find there are better vehicles, there are better cellular vehicles to release those growth factors.
		Therefore, I'd like to hear, as a non-expert at some point during the day, in any case where there is therapeutic effect, how much do we actually really know about whether connections were made, what cytokine is released, and whether you can get the same effect by poking the needle in another direction or just poking a cytokine.
		DR. DRACHMAN:  One other question.  You mentioned that VEGF is produced by these cells.  Will they grow as well without VEGF, do you know?  Have you blocked it all?  That's a question that one might really wonder, the need for endothelial growth factors in order to maintain this sort of function.  Do you have any thoughts on that?
		DR. VERFAILLIE:  We haven't really looked at trying to block that.  So, this data is the last month's worth of data, that the VEGF is there.  So, we haven't really been able to get around to trying to block whatever is being made.  So, they have the receptor for Flk1 on the cell surface.  Theoretically they can respond to the growth factor they make themselves.  
		We don't get endothelial differentiation unless we take away the other two growth factors, EGF and PDGF, and add large quantities of VEGF.  But it might well be that they are maintained based on an autocrine route almost of lower levels of VEGF.  I don't really have an answer.
		DR. SAUSVILLE:  So, one question I have -- and I guess this would be to Dr. Drachman.  One clearly appreciates the concern that the microenvironment into which these cells are being introduced might be damaged as a basis for the original pathology.  Nonetheless, I'd be interested in your thoughts and a comment as to one use of a safe product that we are called upon to advise the FDA in the definition of as actually defining some of these pathophysiologic states as a result of clinical trials.  So, would you require that we actually know this microenvironment before we actually cut off the potential use of a tool to define the disease mechanism?
		DR. DRACHMAN:  No way you're going to know that.  You're hoping that you will know about thousands of guidance factors that might be critical for forming the circuitry.  The answer is no.  We're wondering here whether, if we put clay into this setting, we'll get a nice sculpture.
		(Laughter.)
		DR. DRACHMAN:  That's one way of thinking about it.  We're hoping the sculptor remains there to do the job.  Otherwise, it's just clay.
		DR. SAUSVILLE:  I hope we could do that control somewhere.
		(Laughter.)
		DR. KOLIATSOS:  Actually can I make a comment on that?  This can have a little bit more specific answer.  For example, there could be a legitimate concern that if you put cells in an Alzheimer's brain, you can have more amyloidosis either via the cells themselves or via the secondary inflammatory processes.  You can use animal models to test that.  Some of these questions can be tested.  You can take the familial Alzheimer's disease transgenic mice.  It would be a wonderful model to test the amyloidogenic potential, but many other issues of guidance and recapitulation of development probably will not be addressed.
		DR. SALOMON:  Can we have one brief question and then I think we have to go on to the break.
		DR. AZIZI:  I'd like to make a couple of comments.  Ausim Azizi from MCP Hahnemann.
		I sort of heard the fact of how much we don't know about these cells, and that's very true.  But we don't know much about neural stem cells either.  We can sit and take pot shots for things that experimentally have not been proved.  But the thing that has been forgotten throughout the whole thing is an important issue of how useful these cells could be.  
		For example, these could be aspirated from the patient's own bone marrow.  It can be grown, as Dr. Prockop pointed out, to multitudes of 10 to the 14 in 8 weeks.  You don't have to go to pig, you don't have to go to transgenic animals, you don't have to go to embryos.  And it comes from the patient's own marrow.  If it could be even the slightest bit useful for treatment of any of the neurologic diseases, I think we still have positive things.
		So, those are the two comments I would make.  Thanks. 
		DR. SALOMON:  Well, then my notes of this very interesting discussion, which I'm artificially cutting short just because I think it's time to go to a break and we've got some more time to talk about it, three sort of defining things came out that I got. 
		One was that defining the lineage and function is going to be critical before concluding a cell is a neuronal cell or a neural stem cell for a clinical trial and that simple marker analysis may not be enough. 
		The second was that transplanting a cell that produces reparative factors, such as growth factors, but possibly what we consider now pro-inflammatory cytokines, could be another benefit in addition to just becoming neural stem cells.  I think that came out quite clearly.
		Third, I think this whole issue of microenvironment in the transplant site has been very clearly articulated.  The idea I think stands that if there's enough injury in a site proposed to be a transplant site, due to the primary illness, you may not have the signals any longer.  I think that is an important question that needs to be addressed perhaps for each setting and may have a lot to do with at what point in a disease progression you should be doing these sort of clinical trials and maybe points where you have gone too far and shouldn't be doing it.  So, I thought that was really excellent.
		There's one other theme here that I hope we'll come back to and that is if it's in the bone marrow, is it just the fact that, hey, it was easy, I could find it there and we tend to think about stem cells in the bone marrow, but there's no really overwhelming, total physiologic importance to that?  Or are these bone marrow stem cells, which we know are circulating, of course, feeding these stem cell populations in the different organs in the adults?  I hope that we'll come back to that.  That's certainly an area that I'm interested in.
		So, about a 10-minute break and then we'll start again.
		(Recess.)
		DR. SALOMON:  Welcome back to the second part this morning.
		I'd like to introduce the next speaker which is Dr. Mehandra Rao to talk about the characterization of lineage-restricted, self-renewing precursors from neuroepithelial stem cells.
		DR. RAO:  The title might suggest that all I'm going to talk about is the work that we've done.  I just took advantage of this opportunity to try and sort of make a few specific points about cells present in the nervous system and see whether these points are relevant to how they might be applied for therapy.  So, I'm not going to talk a whole lot about actual data, but most are general points, hopefully what people will agree is a consensus in terms of stem cells.
		This is sort of stem cell 101.  If you go through normal differentiation or you look at development of the nervous system, you can start with what Dr. Gearhart already told you about, cells in the inner cell mass which will give rise to all cells in the adult.  These cells can be either grown in culture as ES cells or you have related cells like the primordial germ cells, which can then give rise to all the differentiated cells in the nervous system, as well as in other tissue.
		These cells don't do it directly, though, at least in the normal embryo.  They differentiate and there are stages of differentiation that have been identified.  You initially have differentiation with three basic germ layers.  You have ectoderm, endoderm, and mesoderm.  It's the ectoderm which generally gives rise to the future central and peripheral nervous system.  
		You get differentiation into the epidermis, and then you have three different components of the nervous system.  You have the central nervous system itself, and you go through several stages of differentiation before you get differentiated neurons, astrocytes, and oligodendrocytes.  
		The peripheral nervous system is derived from two different populations of cells.  It's the neural crest cell which gives rise to PNS neurons and glia.  In addition -- and this is an important to remember -- we also get several non-neural derivatives, which include smooth muscle, cartilage, and bone, which are derived from neural crest.  Placodal cells give rise to the cranial ganglia and in addition give rise to non-neural derivatives. 
		So, you can see that you can get neurons from a wide variety of cells which are there at different stages in development.  They're all functionally similar in the ability to give rise to neurons and glia, but they're clearly different and they have different roles in normal development.
		In addition to the sort of normal process of development, you also have, as you heard already, a process which seems to be more common than we had originally thought and that's the process of transdifferentiation or cells which would normally give rise to other tissue giving rise to nervous system derivatives, or vice versus.
		So, we have neural stem cells, which can maybe perhaps transdifferentiate to give rise to mesodermal derivatives, a thing they normally wouldn't do in development.  We don't know how exactly that would happen. It could be either the process of transdifferentiation or it could be dedifferentiation back to a more primitive cell which then subsequently undergoes differentiation. 
		One thing we need to keep in mind, at least in terms of stem cells differentiating in this direction, is it could be a normal process of differentiation too.  So, you could have CNS stem cells give rise to PNS stem cells like crest, which could then give rise to mesodermal derivatives.
		I want to follow the development for a couple more steps just to emphasize a few points.  As soon as you get ectoderm differentiating into epidermis and neuroectoderm, that's normally present as a sort of plate of cells which subsequently undergo a process of morphogenesis to form a closed neural tube.  At this stage, cells seem pretty primitive.  And people have characterized the properties of these cells, and at least in normal development, it looks like these are functionally neural stem cells.  They can give rise to multiple derivatives.  They can self-renew, and they can give rise to both the CNS derivatives, as well as peripheral nervous system derivatives.
		Development proceeds further in the nervous system so that what you had initially as a homogeneous tube then undergoes subsequent sort of flattening events, and you get differentiation and more differentiated cells which arise.  It occurs in a specific pattern, and I don't want to go into any details except to say that there are specific regions of the neural tube which will give rise to subpopulations of cells which will then undergo further sequential processes of differentiation to give rise to the more mature phenotypes present in the nervous system.
		We and many other groups have been able to isolate cells at different stages of this developmental process.  So, you can, for instance, identify neural stem cells, and we can define them as cells which can give rise to all the major derivatives which are present in the nervous system.
		You can also go in at slightly later stages of development and identify more differentiated cells.  So, for example, you can identify lineage-restricted cells.  In this case, this is a neuron-restricted precursor cell which can give rise to predominantly neurons.  
		I again use the term "restricted" only as a soft term.  It's not an absolute term.  It's maybe lineage-biased and we define that bias as a comparative bias.  If you take two populations of cells, both of which can generate neurons, and if you challenge them to a certain condition, if one is biased towards giving predominantly neurons while another cell gives rise in the same environment to other kinds of cells, we say it's biased towards one fate or the other.
		Likewise, you can identify glial precursor cells.  These are cells, which in the same environment in which neuron precursors would give rise to neurons, will readily give rise to glial cells.  We define glial cells as astrocytes and oligodendrocytes, which are two separate populations of cells.
		Overall, when we've looked at all of this development, there's just one important point I want to make from this slide.  It's that there is a lineage relationship between these kinds of cells during development.  The most primitive cells we've called neuroepithelial cells are present very early in development.  They can give rise to all the differentiated cells that you see on the right here, and they undergo a sequential process.  We and others have shown that you can generate all of these sort of more intermediate precursors from this early precursor cell and that these then are relatively more restricted in their differentiation potential compared to this cell.  Nevertheless, they still have the ability to differentiate into more than one phenotype.
		We've shown the lineage relationship between these sort of particular cells, but I don't want to leave you with the impression that these are the only lineage-restricted precursors that exist.  There clearly are likely to be several other classes of lineage precursors.
		I want to make one more point here and I'm going to use two slides to do this.  Though all of these cells can make neurons, we can identify functional differences or antigenic differences between these cells.  So, they're present in different parts of the body.  They're present at different stages in development, and we have antigenic characteristics which will distinguish between these cells. 
		Here's just an example of differentiation between a neuroepithelial stem cell and a neuron-restricted precursor cell in terms of three different markers that you can look at.  So, both of these cells are similar in that they express nestin.  This cell, however, expresses additional neuronal markers which the early precursor cell does not express.  There are differences between the factor responses to these cells in culture and in terms of their functional ability.  And this is going to be important later in the talk.
		If I can summarize here then, there are multiple cell types that can generate functional neurons and glia.  These include embryonic stem cells, multipotent neural stem cells, more restricted precursors, and also these two things which often get forgotten, crest cells and placodal cells.  
		Placodal cells are quite important because there's been a lot of excitement in the field with olfactory ensheathing cells.  I want to remind people that olfactory ensheathing cells actually derive from a placodal cell.
		In addition, we have cells which may not normally give rise to neurons and glia but can do so by a process of transdifferentiation.  These include -- you've already heard about mesenchymal stem cells, mesodermal stem cells.  I've added others here just because people have also used cell lines as a source of neurons and glia.
		I want to add that this already seems to make life somewhat complicated, and I'm going to say that life is a little bit more complicated even than this.  That's that we don't just have stem cells and restricted precursor cells, but as people have talked about just in the last couple of hours, the environment and the time at which you isolate these cells, the most subtle properties of these cells may be different.  
		One example of that is clearly true, for example, with multipotent cells.  We identified multipotent cells as being FGF-dependent cells which are present at a very early stage in development.  Several other groups actually identified a stem cell which is present a little bit later in development which is present all the way through the adult.  It is not FGF-dependent, but it's EGF-dependent.  Clearly, we know that these are different cells, and several groups have shown that these are different cells.  So, not only do you have multipotent cells, but you have classes of multipotent cells.
		This is just a summary of the differences between at least two classes of multipotent cells.
		The next slide just gives you a listing of the various kinds of multipotent cells that have been defined.  So, we have FGF responsive cells.  We have EGF responsive stem cells.  We have maybe an E-NCAM immunoreactive cell.  That's how people have defined it, which is present in the adult cortex.  It was defined by a group at Albert Einstein.  There are cells which are present in the SVZa in the adult, which may be somewhat different.  Human CNS stem cells seem to be different from rat and mouse in their growth characteristics and the growth factor requirements.  And several other cells.  
		All of them in many ways are functionally similar.  They can give rise to neurons.  They can give rise to neural crest derivatives, astrocytes, and oligodendrocytes, but clearly we can identify differences in self-renewal capability and growth factor responses.
		The other somewhat disturbing thing for us was that this was also true for more restrictive precursor cells.  So, we could go a little bit later and look at neuron-restricted precursors and ask is one neuron precursor cell virtually identical to any other neuron-restricted precursor cell.  The emerging evidence, at least in our minds, is it's not absolutely true.  The properties of a neuron-restricted precursor in the sense of the kinds of neurons it makes or the synaptic connections that it can make are quite different, and these differences depend in terms of where these cells were isolated from.  
		We try and define neurons, and that I guess is a point that came up in the talk here, how do you define a neuron?  You can't define a neuron basically just from the expression of markers, but you really need to look at several properties overall.  In our minds, for neurons at least, you should be looking at electrical activity, the ability to generate action potentials, the ability to receive synapses and the ability to make synapses, and the ability to synthesize and release neurotransmitters.  We normally try and use these criteria to say that this cell can generate neurons and what kind of neurons it can make.
		This is just an example of getting action potentials from a neuron-restricted precursor cell.
		So, when we look at cells and the neurons that they generate, we find you have to look at several different properties.  You have to look at whether they can project axons and whether they can make synapses and use markers to do that.
		We also use criteria in terms of adding neurotransmitters and looking at the response profile using sort of fura imaging, and this just shows you examples of cells responding to different neurotransmitters.
		The importance of this is not just to say that we can do it, but it's to make one additional point.  Even in culture, where we think we have a relatively large amount of control and in some sense they're all in the same environment, if you actually examine the neurons that are formed in a dish, you can look at two adjacent cells here, and their neurotransmitter response is different.  So, clearly there is heterogeneity despite what we think of as a reasonable amount of control in our ability to differentiate cells.  Since neurons are defined by the properties that they have and their ability to make synapses and the kind of neurotransmitter they synthesize, it means we don't know how to regulate this process of differentiation well enough that we can guarantee we have 100 percent of a particular class of neurons.
		This slide just summarizes where different neuron-restricted precursors have been isolated from, different groups of isolated precursors from different parts of the brain.  The importance here is that these differences are actually biologically and functionally relevant, and I'll come to that in a little bit in the next few slides basically.
		The same thing is true for glial restricted precursors, and Dr. Noble made this point earlier where he said there are several types of glial precursors.  And this just summarizes the results from several different labs.  For simplicity's sake -- again, it's by no means complete ‑‑ I've classified glial precursors as sort of maybe three kinds.  There is a glial precursor which can give rise to both oligodendrocytes and astrocytes, another precursor which seems to give rise to predominantly oligodendrocytes, a third precursor which seems to give rise only to astrocytes.  I want to remind people that there are other glial precursors which may be therapeutically important, and that includes a Schwann cell precursor cell or olfactory ensheathing cell precursor.
		Again, there's a huge body of evidence that these cells are different from each other.  We initially looked at these differences in culture and clearly they were there.  But I think that the other important point I'd like to make here is that the response to the same environment can be different, so there are intrinsic biases in cells, and this bias is not just a reflection of a culture artifact, but it's actually true when you transplant cells back in the host environment. 
		A clear-cut example here is simply this, that if you take labeled cells, which have been labeled with GFP, and these are neuron-restricted precursor cells, and you put them back in the brain, if you put them back in one particular environment, they'll only make neurons.  On the other hand, if you take a glial precursor and put it back in the same environment, it will actually make astrocytes.  And you can see that double-labeling here.  
		So, what this tells us is that when we defined these cells as neuron-restricted precursors or glial-restricted precursors, that this is sort of a functionally important classification because, in the same host environment, their response to that environment is going to be different.
		As I pointed out earlier, there are these several classes of neuron-restricted precursors or glial-restricted precursors.  We've also tried to compare whether the behavior of these cells would be the same or different, and these were experiments we did in collaboration with Dr. Marla Luskin.  We just simply asked if we take spinal cord neuron-restricted precursors and put them back in the subventricular zone, will they behave like the subventricular zone neuron-restricted precursors or will they behave differently.  The answer was that they behaved differently.
		Normally if you have subventricular zone precursor cells and either the endogenous precursor cells or ectopically transplanted precursor cells will only migrate down this sort of stream toward the olfactory bulb, the rostral migratory stream.
		On the other hand, if you take spinal cord precursor cells and put them back in that same location, they will migrate up to the cerebral cortex.  They'll migrate to several different regions.  This migration is not random.  It's specified.  So, clearly the environment is directing these cells, but the way the cells read the environment depends on where they were isolated from and what their intrinsic properties are. 
		The low part is just to show you controls to show that we labeled them with GFP, that they look like neurons.  We look at staining to see what kind of neurons they made, et cetera.
		So, if I can summarize this part -- I'm going to cite a couple of other people who have done similar things here -- is that cells show an intrinsic bias in their development even when they are exposed to the same environment.  This bias is seen very early in development.  If you take precursors which have been isolated from the embryo, you can see this difference or bias in differentiation if you take stem cells which have been isolated from different parts of the brain.  It looks like, at least to us, that the bias is retained in culture for several passages, at least for 3 months in culture.  As far as I know, we don't know how to reverse this bias.  I think that it's really important for us to understand this bias because this will enable us to predict the behavior of these cells after transplantation.
		The next point -- and this again became an important point, in fact, in conversation earlier here -- was that not only do cells have an intrinsic bias in their differentiation potential, but the expression of the potential depends on the environment in which the cells are put.  The brain cannot be treated as a homogeneous region and say, well, one part of the brain is the same as any other.  There's clear-cut evidence that the cells will behave differently depending on which microenvironment, so to speak, in the brain you put them back in.  I'm just going to give you a couple of examples where I think this is very true. 
		It looks like stem cells, when they're put in the ventricular zone, behave differently than if they're put into the cortex.  The rate of cell division is different.  The long-term integration is different.  I'm not going to show you a whole lot of data, but there's a variety of data which says that that's true.
		A another very quite important point is this thing about neurogenic versus non-neurogenic regions and injured versus uninjured, which was again a point that came up here.  So, if you take neuron-restricted precursor cells and put them in different parts of the brain, the kind of neurons you'll see will be different.  So, for example, when we put them in the cortex, we see predominantly projection neurons.  However, if you put it back in the cerebellum, you see predominantly glutaminergic cells of a particular kind.  Again, I'm going to skip over all the staining and the markers we used, but clearly we know that this can happen. 
		The other point, as I said, is this neurogenic versus non-neurogenic region.  I showed you all this data where we took neuron-restricted precursors and put them in the brain, and they made multiple kinds of neurons.  However, if we take the same cells and we put them in the spinal cord, a region where you don't see a whole lot of ongoing neurogenesis, we don't see neurogenic differentiation.  So, the same cell responds to the environment differently depending on which environment it has been put into.  I think several other groups have similar data.
		The same point we can make with glial precursors.  Depending on the response of the cell to the environment, glial precursors might make astrocytes or they may make oligodendrocytes.  So, the reflection of the potential of the cell depends on some kind of environmental signal that it receives.
		So, keep this in mind then.  We have many, many classes of cells.  The classes of cells that we have are far more than we thought.  Those cells have an intrinsic bias, which is reflected in the differentiation potential, and the environment can modulate this differential bias.  So, we have this huge population of different kinds of cells that we have to worry about in some sense.
		I want to also say there's one additional complication we need to keep in mind.  We have many different therapeutic targets that we want to use cells for, at least in principle.  That's just a listing and it's just taken from Harrison's Textbook of Internal Medicine, which talks about different neurological disorders.  Different groups have argued at different times on whether we can use stem cells or their derivatives as therapy. 
		I want to say that if you take even one of these single therapeutic targets and ask what do we want these cells to do, we find that there's quite a large number of things that we expect a cell to do.  Because of my interest in spinal cord, I have chosen spinal cord injury as a target and said we'd really like ideally to say we could use stem cells as therapy.  We really want to reduce the scar formation.  We want to maintain synapsis, reduce axonal degeneration.  We want to have remyelination of the demyelinated axons.  If there's a cavity, we want to be able to pack that cavity.  Sometimes we want to give growth factors and reagents, and maybe we want to use cells as a delivery agent to introduce foreign genes or we want to mobilize the endogenous stem cell population maybe because of the cytokines that these cells secrete.
		I want to make a statement, and maybe I'm going to get grief for this.  We'll see I guess.
		(Laughter.)
		DR. RAO:  The strong statement I want to make is that there won't be a single cell for therapy.  It's just not going to work.  There's no one size fits all for any therapy that you want to use in the nervous system.
		The next statement I'm going to try and make is that the choice of cell will depend on the cell therapy or the goal that you have.  For whatever is the therapeutic outcome that you want, you will have to choose the right kind of cell for that therapy.  
		For all transplant therapy, there might be some general rules which we'll have to worry about in terms of doing this, and that's what will be the cell behavior after transplant, the degree of cell death, its rate of proliferation, its ability to migrate, its ability to differentiate appropriately and the lack of inappropriate differentiation.  This is really important to keep in mind.
		You've heard of different sources of cells, mesenchymal cells, transdifferentiated cells, neural stem cells, and how would they respond to the environment.  We always look at the positive and say, well, they did make neurons, they did make astrocytes, but we also have to ask what else did they make?  What is that large group of cells which are differentiated?  What are they going to do?
		There are two other issues which normally don't become major issues when we think about transplants but I think are important in the nervous system, and that's a method of insertion.  It's really quite clear that we have a limited strategy in how we can insert cells or deliver cells, and the method of insertion can be quite critical in terms of survival, how you dissociate the cells, in terms of how you treat the cells beforehand.  I think we need to really worry about that as an issue in combination when we're looking at anything in terms of a clinical trial or looking at animal models. 
		These two things which haven't come up as questions were the immune response that these cells will generate.  And it will become clear why I think that's an important issue.  But I've gone through the literature -- and maybe I'll be enlightened today, but it's very hard to predict the immune response of early cells which are primitive or undifferentiated.  There isn't a sort of standard cell assay that we can do for immune response for a graft versus host or host versus transplant cells, which is a ready, easy assay which we can use.  I think that we have to worry about this and we have to consider what's going to happen when we do long-term transplants with both major and minor histocomparability antigens.
		There's also clearly a growing body of evidence which suggests that a lot of genes which drive expression are shut down.  So, if you use these cells for gene delivery, you have to worry about that as an issue too.
		In addition to these sort of more general properties of cells that we'll have to worry about in any case, I think specialized cell populations will require sort of specialized tests that we'll have to worry about.  I'm going to just give two examples.  
		One is embryonic stem cells.  Dr. Gearhart already raised this as an issue.  What will happen?  Will you get inappropriate phenotypes that may develop?  Will you see tumor formation, and should we be designing specific tests which are relevant to the known properties of specialized populations of cells?  I think in the case of ES cells it's very clear that you have to worry about tumor formation, and that will be an assay that you'll have to run.
		With the issue of transdifferentiation too, we'll have to worry about a couple of things.  I just want to make the point about numbers and cytokines.  The issue of numbers is that we clearly know -- at least the data is quite compelling -- that you can get transdifferentiation and you can theoretically use, for example, mesenchymal stem cells to give you neural derivatives.  But what sort of gets lost is what is the percentage.  What is the degree of efficiency?  Are we going to get all the cells becoming the kind of cell that you want?  Are the numbers going to be something which are useful?
		The other for me conceptually that is a major worry is that when you go back and look at the cytokines that regulate differentiation -- you heard today EGF and FGF seem to work to get you bone marrow formation and you get stem cells proliferating which can give rise to mesodermal derivatives, and the same growth factors will cause a stem cell to differentiate into neurons.  We know that from published data.  So, how will the cell know what to differentiate into in terms of an environment, and why will you not get inappropriate differentiation when you put a cell back in the brain?  I think they're important issues that we need to design specific tests for.
		But these are beyond the scope of what we can do because we don't have a specific cell that we're going to think about.  So, I'm going to just try and recast these issues in terms of tests that one might possibly consider and leave that open for discussion.
		We have source control.  I know there are these privacy issues and we have to really worry about them, but in some fashion I think we will need to know something more about the cells in terms of the age of isolation because we know there are differences in self-renewal potential.
		Perhaps there might be dimorphic differences.  We know parts of the brain show sexual dimorphism.  
		Maybe we'll need to know about the sex of the cells that we're going to transplant. 
		I think it's going to be quite important for us to know the region of isolation because there's clearly now a lot of data which suggests that there are regional differences in cells.  
		Viral testing.  I think maybe in terms of the CNS, we may have to think about prion diseases, and that may be an additional test that we may want to think about rather than just sort of a standard organ and tissue transplant test that we do.
		As this point has come up several times, I think sample profiling.  We can't look at single antigens to classify a cell.  We will need to look at multiple criteria, and perhaps we'll have to think about profiling by some sort of standard criteria for any kind of cell that we use for transplant.
		This is an issue which I think may be quite important.  We are deriving lines and we are hoping that we can get these cells and we can use them for a large number of people.  Perhaps soon after these cells are isolated, there should be some mechanism to deposit these as a reference aliquot that you can compare your cells with when you're going to do any kind of tests or transplant back in the brain.
		In terms of a manufacturing process, I think there will be issues that we should definitely keep in mind since we're taking dividing cells and putting them back in the brain.  One issue that's sort of become clear from the data that's been out there is that the response of cells changes with multiple passages.  For example, with neural stem cells, at least with the EGF-dependent cells, it's clear that the degree of astrocytic differentiation is far higher from late passages than it is from early passages.
		The issue of karyotypic stability has been raised before, I think that's going to be very important.  Anytime you passage cells, one should think about these as cell lines and maybe we should have some measure of mutation rate.  Maybe we should be looking at p53.  Maybe we should be looking at some other measure.  
		I think we should look at telomerase activity because it's clear, at least in human cells, it seems to be  a measure of degree of self-renewal potential that these cells have.  
		I think we should be comparing at each passage with a reference aliquot the properties that's been defined for that population at each stage.  
		And I think in any culture you need to worry about viral testing and antigen testing.  We should be doing that.
		I want to remind people here that the other thing we need to think about is that if you take cells which have many, many sort of potential outcomes because they're stem cells -- so, in this case, they could die, they could make crest cells, they could make CNS derivatives, or they could convert from one stem cell to another cell, as has been shown -- then we should really be getting some numbers on the frequency of these sort of things happening.  
		So, I think we have to worry about specifications of the cells when you're ready to transplant them.  Given what we've heard so far, I think the purity of the cells that you hand over for transplant is an important consideration.  Is it a pure population?  Is it a homogeneous population?  What fraction has already differentiated?  What are the detailed characteristics of this clump of cells that you're going to give?  
		I think we need to know something about cell division after transplant.  And the reason I think this is quite important is that if you get inappropriate cell division, it doesn't have to be a tumor, it just has to be a mass, and that's going to have effects in the brain.  If these cells divide inappropriately, then we need to know that.  We need to know how long they will divide for because this can have important functional consequences.
		This is a point that came up earlier I guess in conversation:  functional assays that reflect therapeutic use.  I think if you're going to put cells back in the brain, you want to have a functional assay which will tell you that these cells actually give you that right kind of function.  So, we have to really define which functional assay we will use depending on which cell type we use and which therapy indication we are using them for.
		I think at this stage too you need to profile that specific lot of cells that are going to be used for therapy.  We need to always compare them with some kind of reference aliquot. 
		This last issue is numbers in a lot.  That also came up earlier.  How many of these things can we do?  If it turns out that each cell that we use is unique to that population, if we have to isolate cells in an autologous transplant, then maybe we don't need to do as many different tests.  On the other hand, if we want to have cells which are going to be used only in 10 patients, because that's the total number of cells we have at any time and we have to go back to primary tissue to get cells, then I think a lot of this criteria of what we're going to do will change.  So, what we really need to know, when we're going to use cells for transplant, is in what numbers of patients will that particular lot of cells be used.
		I'm just going to briefly go through a couple of other issues which I think are important, and that's implantation control.  I don't mean to say that we should be deciding which device, but I think that what's important here is to be thinking about cells and knowing that they respond to the device in a certain way.  We need to have some parameters because this is quite a critical issue in terms of the number of cells you can get into the brain.
		For example, in Parkinson's when you do a transplant, the total volume that you can put in into a site through a single injection is limited.  You have to make a decision as to what the suspension will be like, how many cells will that mean.  If you dissociate them, should it be in clumps or whether it should be single cells.  We need to know exactly what percentage of cell death there is because otherwise we won't really have a read-out which will be reproducible.
		These are other issues that have already been brought up, and I don't want to go through them.  But I think that these are some things that will have to be considered at some level by the committee.  Should we be thinking about noninvasive read-outs when we put in transplants?  Because there are a lot of things we don't know yet.  Should we be putting in some "what-if" controls?  Things may go wrong.  Should we have to be thinking about engineering suicide genes?  Should we be thinking about how we can kill cells?  Should there be some other kind of noninvasive read-out which tells you that something is going to go wrong?
		I'm going to end here and I'm going to show this picture from National Geographic.  It's from bridge building in a country that will remain nameless.
		(Laughter.)
		DR. RAO:  But you can see that they know how to build bridges.  They know how to pour the concrete.  They know how to extend it, but clearly there wasn't an overriding control which said, look, there's a slight problem here and you probably knew about this well in advance and you should have taken care of it before.
		I think the FDA's role here is exactly that.  It's we know how to do all of this, but we want to make sure that somebody looks over this and controls the fact that we don't have this slight miscalculation here. 
		(Applause.)
		DR. SALOMON:  Thank you very much.
		I think because the two talks in this group are so similar, what I'd like to do -- also because lunch is looming, and in the end, if we're way off time, it's always the chair who gets all the grief -- is to go to the next talk.  Then we'll stop and discuss both of them together.
		So, Steven?  Dr. Goldman is going to talk about the isolation, identification, and characterization of adult human neural progenitor cells.
		DR. GOLDMAN:  I'm a little less optimistic I think, at least less sanguine, than Mehandra in terms of what we do and don't know.  I think we still don't have the parts of the bridge sorted out.  I don't doubt that it's not too early for the FDA to be involved with respect to that one slide of Mehandra's that was presented for subliminal comprehension only --
		(Laughter.)
		DR. GOLDMAN:  -- simply because there are proposals out there now to use these cells clinically.  In fact, there have been trials already initiated.  So, by definition, it's not too early for regulatory involvement. But I think in terms of the basic science, there's still quite a bit that we don't know.
		I'm just going to be focusing not on stem cells here but more practically speaking, looking at what the human brain actually has because that in some way has to be the fulcrum upon which the rest of the discussions revolve because unless we know what the adult human brain actually harbors, it's hard to know how to perturb or influence those endogenous neurologic processes and the cell types that subserve them. 
		Now, without reviewing history, there are several neurogenic populations that have been described in the adult mammalian brain.  As far as back as the early '60s, in fact, neurogenesis within the olfactory bulb and hippocampus where described by Altman. 
		It had been thought literally for a couple of decades that these were locally neurogenic populations, and what became clear in the early '90s with the work of Marla Luskin and Alvarez-Buylla and others was that the olfactory bulb in particular -- the neurons being generated and migrating to the bulb are forming from and arising from progenitors within the ventricular zone.  This is a process of long-distance migration, similar in some ways to work that Fernando Nottebohm and I had done years before that in the adult bird brain where long-distance migration of newly generated neurons from ventricular zone progenitors had been found.
		Now, the hippocampus remains a population that is largely locally neurogenic with some migration from the subgranular zone, a still undefined degree of migration from ventricular zone progenitors.  That's what had been known.
		Actually on the model in the adult bird brain, there are progenitors lining the entire ventricular system, and these cells are only being utilized in a few discrete loci.  With that thought in mind, we hypothesized that perhaps the progenitor population of the adult mammalian ventricular zone was more widespread.  This is work of Barry Kirschenbaum in the mid-1990s.  We found that, in fact, neuronal progenitor populations were quite widespread throughout the adult rodent ventricular system.  These are subependymal chains of newly generated neurons.  Chains like this can be found throughout the ventricular system.  These include mitotic and neuronally restricted progenitors, as well as less restricted, uncommitted, multipotential neural progenitors.
		Using that as the conceptual model, we then looked at the adult human brain to see whether humans continue to harbor a persistent neurogenic population within the ventricular zone.  For those studies, we used temporal lobes resected from adult epileptics who were having a temporal lobectomy for medication refractory epilepsy.  That provides a nice source of temporal ventricular zone ventricular wall lining. 
		When we cultured these in explant cultures -- actually the same types of culture methods that were established years before for canaries.  The factor controlled in these processes is quite conserved across species.  
		In any event, we saw neuronal migration and neuronal generation from the ventricular zone explants.  These are neurons labeled for several markers, MAP-2, NCAM, MAP-5.  And some of these neurons are generated in vitro.  The silver grains indicate thymidine incorporation from a mitotic marker introduced in vitro.  So, we knew from this work that there was a population of neuronal progenitor cells that, indeed, persisted in the adult human brain. 
		From the work of Rusty Gage and others in the mid-1990s, it became clear that FGF was a strong mitogen for the ventricular zone population in rats.  We then applied that knowledge in our own observations of the role of BDNF in driving differentiation and survival of neurons generated from these precursors to do the following experiment. 
		We took adult human ventricular zone explants and cultured them for several months in vitro under the serial control of FGF-2, followed by BDNF, the idea being to drive mitotic expansion of the neuronal progenitor population and then differentiate that pool using BDNF and assuring their long-term survival.
		You see networks of neurons generated in these cultures, lying upon an ependymal substrate.  This is simply a high power of this sample clump.  When we look at these cells viably in terms of their function, we see that this is with calcium imaging using fluo-3.  In response to glutamate, most of the neurons within these clumps light up.  In fact, they generate action potentials as well.  So, these are functional neurons. 
		After fixation, we can show that they express typical neuronal markers.  This is MAP-2.  And most of them have been generated in vitro during the period of FGF-2 exposure which is in the first week of this culture 2 months before its fixation.
		Now, this was a reasonable demonstration of the neurogenic potential of the adult human subependyma, but the numbers, really when you get down to it, aren't all that impressive.  We can generate thousands of cells, perhaps tens of thousands, from a given ventricular zone sample, but that's spitting in the wind compared to what we need in terms of clinical implantation or clinical utilization.
		We needed to get a better idea of how many of these cells actually existed in vivo.  Luckily in the mid-1990s, a number of markers became available that allowed us to at least make guesstimates of the density and frequency of these cells. 
		These are two markers:  Musashi protein, which was initially identified by Hideyuki Okano, which is an RNA binding protein expressed by early neural progenitor cells.  Here we see in an adult human ventricular zone specimen a number of Musashi expressing cells.  When we look at another RNA binding protein called Hu, which is reciprocally related to Musashi -- it's expressed upon neuronal differentiation concurrent with Musashi shutdown ‑‑ we see a number of Hu expressing cells within the ventricular wall.  These are essentially the neuronal progeny of these cells. 
		The problem is that there aren't very many of them.  They're lying in essentially a cellular monolayer within the ventricular wall, and this is the kind of tissue piece.  It can be anywhere from 50 to 100 grams that we have to isolate what are essentially less than 10,000 cells from.  We're starting with tissue populations, cell populations in the 5 times 10 to the 10th range, and we're trying to sort from that pool cell numbers of 10 to the 4th to 5 times 10 to the 4th.  This is a level of enrichment that is even beyond that which is required for the isolation of the hematopoietic stem cell, whether from marrow or peripheral blood.  
		Making the purification issue all the more difficult is that there are no truly specific surface markers for neural stem cells, neural progenitors, as we've seen from the earlier talks.  So, we utilized a different strategy.
		This is as dense, by way of reminder, as these cells ever get.  This is a stain from Musashi.  
		So, to pull that relatively sparse population out, we took advantage of the known selective expression of a variety of cytoskeletal and regulatory molecules in neural progenitor cells and neuronal progenitors.  These are not surface markers, but rather skeletal and regulatory proteins, for which the promoters had been identified.  In other words, the genes had been sequenced in total and the regulatory sequences controlling the expression of those genes had been identified.  
		As our prototype, we took the promoter for T-alpha I tubulin, or a tubulin that's made within the ventricular zone by neuronal progenitor cells and also by very young neurons as they migrate.  This is a promoter that we obtained from Freda Miller.  We took the T-alpha I tubulin promoter and coupled it to the gene encoding green fluorescence protein and then took the resultant T‑alpha I:GFP construct and asked whether we could identify progenitor cells while they're still alive, as opposed to in fixed material after the fact.  For this purpose, we used dissociated ventricular zone, transfected all the cells in the cultures with the T-alpha I:GFP plasmid construct.  
		The question is, who's the progenitor?  It's always the cell in the middle.  That's the progenitor.  So, the T-alpha I tubulin:GFP construct allowed the selective expression of the GFP, the fluorescence reporter gene, in the progenitors of interest.  When we followed those cells under FGF conditions, we get expansion.  When we switched to BDNF, some of the cells die, but others become neurons. 
		Given the presence in these cells of a fluorescent marker, that allowed us to use fluorescence-activated cell sorting to pull that population out.  This is a typical sort graph in this type of selection.  Here we've taken the T-alpha I tubulin-driven:lacZ as a control against GFP.  You see nothing gated in the fluorescent fraction.  This is a typical fluorescence versus forward scattered, in other words, versus cell size type of graph. 
		In this plot here, with the T-alpha I:GFP transfection, we see a very discrete, very small population of cells from the adult ventricular zone dissociate.  It was a 33-year-old who's down to one-tenth of his ventricular zone population being recognizable as a neuronal progenitor pool.
		This is what the cells look like after FACS.  And at 1 and 2 weeks thereafter, we're seeing the maturation of neurons, all of which have incorporated bromodeoxyuridine in vitro in the first couple of days after sort.  So, these cells, when they're being harvested, are still mitotic.  They go on to express fully matured neuronal markers, and as we'll see, they become functional.
		Now, we can pull out different cell types to find at different stages different phenotypes, stage-defined, by virtue of using promoters that are transcriptionally activated at those different stages.  So, it's a powerful technique. 
		Here we've used the enhancer controlling in the expression of nestin.  Nestin you've heard mentioned before.  Nestin is expressed by neural progenitor cells.  It's expressed by other cell types too, so it's not entirely specific.  We've also found it's not as early as we might like for true stem cell derivation, but at least we're pulling out multipotential neural progenitor cells.  It's sufficient.  
		So, we've used a nestin enhancer driving a basal promoter, the heat shock protein-68 promoter.  What that does is target nestin expression.  This is the second intronic sequence of the nestin gene which targets the expression of the gene at two neural progenitor cells -- this was the sequence first identified by Ron McKay some years back -- regulating here a basal promoter, in turn regulating the expression of GFP.  When we transfect that plasmid into adult human ventricular zone, we see a very discrete pool of nestin expressing cells.  Again, it's a rather small pool; 1 cell in 1,000 here is being labeled.  These are dissociates not of the whole temporal lobe, but actually of the ventricular zone, of the ventricular wall.  So, the dissection itself is allowing some degree of enrichment.
		Also, I should mention that the transfection efficiencies here are in the 10 to 14 percent range.  So, effectively you can multiply these numbers by about 7 to estimate the endogenous frequency of these cells.
		These cells are nestin cells.  The nestin sorted cells, unlike the T-alpha I tubulin sorted cells, when we raise them in suspension culture in a typical neurosphere prep, they will generate neurons and astrocytes both.  The blue cells are neurons, the green cells are astrocytes.  These are cells that have been plated after neurosphere expansion.  The nestin expressing cells are still present.  So, the progenitors are dividing and at least some fraction of them are remaining in a multipotential, still mitotic state.
		Now, we wanted to see whether these cells were engraftable, whether they were functional, and whether they would achieve the range of phenotypes that we wanted after in vivo implantation.  
		For this purpose, we used a developmental model.  We purified from the human ventricular zone the nestin:GFP defined cells.  After FACS, we implanted them into the ventricular system as a transuterine xenograft into embryonic day-17-aged fetal rats.  We allowed the mothers to then give birth, allowed the babies to grow up, variable periods of time, anywhere from 2 to 8 weeks, and sacrificed them, and looked for the human cells in the overall rat brain to see what those cells had become. 
		This is what the 17 rat looks like.  This is after injection of a marker dye.  
		This is a typical section at a couple weeks after delivery.  Here the green cells have been stained for GFP, so we're looking for persistent GFP expression.  GFP actually persists for a couple weeks, even after down-regulation of the promoter expressing it.  It's relatively stable in these cells.  So, we're looking at double-labeled green and red.  Therefore, yellow cells, which are human-derived, and the red cells are the host rat cells, rat neurons, which have been identified by Hu.  So, essentially we're generating chimeric cortices here of rat and human using the adult human-derived multipotential neural progenitor cell.  So, we know these cells are competent to integrate into developing cortex.  
		I'm not showing the data here, but we've implanted at later time points and seen differentiation of these cells into subcortical populations that are predominantly oligodendrocytic as well.  So, we do think that at least the nestin defined pool is multipotent.
		Now, it's still a relatively unusual population, the ventricular zone pool, unusual in terms of numbers.  So, we wanted to look at something a bit more abundant and something that might be a bit more active in vivo.  
		This is a slide of Rusty Gage's two years back where he and his group identified mitotic populations in vivo in the adult human hippocampus.  This is a BrdU-labeled granule cell neuron within the adult hippocampus.  So, we focused upon the hippocampus with the same types of protocols, taking normal hippocampus, dissociating it, and in fact, we were easily able to demonstrate mitotic populations of cells that gave rise to neurons.  That in itself wasn't a surprise.  We found that the cells were able to drive the T-alpha I tubulin promoter and differentiate as neurons.  So, that gave us the technical wherewithal to be able to sort that population.  We utilized exactly the same protocol now as we did with the ventricular zone, taking out normal dentate gyrus. 
		Now, I should mention actually for the clinicians among you that the ventricular zone preps, by and large, were down with patients with medial temporal sclerosis who were having temporal lobectomy for that reason.
		These cases were patients either with focal cortical epileptogenic foci.  They required corticectomy and subtemporal lobectomy despite normal hippocampi or were non-epileptic patients who were having decompressive lobectomy in the setting of trauma and a couple of aneurysmal resections. 
		So, this is normal hippocampus.  Dentates dissociated, T-alpha I tubulin is transfected into it.  We wait for expression and then FACS the sample. 
		Basically this is the population that results. This is 4 weeks after FACS.  Pure populations of neurons generated from the hippocampal progenitors.  This we believe corresponds to the population that Rusty had originally identified in vivo.
		The numbers are between 10- and 20-fold higher for a given dentate gyrus sample relative to a given ventricular zone sample.  So, there are a lot of these cells in the adult human hippocampus. 
		We've looked at patients as young as 5 and as old now as a woman in her 80's, and we see very little fall-off.  This is simply comparing a 5- and a 20-year-old.  But all the cases have shown FACS sorted pools of at least 1.1 percent of the population.  Again, taking into account the transfection efficiency, we're looking at potential progenitor populations of at least 7 percent of the total dentate gyrus, which is really extraordinarily high.  It suggests either the presence of an abundant population or, for that matter, of a persistently mitotic dentate gyrus granule neuron phenotype, in other words a neuronal phenotype that's potentially competent to dedifferentiate to a mitotic pool.
		These cells are functional.  This is the work of Maiken Nedergaard after loading with fluo-3, the calcium imaging dye, and the same population after glutamate exposure.  The cells all light up.  These represent at least 4-fold increments in cytosolic calcium in response to glutamate, typical of voltage-gated calcium channels in mature neurons.  
		You can actually target the GFP-expressing cells in vitro and record from them.  This is a patch-clamp analysis of Jian Kang.  This is in a voltage-clamped configuration.  We see current injection related current increments, very typical again of the fast sodium channels of neurons.
		Now, I'd mentioned before that we can target essentially progenitor populations of interest at will, as long as we have a promoter or regulatory sequence that allows us to identify that pool.  When we culture adult ventricular zone, we see not only neurons generated, but also oligodendrocytes. 
		In rats, the oligodendrocyte or at least the glial progenitors have been looked at for many years, and it's a very highly abundant pool and a very active pool.  
		In the human, it's been a much more controversial story through the years.  Adult human oligodendrocytes appear to be every bit as postmitotic as neurons, and yet oligodendrocytes are generated to subserve remyelination in a number of acute demyelinating diseases.  It's never really been clear where those cells are coming from.  A number of attempts have failed at isolating human oligodendrocyte progenitors, it turns out, because some of the markers really are different in the human population.
		In collaboration with Peter Brown and Michelle Gravelle in Montreal, we took the early promoter for cyclic nucleotide phosphodiesterase.  This is a protein that's made by oligodendrocytes but also by their progenitors.  There's a segment of the promoter of the CNP gene that is expressed differentially in the oligodendrocyte progenitor.  That was work that Peter Brown had established through the early '90s.
		So, we were able to take the CNP-2 segment, this early segment of the CNP promoter, couple it to GFP and then transfect adult human white matter.  So, these are white matter dissociates.  The question once again is, who's the progenitor?  For that matter, in this case, we didn't even know whether a progenitor existed.  So, this was a bit empiric.  
		And these are the cells that express the CNP-2 driven GFP.  They're very, very small bipolar cells.  In fact, it would be very easy to lose in a culture otherwise.  Very small, very undistinguished. 
		When we sort them -- actually it's a fairly large number.  We're not showing it here, but this is about four-tenths of a percent of the adult white matter dissociate, translating to about 3 percent of the white matter pool.  These cells at the time of harvest are almost entirely mitotic.  It's an extraordinarily mitotic pool after they're removed from the brain.  At 6 hours and 24 hours in vitro, they are all incorporating bromodeoxyuridine and are expressing at the time of harvest the A2B5 antigen of the GQ ganglioside, which is a marker for oligodendrocyte lineage cells.  But many of the other markers that have been used for identifying oligodendrocyte progenitors in the rat brain, particularly O4/O1, are not expressed by these cells at this point.  They're solely making A2B5 among the early oligodendrocyte markers.
		When we follow those cells over 2 weeks, 3 weeks -- we've taken them much further out than this -- they go on to develop mature oligodendrocytic antigenic expression.  We can show that those oligodendrocytes generated in vitro were, in fact, generated mitotically.  They've incorporated bromodeoxyuridine in those first few days in vitro.  We can actually see layers, carpets really, of purified oligodendrocytes generated from these cells.  
		Starting with a couple of grams of adult human white matter, if we pull out 10,000, 15,000 CNP-2:GFP defined progenitors, we typically have at least 10 to the 8th after a month in vitro.  So, we can expand this population considerably generating relatively pure populations.
		Now, before moving on to the utility of these cells, I just inserted this in the break.  With regards to the conversation before, the issues arose with regard to the lineage restriction of these cells.  Well, this is a parenchymal progenitor.  By any criteria that we can use, it's biased very strongly towards generating oligodendrocytes.  In basal culture conditions, 93 percent of these cells will generate oligodendrocytes.  We see only 7 percent astrocytes and essentially no neurons at a month in vitro.  
		But when we played the cell sorting as a function of cell density and plated cells at different densities, we found that when we went to very, very low densities, post sort -- so, now we're dealing with a highly artificial situation of very, very pure cells of a given phenotype at very low cell densities -- 10, 100 cells per ml, no greater -- we saw a diversification or a degradation, depending upon your point of view, of phenotype.  So, now the CNP-2 defined cells started to express both neuronal and astrocytic markers.  In fact, we can generate neurons and astrocytes, as well as oligodendrocytes, quite freely in a density-dependent fashion.  It's only at relatively high sorting densities of at least 5,000 cells per ml where we see essentially an oligodendrocytic phenotype exclusively being generated.
		So, these cells, even though they're parenchymal and in the adult human, actually have quite a bit more in the way of phenotypic potential than we had realized.  They may have every bit the degree of phenotypic potential as their ventricular zone counterparts.  It's simply that they're being restricted by the regional environment in a bit of a harsher way. 
		Now, obviously, we wanted to see whether or not these cells were engraftable, whether they were potentially utilizable.  Martha Windrom in the lab established a lysolecithin lesion model.  This is a standard technique for demyelinating adult brain.  We modified a technique of Jim Goldman by putting lysolecithin -- it's a detergent that causes reversible demyelination -- in as a very slow infusion bilaterally into adult rat brain and allowed the detergent to very slowly diffuse in a longitudinal fashion, to follow axon tracks in the corpus callosum.  And by putting injections at the same depth level bilaterally, we end up with these centromedian plaques that are in the depth of the corpus callosum.  It provides a very nice model for reversible demyelinating placque lesions of the adult nervous system. 
		Now, we injected the adult human oligodendrocyte progenitors.  Now, these cells -- this was early in the process.  These were simply di I labeled.  We looked at a week and saw that the cells -- here's the injection point -- migrated within the lesion, very, very rarely beyond that.  So, they're following cues to stay within the lesion.  They migrated extraordinarily.  Here's a midline.  It's a bit off kilter photo.  But this lower power montage, you can see the cells migrating right across the midline right over to the other hemisphere.  Again, they're remaining within the lesion.  We see some departure from the lesion to follow blood vessels, here tangentially, here coronally, but otherwise, the presence of normal intact myelin seems to be nonpermissive for oligodendrocytic progenitor migration, which for these purposes is very adaptive.
		Now, to try to make these cells again more useful, we wanted to take advantage of their abundance -- this is a far more abundant population than the ventricular zone or the hippocampal pool -- and at the same time not lose the 80-odd percent that we were losing up front by virtue of transfection inefficiency.  Since we had identified A2B5 as being expressed by these cells, we sorted on the basis of A2B5, looking for A2B5 expression by these cells as a sufficient marker for their extraction.
		This is a young brain where the percentages are a bit higher than in the normal adult.  But here you can see seven-tenths of a percent of the population of the actually juvenile white matter expressing the CNP-2 driven GFP after transfection, while almost 8 percent expressed A2B5.  Now, in the adult, our average is now just over 3 percent of the population by A2B5.  So, we can increase our yield substantially.
		We took the A2B5 sorted cells and injected those into the adult lysolecithin model.  Here's a control at 2 weeks.  The lesion tends to remain a lesion for roughly 5 weeks with this model. 
		This is another animal.  Here is a corresponding region after engraftment and migration of the adult human-derived progenitor. 
		The red stain is for CNP protein, the early oligodendrocyte protein.  When we look at the green cells ‑‑ and here we're targeting the human cells using an anti-human histone that is specific for recognizing human cells.  Vis-a-vis the issue that came up before with regards to recognizing human cells in the rat context, we have a few ways of doing that now. 
		The other that I'm using interchangeably here is in situ hybridization for human alu sequences.
		In any event, looking at the double-labeled cells here, we can see that the human cells are starting to make CNP protein.  In fact, within a week thereafter -- this is now 3 weeks after the lesion, 18 days after the implantation -- they're starting to make myelin basic protein as well.  The red stain here is for myelin basic protein, a mature myelin protein.  You can see within the lesion bed, there's diffuse expression of MBP.  When we confocal and actually stain for a human-specific MBP, we see myelin basic protein expression by these clumps of human oligodendrocyte progenitors in the engraftment model.
		Now, there's still a lot we don't know.  We don't know whether this remyelination process is functional.  We do know now that the axons are being enwrapped, but we don't know whether or not compaction is appropriate.  We don't know whether or not these are physiologically functional remyelination processes that are occurring.  That's work underway now.
		Now, I just wanted to close.  Lest an alternative not be mentioned at the meeting at all, beside implanting cells, implanting stem cells, implanting lineage-restricted progenitors, I think it's important for us to be able to utilize the endogenous progenitor populations that are present to understand their role in disease, what happens to them in disease, where they may contribute to disease, where they may contribute to endogenous or spontaneous recovery.  One way to approach that is to use the very limited knowledge we have at this point in terms of the growth factor control of these cells to try to induce endogenous progenitor pools to be neurogenic and in fact to be neurogenic in otherwise non-neurogenic regions.
		Now, there are a number of ways to tackle this.  This is one approach that we used as a result of a collaboration with Ron Crystal's group at Cornell.  We took an adenovirally delivered, in this case, just a marker gene, GFP driven under CMP promoter control, and injected it into the ventricular system to see, as a first level of analysis, where an adenovirally introduced transgene would get to after ventricular injection, the logic being to try to turn the ependyma or the ependyma/subependyma into an endogenous source of secreted neurotrophin introduced by the viral transgene. 
		Here is simply a low power sagittal section of the adult rat brain after the adenoviral GFP introduction, and you can see that the tag is remaining restricted to the ependyma and to a much lesser degree the subependyma.  There's some immigration of cells into the corpus callosum, but nothing into the gray matter parenchyma of either the striatum or the cortex.  So, we know that if we give an adenovirally delivered transgene, that the adenovirus itself and the cells expressing those transgenes will remain restricted to the ependymal surface.  
		So, with that logic, we constructed an adenoviral BDNF vector that has GFP and IRES association.  So, it's expressing both a marker and BDNF.  BDNF we and others have described as, again, a differentiation survival factor for neurons generated from the endogenous ventricular zone progenitor pool.  
		Here's the virus.  We used a protocol of serial injection with bromodeoxyuridine for essentially 3 weeks, followed by sacrifice 3 weeks following virus injection.  So, these animals are producing high levels of BDNF in response to the viral injection, which is restricted to the ependymal surface again.  This is in situ hybridization showing BDNF and GFP message against the control.  Nothing in the parenchyma.  The idea again being that the ependymal cells are making the BDNF.  The subependymal progenitors are then being exposed to the BDNF without being infected by the transgene or infected by the antivirus, rather, themselves. 
		That results in very high levels of BDNF expression both parenchymally and here in the CSF.  We're looking at a couple of nanograms per ml, which approximates what we actually had in vitro to study these cells.  So, the CSF is achieving very high BDNF levels.
		In response to that, we see a tremendous increase in neurogenesis to the olfactory bulb.  Remember, the olfactory bulb is undergoing neuronal recruitment normally in an ongoing fashion from the cell population.  Now, if one looks at the olfactory bulb, you can see double labeling for the neuronal marker in red, beta-III tubulin, and the green indicating bromodeoxyuridine incorporation.  These cells are being recruited as neurons, here double confocals for beta-III tubulin and MAP-2.
		This is what the comparison of the BDNF versus a null injected set of animals shows.  If one does a low power camera lucida reconstruction where every dot represents a BrdU incorporating neuron, you can see a substantial increase in density of neuronal recruitment, neurons being added to the BDNF-treated olfactory bulb.  So, we're inducing neurogenesis from the endogenous pool here. 
		This is what it looks like quantitatively, BDNF versus an AdGFP, no BDNF insert control.  We're looking at numbers here, several thousand newly generated neurons per cubic millimeter, so very high recruitment numbers in response to BDNF.
		Now, the issue that actually I want to close this with then becomes can we use this approach to cause neuronal recruitment in regions that are otherwise non-neurogenic.  So, we scored the addition of newly generated neurons in the cortex, the striatum, the septum, as well as the olfactory bulb in these animals.  
		I should mention that we actually saw very low degrees of neurogenesis in the cortex in response, too low to count, but is there.  We saw nothing whatsoever in the septum.  But in the neo-striatum in the caudate putamen analog, we saw very high numbers and densities of newly generated neurons in the striatum and only in the BDNF-treated animals. 
		These are simply confocals where we've serially reconstructed Z-dimension stacks.  The red is a neuronal marker, beta-III tubulin.  The green is bromodeoxyuridine.  This is simply to be sure that the newly generated cells as marked by BrdU really are neurons, that they're expressing neuronal markers throughout their extent.
		When we look at this quantitatively, we're generating on average 140 neurons per cubic millimeter within the neo-striatum.  This is a fraction of what is generated in response to the AdBDNF factor in the olfactory bulb, but nonetheless, it's really a significant addition.  If one accumulates that number over time -- Yuesh Melniki in the lab has done this stereologically in a large sample at this point of rats looking at total striatal number and the newly generated fraction thereof -- we can predict essentially a 10 percent replacement of the normal adult rat neo-striatum every couple of months.
		What's particularly interesting here and germane to this morning's discussion is that these appear to be functionally relevant, or at least functionally relevant, neurons.  The striatum is a gemisch of different phenotypes, but the cell type, in many ways, of the greatest interest to neurologists, at least, as a target of disease is the medium spiny neuronal population, which is largely a striatal/pallidal projection pool as well as containing some striatal/cortical cells.  
		Now, the medium spiny population is, of course, the population lost in Huntington's disease.  It's characterized by expression of a number of markers, the calcium binding protein calbindin, as well as GABA and its synthetic enzymes.  We found that the cells being generated in response to the AdBDNF factor express almost exclusively calbindin.  Most of them are GABAergic.  They don't make any DPH or acetylcholine or the other markers of striatal interneuronal phenotypes.  So, we think that the cells being generated correspond to the medium spiny neuronal pool.  In fact, this is work we're now doing in Huntington's mutants to see whether or not we can, on any level, not necessarily restore function, but at least prolong the degree of life, the life expectancy of animals that develop the disease phenotype.
		I think for this audience, the take-home points are unnecessary.  The real bottom line is that we have a number of different lineage-restricted pools in the adult human brain.  Some of them are multipotential.  I say they're multipotential and lineage-restricted at the same time in that we have not shown that they are competent to generate non-neural phenotypes at this point.  So, these are relatively restricted, as least as defined by nestin and, for that matter, other early promoters that we've used, relatively restricted neural phenotype.  
		Then we have more restricted populations vis-a-vis the T-alpha I tubulin defined neuronal progenitor pool, the CMP-2 defined oligodendrocyte progenitor pool, that nonetheless may have some transdifferentiation or diversification capacity under the appropriate conditions. 
		The abundance of these cells is variable.  It varies by age.  It varies by region.  We still don't understand the lineage, the lineal relationship or the genealogy of these cells with respect to one another. 
		It's fair to say that the most abundant progenitor pool of the adult human brain we've been able to identify is that of the adult white matter.  Whether we can use that pool for purposes other than remyelination still remains to be seen.  
		Again, not to forget the viability of approaches directed towards inducing the endogenous progenitor pool and, for that matter, to combine these approaches to potentially induce endogenous progenitor pools in the context of exogenous progenitor implantation or co-implantations.
		Thanks for your attention.
		(Applause.)
		DR. SALOMON:  Thank you very much.
		Well, I think just so that we have some sort of framework, I'd like to have some discussion of these two very important talks.  We're also a little bit off time.  What I'd like to do is have about a 10-minute discussion of this, which isn't going to adequately cover it, and then break for lunch, which will be about 12:30.  Then instead of restarting the meeting at 1:05, we'll restart at about 1:30.
		So, what I'm opening up for discussion is Dr. Rao's and Dr. Goldman's talks.  I took notes with some questions that I wanted to ask.  
		One question I had for Dr. Rao was that he brought up this idea that the different precursors could be defined by different growth factor dependence.  So, you had FGF responsive and EGF responsive and FGF and EGF.  So, the question I have for you is how different are these different populations.  Are they possibly overlapping, or they actually just unique, distinct populations?  Then it gets quite complicated, as you said.
		DR. RAO:  It's not our data, but I think Dr. van der Kooy's lab actually did a series of very nice experiments which showed that these are distinct populations.  They used actually chimeras from FGF receptor knockout animals and showed that these two cells overlap in developmental time, but they're clearly distinct and you can actually isolate both of them at certain specific stages and at early stages isolate one.  
		The knockout data from FGF receptor knockouts and EGF receptor knockouts also clearly shows that at early stages in the ventricular zone, there's only an FGF receptor dependent cell, and at later stages, there might be an EGF receptor cell. 
		So, both of those say that they're clearly overlapping in developmental time and that two precursor populations, at the very least, exist.
		In terms of functionally their being different, I think there's quite a lot of data in terms of the frequency of neuronal differentiation at the very least.  So, the FGF dependent cell seems to have a much higher frequency of both after the short-term and long-term culture into making neurons, while the EGF cell seems to make at a lower frequency.  The phenotype of the neurons is different.  So, mostly what you get from EGF dependent cells are GABAergic sort of interneuron phenotypes, while what you get from the FGF dependent cells are really these sort of much larger glutaminergic type of neurons.
		DR. O'FALLON:  Professor Rao had a list -- I think it filled up the page -- of potential neurologic problems which we might be trying to address.  At top the list were cerebrovascular accidents.  Ischemic strokes come in a variety of different environments.  75 percent of people with those accidents will be hypertensive.  Some substantial number will have diabetes.  A whole bunch of them will have had cardiovascular situations.  Are we going to need different cells in every one of those contexts?  And does the same example persist for all of the other problems that you raised?
		DR. RAO:  I think for all strokes perhaps you might need certain classes of cells, but it will also I think depend on the region involved.  I think, for example, brainstem strokes will be very different from cortical stroke in terms of therapy.  I'm absolutely convinced of that.
		DR. REID:  I wanted to make three comments and questions.  One is in other fields it's been shown that when there's very striking density-dependent effect on cells with the same growth factors, the most common matrix molecule that's involved in that are the proteoglycans.  The proteoglycans are probably the major or one of the major matrix molecules of the brain.  So, there are a number of groups who have done a lot of work on identifying the particular proteoglycans that are in the brain and analyzing facets of their functions. 
		So, one prediction would be that if you take your cells at the low density but add a proteoglycan that is known to be generated by the cells at high density, that you would get the same result.  If that's the case, then proteoglycans could be one of the factors that you might want to utilize in order to drive cells in one particular way.
		The second issue is one raised about the immunogenicity.  Certainly in a number of fields, particularly in the pancreatic islet field, they have made use of fetal pancreatic islets to try to overcome immunological rejection phenomenon.  What they found was that they could actually buy some time when they would have not a significant amount of rejection phenomenon in the patient, but with time, those cells acquired the immunogenicity that adult cells would have. 
		We have just done the same thing in the liver stem cells.  Hepatic stem cells have virtually none of the immunogenic markers that might elicit an immunogenic response, but they very quickly differentiate into cells that do.  
		So, I think we're going to probably have to adopt the procedures long used by the hematopoietic field in which they tissue type the cells, and that's probably how we'll have to overcome immunological rejection phenomenon.
		The last point is that at least in the liver one of the critical variables on the extent of expansion of the cells is whether there is a cellular vacuum.  That is, it turns out to be in the liver the majority of the cells are polyploids, they're tetraploids.  I don't know if the same phenomenon exists in the brain.  But you have to eliminate a significant percentage of those tetraploid cells in order for donor progenitor cells to expand significantly.  
		So, it means for us that, when we start to treat, for example, children with inborn errors in metabolism where the they have an intact liver acinus, we will probably have to give higher numbers of progenitor cells in order to get a requisite response.  Whereas, if you have a patient with liver failure, in many cases it's most commonly due to loss of the polyploid cells.  You can probably inject smaller numbers of cells because they have a greater expansion potential.  So, the bet would be that in the brain, the same phenomenon may be true. 
		DR. RAO:  Maybe I'll answer the last part and see if you can tell me whether you agree. 
		So, Steve showed these cells which were A2B5 positive and he transplanted them in an intact brain, and what you see there is that they migrate quite extensively.  So, it seems to be dependent on the cell type that you put in and what its behavior will be.  So, it may not be as difficult as the liver where you necessarily have to lose a large number of cells before you see differentiation.  
		But by the same token, Steve pointed out that the cells seemed to be located in that part where there was injury, so that they were in the lysolecithin lesion, for example.  That's true even for neurons.  When you have a cavity, then you will see that the scar will look like the cell.  
		So, certainly both issues may be true.  We just don't know enough to be able to make a clear-cut prediction of what each cell type will be doing.
		Your point on the matrix and density is also very well taken.  We know, for example, that the effect of FGF is modulated by heparin, and heparin cells make proteoglycans.  In fact, when you grow them in culture, if you add that, you can reduce the dose of the cytokine 10-fold.
		It's also true that there are several other inhibitory proteoglycans which are quite critical in their response in terms of migration.  So, there's chondroitin sulfate proteoglycans which are also quite critical.  So, that point is absolutely true both in culture and in vivo. 
		However, I also think we just don't know enough about all of these extracellular matrix proteins to use them in the way you suggested in terms of being able to direct differentiation in any fashion.
		DR. NOBLE:  It seems that one of the things that's already clear from this morning is that the pace of discovery in the biology of the cells is very different from the actual pace that will be followed in clinical trials because while we discuss this complexity, the fact of the matter is that each individual group or company that's going to move into clinical trials has its particular population in which it has an invested intellectual property position, and it doesn't have the freedom to choose from this great panoply of cells and say, well, which one is the best.  They're going to go forward with what they have.  
		So, one of the things that I thought was very important in Dr. Rao's presentation was this list of information that perhaps needs to be collected in every clinical trial about the kinds of cells that are used so that at some point in the future, where we're able to make sense of this great complexity, we actually have this information to go back to rather than to have it be done in a slipshod manner that these things appear to sometimes be being done.
		DR. SALOMON:  Steven, when you started off, you actually introduced yourself saying you're not quite as sanguine as Dr. Rao.  I was waiting for the non-sanguine part of the talk.
		(Laughter.)
		DR. SALOMON:  You ended up using adenoviral gene therapy.
		(Laughter.)
		DR. SALOMON:  I'm thinking this guy is pretty optimistic on my part. 
		DR. GOLDMAN:  Adeno, of course, was intended as proof of principle only.
		(Laughter.)
		DR. GOLDMAN:  Nonetheless, I'm differentiating between the basic biology and the clinical utilization thereof.  I think that's obvious. 
		One thing that I thought was a take-home point of this morning's discussion was that all of the debate, with regards to the relative priority of importance of understanding lineal relationships, the microenvironment, the interactions between the two, ultimately will be meaningful only in the context of discussion surrounding each disease choice, each disease target.  The priorities that obtain in designing therapeutic strategies will very much be a function of what disease target one is trying to use these cells for, "these cells" being a very generic term in and of itself.  I don't see any of the disease models yet being at a point where we can move very quickly with any assurance towards clinical trials.  
		I've focused a bit in my own work on the demyelinating diseases simply because, in my simplistic way of thinking, those are the most simple and will be the most readily addressed.  I think when we start thinking in terms of stroke, traumatic brain damage, at least as a neurologist, I'm very pessimistic in terms of utilizing any single cell type towards any meaningful treatment, at least at this stage in our current ignorance.  It's going to be years before we develop the degree of understanding that will allow us to do so safely, at least again in my own opinion.  
		Whereas, with some of the more phenotype-defined and phenotype-specific diseases such as the acute demyelinating illnesses, we may be able to at least attempt therapeutic trials, recognizing that we still have a substantial degree of ignorance but also working on the assumption that we're likely to do very little harm in trying to address these more simple problems. 
		But the sanguinity comes from contemplating Mehandra's list of diseases, and thinking in terms of meaningfully treating any of them at our current level of understanding I think is premature. 
		DR. RAO:  In my defense -- 
		(Laughter.)
		DR. RAO:  -- I'd just like to add that, in general, whenever I've given a talk like this, people have told me I'm too pessimistic.  I didn't take that list as a specific indication of therapy.  I just abstracted the list from Harrison's Textbook of Medicine --
		(Laughter.)
		DR. RAO:  -- simply to point out that this is the list of neurological disorders in which people have said that they would like to use stem cells in therapy.  Certainly I am not suggesting that we should use stem cells in any of these things. 
		DR. GAGE:  Listening to this morning and this talk and then hearing what the topic of the conversation is, stem cells, at some point we should have some discussion about really are we talking about stem cells.  Is it really useful to even talk about stem cells if we're talking about cellular therapy?  Because in a broader sense, we're looking at harvested cells that have therapeutic value.  While there's, I think, a lot of attention surrounding the conceptual problems associated with the term "stem cell," I'm hearing in the talks that it's more important to define the cell type that's appropriate for the disease and you may isolate the cell at different time points depending upon when it's needed. 
		One of the key features that separates out, it strikes me, this idea of stem cell progenitor population from what's been going on in fetal tissue grafting for a long time is the ability to propagate the cells, this ability to propagate the cells in some immature state so that it can retain the capacity for differentiation down the lineage that one is interested in.  Whether or not you're propagating it was a stem cell or as a totipotent, pluripotent, multipotent or progenitor cell doesn't really make any difference from the perspective of the clinical application so much as that you can retain the plasticity of the cell so that it can effectively and efficiently generate the cell type that will be effective down the road. 
		I think sometimes this idea of having to fall back on the concept of stem cells and the definitions associated with stem cells is a bit of hindrance in our ability to maybe think about applied problems associated with cell therapy.
		I was wondering if the speakers had any -- since they were talking about lineage-restricted cells.
		DR. RAO:  I think the emphasis was more a function of classification of what the cells can do when you call them lineage-restricted or we call neuron-restricted or biased because that is of some predictive value in which cell you use.  It's a function of classification.  That's really critical.  So, I absolutely agree with you that you really need to know what the cell can do and what it's properties are going to be when you use it.  That's why you classify them in some fashion.
		DR. GOLDMAN:  Well, there are operational consequences to that.  To the extent that there's an inverse proportion between the profligability and the lineage restriction, which at least has been the case with most of the models thus far presented, thus far published, we may find difficulties in terms of harvesting -- I think this is axiomatic.  It's not surprising -- lineage-restricted, more functional, more competent lines that can be maintained as lines.  I think that's the situation.  We're getting back to the earlier discussion.  We're going to be the most dependent upon going back to new source material.
		DR. CHAMPLIN:  Just reflecting that one needs a stem cell deficiency disease ideally to test this, the neurologic equivalent of aplastic anemia, and at least in the hematopoietic systems, if you treat stem cell disorders like myelodysplasia just by giving identical twins stem cells, of course, you don't cure those patients because the disease itself is suppressing the growth and differentiation of the normal stem cells.  So, in these situations, your endogenous stem cells are being suppressed and/or you've reached a regulatory state where the nervous system no longer is providing a proliferative stimulus at the stem cell compartment.  So, one needs to modify the local microenvironment to allow the stem cell transplant, if you will, to proliferate and restore neurologic function.
		DR. GOLDMAN:  Again, that's a function of disease.  It's very likely that there are diseases where the stem cell pool is functionally knocked out and others where it's very likely to be adaptively involved.  Unlike the hematopoietic system and unlike hematology, I think the problem in neurology is that we simply don't even know what the diseases of stem cells are, or at least of the diseases that we might presume to exist of resident neural progenitor cells, not to say stem cells.  The fact that these progenitor populations exist and are abundant suggests that there is very likely some pathologic manifestation in disease of their dysfunction, and we don't even know what they are yet.  So, again it's premature to draw that parallel I think to the hematopoietic system.
		DR. MULLIGAN:  Just to go back to Rusty's point, I was struck by John's talk when the stem cell-ness of his cells was looked upon as almost a manufacturing concept.  So, I think that one very simple concept is how do you make a batch of the cells.  
		But I think the uniqueness of the stem cell approach is definitely the migration capacity, the trafficking capacity.  Although, while it's very true that the natural environment doesn't occur, certainly from our work from, say, endothelial cell specification after bone marrow transplantation, there is such exquisite trafficking in response to bone repair.  So, for instance, if you ligate a coronary vessel, after doing a bone marrow transplant, the only situation where you'll see endothelial cells is right in the infarction zone.  
		So, I think that we should keep in mind that, in addition to what I would call the simple kinds of applications, which is the just manufacture and amplification, that understanding, albeit in an abnormal, maybe wound-healing context, these stem cells, depending on exactly what they are, may have very different kinds of properties.
		I had a gene therapy question on the adeno case.  It gets back to the hematopoietic system, the fact that when you treat animals with 5-FU, which kills off a lot of cycling cells, you see a rebound in terms of cells that have some sort of reconstitution potential.  Is it possible that the mechanism of the AdBDNF is more destruction, adeno-mediated destruction of those cells, with perhaps BDNF providing some protective effect over the destruction?  I noticed that from the bar graphs that you had, it looked as if even the AdGFP seemed to induce some increase in the proliferation.
		DR. GOLDMAN:  In the striatal neurogenic population, that's right.  That's why we did that control was to see whether or not there was an adeno-mediated cytokine release.  
		What we've actually parenthetically observed in adeno-dependent endothelial production or in adeno stimulation, it consisted of endothelial production of BDNF itself which would directly relate to your question.
		I don't have a straightforward answer there in that we have done a no adeno control and see a smattering of striatal neurons being added.  That surprisingly is not new.  A number of groups through the years, as far back as Caplan in the '70s -- van der Kooy had a couple of reports in the mid-1900s on this -- in the context of looking for ‑‑ in various controls looking for neurons being added to adult brain, at least in the rat, there are consistent reports of occasional neurons being generated and recruited to the adult neostriatum.  
		So, here we see an adeno BDNF associated increment, a substantial increment, in neurons being recruited to the neostriatum.  We see a very, very low number of neurons being generated and recruited in the adeno no condition.  Those numbers are so low that I don't know that they're any different from that which we would see in the absence of the adeno.  
		So, the quick answer is I don't think that the adeno per se is affecting the natural history of these cells.  I think the more important issue here is whether an adeno-dependent cytokine effect might be acting synergistically with the BDNF.
		DR. MULLIGAN:  Yes, that was really what I was getting to, but even whether the BDNF was acting synergistically with the adeno.
		DR. SALOMON:  One last question.
		DR. DRACHMAN:  Dr. Rao raises a real paradox, that is the more restricted the neurons, the safer, the less restricted, the more they're capable of doing a variety of things.  From the point of view of a neurologist, it's always worth remembering the brain is not a liver. 
		(Laughter.)
		DR. DRACHMAN:  Santiago Ramone y Cajal and others pointed out how many thousands of different types of neurons we deal with.  We as neurologists live on the notion of selective vulnerability, that is, all these diseases which pick out tiny groups of neurons that characterize the disease itself.  So, the attractiveness of stem cells, meaning the tremendous capability of doing almost anything, is sort of contradicted or balanced by your need for defining what they are and using just the right ones.
		DR. SALOMON:  When you have this many intelligent people with an interesting subject, it's never a good time to stop but I think for biological reasons --
		(Laughter.)
		DR. SALOMON:  -- that are short-term rather than long-term I think we'll stop here.  See you all at 1:30.
		(Whereupon, at 12:50 p.m., the committee was recessed, to reconvene at 1:30 p.m., this same day.)

	AFTERNOON SESSION
	(1:48 p.m.)
		DR. SALOMON:  The one thing I wanted to do this afternoon, just because I think that the discussions that we're having -- and I've gotten feedback from a number of people at lunch -- are really excellent and where a lot of the meat of what we're going to provide the FDA in this is going to be -- is to just ask the speakers to try and stick to like a 25/30-minute talk more than the 35-minute/40-minute talk, which allows us to stay a little bit more on track because I really do hate cutting off these discussions, as they're really I think very, very valuable.  So, usually somebody decompensates whenever I say that.  It's, oh, my God, I've 10 slides too many.
		The first talk of this afternoon is Jeremy Sugarman from Duke University, Anticipating Ethical Issues in Clinical Experiments Using Stem Cells.
		DR. SUGARMAN:  Well, I appreciate the opportunity to think about these issues with you, and it is always, I think, important to bring the ethics in from the beginning of when deliberations about thinking about moving forward to clinical experiments with people are being contemplated, to think about the ethics early rather than waiting for something wrong to go on or to try to address what might be a political or a moral debate.
		I want to thank especially Don Fink for his help in getting materials ready for this meeting in preparation for it. 
		Starting this, even though I do have a Power Point presentation, I have serious slide envy here.  I don't have an ethics gel or I don't have any ethics fluorescence.
		(Laughter.)
		DR. SUGARMAN:  I really am feeling inadequate to this task.  I could build on the word "potency" but I won't.
		(Laughter.)
		DR. SUGARMAN:  So, I'll just move forward with what I hope to do. 
		What I'd like to do is give you an overview about the focus and background conditions of the area of ethics that I'm going to address, and then talk about what are some of the issues that might be important when we move from bench to bedside or thinking through that, and then some additional considerations that are going to be of relevance when thinking about cellular stem cell therapies.
		The focus of my comments today are going to be on clinical trials, not the collection and storage and all the wonderful things people do to stem cells.  I think a lot of these issues have been discussed, debated, deliberated, created lots of emotional statements on either side.  There are some critical issues at hand, but there's plenty of scholarship out there and plenty of places to go for work in that area.  I think we're at another threshold here and those are the kinds of questions I want to take on.
		In addition, I don't want to try to tackle what the issues might be for using stem cells for anything, from diseases outside the range of neurologic disorders.  I don't want to go into the treatment of cardiac disease or all the other chapters in Harrison's.  What I'd like to do is just focus on sort of the candidate diseases here, staying away from the use in angst which I think would be useful in the future.  Parkinson's disease, amyotrophic lateral sclerosis, ALS; and spinal cord injury.
		I'm also going to make an assumption about adults as potential subjects rather than kids.  Basically a spinal cord injury obviously can happen to children.  It raises different issues for consent and proxy decision making, which we can talk about if it becomes an issue, but I just don't want to complicate it at this point.
		Here are background conditions that I think are of great relevance.  There is a huge amount of trust in the scientific enterprise and its oversight.  Despite the scandals that take place from now and then, all the empirical work in ethics shows that folks trust us.  They trust investigators.  They trust institutions and they trust the oversight mechanism as a whole to get this right.  There are folks who don't trust us, but when you talk to patients in hospitals around the country, they trust us.
		The trust, when you hear it expressed through a variety of empirical work, is very humbling.  The expectation of looking at somebody in a white coat taking care of someone who's sick is a very powerful symbol that clearly can't get dismissed.  And the thought about what it means to get this right becomes magnified for those of you that have the honor of working in the clinical setting.
		At the same time, there are huge arguments for access to investigational drugs.  This debate got started, obviously, with the AIDS epidemic and clamoring and changes in drug approval mechanisms and approval processes.  And especially for devastating disorders, it's hard to argue in the face of folks who are suffering and saying we want access to this.  We don't care if it's going to hurt us. We're dying, we're suffering, we're in pain.  Whatever the condition is, it is really hard to say we're not ready to give you something.  We just don't have it.  It's difficult.
		The scientific enthusiasm is enormous.  We saw some great science this morning.  I'm sure we'll see some more great science this afternoon.  Scientifically these are great ideas.  Gene transfer experiments, sometimes called gene therapy, exciting ideas, novel.  They build on what we assume.  We like it.  So, the science here is high.
		The financial and moral stakes here are high as well.  It's no kidding that there has been a lot of money expended on doing this science.  The moral stakes of messing with folks and messing with people's wallets and lives and things like that are pretty high as well.  So, it's important that we get this right, especially in light of all the trust.
		That's sort of the preaching part about ethics.  Let me just switch gears here and give you some principles.
		We have well worked-out principles once we get into clinical research.  The Belmont report that came out of the National Commission for the Protection of Human Subjects and Biomedical and Behavioral Research -- there's no test on the name of that commission -- outlined principles and outlined rules that we have once clinical trials start.
		But what goes unaddressed is the really crucial step of doing things in people for the first time.  It's not to say deliberations don't go on, but they're important.
		I'm going to draw here on an article that I did in Science a few months ago that lays out the rationale for these principles.  After lunch, I'm not going to try to burden you with that.  But let it be said that there are four sets of considerations, ethical principles that need to be met.  And I'm going to go through each of those from the little that I know about the basic science to date and the preclinical studies.  What I'd like you to do over the next couple of days and in continued conversations is to think about what's the science, what's the clinical problem, and how do we bring those principles to bear.
		The four are safety, the possibility of benefit, what the experimental design is going to be, and consent.  Let's move through these one at a time.
		In terms of safety, this is the crucial element.  This alone, not so much that there is a consideration of a calculus between risks and benefits.  They're not commensurable.  You can't equate them.  The calculus doesn't work.  A risk/benefit calculus isn't meaningful in this first step.  What's really critical here is that preclinical studies tell you that it is safe to proceed.  The idea of doing something in a person for the first time, safety is paramount, not harming. 
		Here you could make an argument that there needs to be near unilateral consensus in the scientific community about safety.  We've heard different issues raised this morning about what those safety issues are.  There are concerns about tumorigenicity.  Where is this stuff going to go?  Where is it going to grow?  Will it not grow?  Will I hurt the person in the process of putting it in?  Will they be infected with something else?  The preclinical studies should give you sufficient information about safety to say that it's okay to move forward. 
		If you can get assurance or consensus that there is safety, the next step would be the possibility of benefit.  In an early phase experiment, it's critical that safety is met.  It's desirable that there's benefit, but it is clear that the reasons why we do things in people for the first time is we're just not sure from our preclinical studies about whether this is safe in a human being.  It's nice if you can get some benefit out of this. 
		Now, originally the paradigm that most ethics folks in the regulatory scheme work on is a little out of date for biologics.  It was put into play for chemotherapeutic agents and the like in which phase I was dealing with toxicity and just checking for toxicity.  That model doesn't seem exactly right, but it's the best we have to date.  So, the question is, it's desirable to design an experiment that can show some benefit, but really what you're trying to get at -- and this is important in the scientific design, as well as getting consent and moving forward -- that you can say that we want to get there, but truthfully this study is not about this.  This study, the first time we do this, is not about curing you or the disease.  It's about figuring out whether we don't hurt folks.
		Here a thing called clinical equipoise is a sufficient metric.  There doesn't really need to be consensus about whether there's going to be benefit.  People are going to disagree.  Is it 1 cell?  Is it 2 cells?  Is it the microenvironment?  Is it this kind of cell or that kind cell?  There is going to be disagreement, and science will give you the answer.  As long as you're not harming people in the process, in some ways it doesn't matter quite as much.  You want to get it right, but the history of science and medicine has shown us that our predictive abilities are not that great.
		Here the word clinical equipoise is one used when you're in the middle.  You don't know.  It's a great word for Scrabble because it has a Q in it.  So, you can use that.  People will say that's an equipoise word.  It doesn't mean that every scientist is going to be directly on that tight rope saying, yes, I'm 50/50 on this.  I don't know.  Every one of us, when we want to move forward in the clinical experiment, believes that this cell, this approach, this remedy is a home run.  And we're wrong statistically.  If you don't think you're hitting a home run going into clinical experiment, you might not as well try.  The numbers are just dismal about how well we do in this.  But there should be some equipoise in the entire community of scientists about whether there's a possibility of benefit.
		Now, design here is really crucial.  It's not just about putting something into somebody, but the design itself has to be sound.  There have to be ways to assess it, and there has to be fairness in the selection of the conditions and subjects.  And I'll talk about each of these in turn. 
		For science to be sound, there obviously has to be some theoretical justification for it.  You would like, with all of these preclinical studies, animal models and the like, to be able to explain, at least with the current theory or theories that you're working under, that the science makes some sense. 
		You also want to minimize risks and maximize benefits.  Minimizing risk here means to use a safe product, and the question of the source becomes really critical here.  At this stage of the science, from an outsider looking in, it would seem that it makes considerable sense to know exactly what that source material is.  Dr. Rao mentioned some of this in his talk.  Those may not be the final set of criteria which you use to address this, but it's clear that some of that information is going to be vital at this stage of science.  
		What are the implications of that for donors?  Well, obvious.  Their privacy might be violated.  People might want to go back and get more cells.  We know that that problem is there.  We just have to build appropriate fire walls and protections and mechanisms of protecting the privacy and confidentiality of donors.  That's possible.
		The key here is going to be to know whether the source is safe and then, again, another set of scientific considerations regarding what kind of tests we can do now.  No matter what kind of tests we dream up now, within five years we're going to think of new tests that we can now do, that we want to do.  Anticipate it now.  We've done this over and over and over again where we've got a pot of stuff, whatever it is.  If it's blood, if it's stem cells, if it's cord blood, it doesn't matter.  We find a new test.  We want to do it for everybody.  So, we need to anticipate that in the donor recruitment and selection process.  Again, I don't want to get diverted there, but again, you're going to want to think through those issues now if you're anticipating doing clinical trials in the future.
		Linkage in testing, obvious results.  The hazards here are not maintaining an appropriate fire wall so that people's privacy and confidentiality aren't inadvertently violated.  Coming up with sort of standard procedures that hematologists, blood bankers, and tissue bankers of all sorts are well accustomed to.  These are not new issues about what the procedures are going to be for notification, what the notification procedures are going to be provided something goes wrong, provided you discover something.  When will you warn?  These are well rehearsed problems. 
		In terms of maximizing benefits, obviously you want to pick the best science.  You want to try to hit the home run.  This is obviously the intent of most, but that may mean some distinctions and differences between which product gets tested first in people.  That's when the science gets a little interesting.
		Outcome measures.  This is critical in biologics.  What's measurable and what's meaningful?  How are you going to measure it?  It's one thing to do this in a person and put it into brain, a black box.  How are you going to get it?  Are you going to be able to image it?  Are you going to have to take a piece of it, a chunk of it, a chunk of it periodically, and what the implications will be about where you're putting certain cells and what they're doing and how you're going to model that.  That's going to be crucial to figuring out whether this is the right condition to start with.  You might be able to do the same kind of learning somewhere else.  The question is how are you going to get there.  Is this going to be a meaningful result, not just something that changes over time?
		Adverse events, another area that's received just a little bit of popular press and attention.  Adverse events in biologics, gene therapies, vaccines, and the like.  Again, we've got to know what we're measuring and we've got to figure out how we're going to report it, and when we're reporting it, where does that mass of information go and who's going to look at it?  
		IRBs are not equipped to look at adverse events.  DSMBs may be equipped to look at adverse events, but may not communicate with IRBs.  This is a problem across clinical trials.  But as we're moving forward, especially with high profile trials, it would be important to lay out those mechanisms in advance about who is going to do what.  
		It may be that you construct your DSMB, if you have one, even though you might not construct a DSMB for a phase I equivalent sort of trial, but in a case like this, there might be good reasons for oversight, especially because it's sort of devoid of what once was the RAC mechanism and the like.  And we can go into any of this in detail.  But you're going to need some folks who are pretty savvy with the science to help sort this out.
		Fairness in selection is another criterion of doing good science.  First, of the condition.  In this country we have people who were effective at lobbying hard for their disease condition.  AIDS and cancer are the most notable about being able to lobby political interests.  It affects a lot of people.  There were clamors for access to experimental designs.  Those are legitimate claims.  The question is early on what's the appropriate candidate disease to learn about the technology or the approach.  
		One criterion here may be what best advances a scientific goal.  If one disorder requires the intervention of 20 things and another disorder might require the intervention of 1 thing, it's going to be a whole lot easier to figure out the 1-thing science than the 20-thing science.  You can figure out which diseases and approaches you're thinking about, but the elegance of the science will probably help inform future efforts, even if that flies in the face of some pretty intense lobbying efforts.  It's not to say that those diseases aren't devastating, but if you take this approach and there are bad outcomes and they're uninterpretable, we don't do well in the long run.
		One other condition that's often advanced is there is a lack of available alternatives, and there's a plus and a minus to this.  I'm sick.  There's nothing for my condition.  Treat me first.  It's a legitimate claim as well.  It's a very powerful claim.  The question is, though, from a position of consent, where you maximize the ability to sort of say there are no other alternatives, and so the risks and the benefits seem to not make a lot of sense, folks can't very well be positioned to give a meaningful, voluntary consent process.  They're pretty vulnerable folks.
		Among that, you're going to get questions of fairness in selection of subjects.  Who gets to come in first?  The stockholder, the first person in line, the first person who expressed an interest, someone who will donate to the lab, build a new building, has basketball seats of the major university?  Whatever the major goal is, I think it's important to think how you're going to choose among subjects, and some of those criteria aren't just who got there first but a question about how that selection process is going to take place, and again, how you can learn the most from the science, especially early on.
		The final part.  No ethics person can get up here without talking about informed consent.  It's our favorite and one of the reasons it's our favorite is because most scientists recognize that you have to do that, and so it's our way in.  We know you're going to fill one out.  We know you're not going to like whatever the IRB says about the font, your language choice, and consent documents, but there are some other parts here. 
		Let me go through an informed consent, the standard approach, some challenges that are special to these sorts of things we're talking about, and avoiding something called the therapeutic misconception.
		Standard consent.  This is ethics 101.  Informed consent is a process, not an event.  Three major steps:  threshold, information, and consent.  Threshold means if you don't pass that threshold element, you're not allowed to play informed consent.  It has to do with decision making capacities, sometimes termed competency.  Competency is a legal determination.  Most of us are not judges.  Most of us don't want to be judges.  So, we can make an assessment in the clinical or research world about decision making capacity, people having an ability to make a decision, take in new information, process it, use it to make a decision, and evidence that choice.  
		Critical components.  Think about the diseases.  Some folks for some of the neurologic diseases may have impaired decision making capacity or competency.  Now, some of the ones bandied about today, not so much, but the question is going to be very important to think through what kind of capacity people have to make that decision.
		Another threshold element is voluntariness.  If you really have nothing else wrong with you, anything else to be considered in that situation, you may not be positioned to make a voluntary choice.  I once gave an ethics talk and a woman came up to me.  She raised her hand and she said, you know, I can never get people to sign the consent form.  And I said, well, why not?  I was thinking there must be some kind of strange science that they were going to put something in and it was like an X Files case or something.  I didn't know what it was.  She said, I can't get them to sign it.  I said, why not?  And she said, well, their arm is always tied to that arm board.  I said, when do you recruit subjects?  And she said, oh, in the pre-op holding area.
		(Laughter.)
		DR. SUGARMAN:  Now, most of us are uncomfortable buck naked with 16 gauge IVs and our arms like this.  It's definitely not a position of voluntary choice.  You may volunteer to be there, but that's not what was intended by here.  Some clinical circumstances position folks not to be able to make a voluntary choice, and we need to think through that. 
		Those may be pieces.  You don't put those on your consent documents that the person has decision making capacity and that they're in position to make a voluntary choice, but that's the spirit of the process of informed consent.
		In considering and designing these, we should be talking about the consent process as a whole.  Now, here this stuff is going to be more familiar.  
		Information.  Right?  Part of it is informed. 
		Disclosure.  Disclosure has nothing to do with the example I gave you in the pre-op holding area.  Disclosure has to be the kinds of information that the investigator gives to the potential subject.  This stuff everyone is familiar with.  Right?  It's in the common rule.  It's in the FDA regs.  It's everywhere.  
		Procedures to be followed.  This involves research, the risks, the benefits, the alternative, procedures to protect confidentiality, who to be notified in case you're a research subject, who to talk to if you're injured as a result of research, that you can opt out anytime.  Those elements are clear.  And, you know, they're pretty sound.  It's hard to argue against any one of them, especially in new technologies.  Just follow the rules.  Check the boxes.  Make sure they're all there and make sure you get them right. 
		Understanding here is really key.  Now, understanding these elements.  It's one thing to walk up to someone -- and I'll choose a non-neurologic disease -- and say, hey, you've got x-linked hypophosphatemic rickets and I'm interested in 1-alpha-renal-hydroxylase.  Do you mind giving us 24-hour urine specimens so we can consider the possibility of gene therapy?
		(Laughter.)
		DR. SUGARMAN:  Now, numb.  Right?  It doesn't mean a word.  You have to give this information in a way that people can understand it.  
		This is an embedded process, and if I did bring my ethics gel, it has a bunch of words that we use to describe research.  It is clear from empirical studies that the very terms that we use to describe what we're doing are critical to what's going on.  
		The work with which I'm most familiar is some that we did for the Advisory Committee on Human Radiation Experiments.  Notice it was the Advisory Committee on Human Radiation Experiments.  At the same time, researchers at Johns Hopkins and Duke University announced a cure cancer in mice.  Right?  
		So, here we have research and experiment.  When you talk to patient subjects around the country, they hold these terms to be very, very different.  Experiments are scary and risky and you're a guinea pig.  It's the first time.  Medical studies, a phase I study, for instance, very benign to most patients who might be recruited for research.  A study is when the doctors and nurses look over your medical records.  They study up on you.  Who wouldn't sign up?  So, the terms themselves are quite important. 
		And just so you know, the terms "clinical investigation" and "clinical trial" -- people don't have a clue what they mean.  "Clinical investigation" is they wonder what went wrong.  And "clinical trial" is like time for Wapner.  There's no sense of what these terms are that we use when we bandy about. 
		Now, the other piece here that's going to be key with biologics is that the perception that biologics are natural and natural is better.  Sort of like your poison ivy, right?  Now, poison ivy causes problems, but when we understand natural or biologic, this is a difficult concept to communicate about the fact that this is something medical.  It may be like a chemotherapy that there's some associated risk.
		Get through that.  You've got someone with decision making capacity who's been able to make a voluntary choice.  You've given them information that you're required to give them in a way that's understandable to them.  And then they make a decision and then they authorize their decision by signing one of those forms.  That's basic consent. 
		The special challenges here I think are going to relate to the source of stem cells.  I would add -- I would be hard to convince not to believe this.  Truman talked about the economist, you know, the one-handed and the two-handed.  My friend Lance Dell at Davidson College talks about two-handed or evenhanded ethicist.  The two-handed ethicist weighs all the alternatives on this side and all the alternatives on this side, and then washes his hands and lets you make you make your own decision.  The evenhanded ethicist sort of says, there are some options on this side and there are options on this side.  And you know?  That one is just not right.  
		I tend to be evenhanded when I can be.  Sometimes I can't know.  I don't have a horoscope.  I do use an 8 ball on occasion, but not for matters of real scientific importance. 
		But here the source of stem cells is going to need to be included in the informed consent process.  The area that is charged in this area -- say it out loud, say it explicitly -- is the source of stem cells.  If they come from embryos, it scares people, not all people, some people.  Tell them.  Some people will say, thank you very much, I don't want that.  Other people will say, I don't care, it doesn't matter to me.  But they need to know.  Just include it.  Figure out you're going to include it.  These were derived we don't know.  These were derived from whatever you need to tell them. 
		There is a lot of uncertainty about risk, and I think that's going to be hard to communicate.  It's always hard to communicate this and especially the first time you're going in.  This isn't a novel, but the part that I mentioned about a biologic and what that means is going to be the trick here about communicating that kind of risk.
		The other is the alternatives or lack of alternatives.  It is no surprise that the list of candidate conditions is devastating.  They're awful conditions with a lack of good alternatives in many cases.  But in other cases, there are.  If you're thinking about Parkinson's disease, there are pharmacologic interventions.  There are fetal surgery -- I mean, we can debate and quibble about whether that's an effective alternative, but there are alternatives.  And those alternatives need to be described in detail so people can make decisions because sometimes we've got something to offer.  Those need to be incorporated even though there's going to be this move to move the science forward quickly.
		Well, here the piece on biologics again plays into something that's common in clinical trials of all sorts.  It's known as the therapeutic misconception.  It's the belief that experimental procedures are directed primarily at therapy.  There is a distinction.  When a doctor is taking care of a patient, the overriding goal in that case is the primary care of that patient.  Something is not working.  You shift gears.  Right away it's about you, the patient.  In a clinical trial, part of it's about you, the patient, the very critical part about it, but there's also another tension and that's of sticking to the science and finding out what happened.
		There are additional procedures, even if both of those interests are aligned, that just aren't part of clinical practice.  There's extra sampling to check for biologic effects for outcome measures.  There are extra visits.  That could be good or bad.  There is going to be everything from questionnaires to urine and blood samples.  Whatever it is you dream up, there's going to be something else.
		When you talk to patient subjects on trials, they believe, even in phase I studies in oncology, which are about toxicity -- they can tell you the scientific rationale of the study, but they believe that they're going to get better as a result on this trial.  Now, balancing candor and hope in these situations is very difficult.  The challenge, though, is so that folks can make a decision to know what the stage of the science is and early on -- later on phase III, randomized phase IV, whatever -- the differences are hard to flesh out and you don't need to quibble there.  But early on it's real critical to know that therapeutic misconception exists. 
		Serving as a subject can be challenging for patients.  We just don't.  The phenomenology of illness tells us that we don't want to be treated as a subject.  We'd rather not be treated like a patient, but it's better than being treated like a subject.  We just don't like it.
		An investigator's interests are, unfortunately, aligned with this.  We want and need to personalize.  We don't want to tell folks that this is about the science and not about them.  It's tough.  For those folks that are clinical investigators to walk into a room and say, this is about the science and not those things I wrote on my essay to get into medical school is a hard transition.
		Moreover, it enhances enrollment.  If people believe this stuff, this is a perennial issue in clinical trials.  None of it is bad intentions, I don't believe, but it's very present and I think critically important in early phase trials.
		Finally, these are additional concerns outside of the sort of box that we've been thinking through.  
		There are going to be some pressures to delay progress.  I don't know why you decided to have this meeting out here in this outer galaxy of the beltway, but we're not downtown.  We're not in the fray.  There are going to be pressures to delay.  You say stem cells.  People panic.  There are going to be some pressures to delay.  
		But there are also going to be huge pressures to move forward.  And the pressures to move forward are going to have to be balanced in these ways about the excitement and when it's appropriate to move forward.  Again, it's just important to say these things.  It's like the emperor's new clothes.  It's just what it is.
		So, in conclusion, the scientific and commercial interests, along with the hopes of patients, need to be considered in light of the scientific realities and not merely the aspirations.  There is a clear set of ethical issues and considerations that we need to think through in moving to clinical trials, and I think it's critical to deliberate about these things explicitly as the process moves forward.
		Thanks for your attention.
		(Applause.)
		DR. SALOMON:  Thank you.  That was really excellent.
		So, to start the discussion, there were many points you made that I think are worthy of discussion.  One that sort of picks up on a theme that we were discussing when we walked in you brought up under outcome measures.  I thought you put it very well by saying there was a contrast between what was measurable and what was meaningful.  I'd like to hear some comments from the experts on that since outcome parameters in terms of these first clinical trials in these transplants is going to be obviously critical to everybody.  That's not saying anything surprising.  
		So, there are people here at the table who have got their own disease stuff.  Right?  Some of you are doing Parkinson's disease, some Huntington's, ALS.  So, could you guys maybe make some comments from your own area on what would be a measurable outcome parameter versus what would be a meaningful outcome parameter?
		DR. FREEMAN:  I think the first question is what's tried to be addressed scientifically in the trial, and if you've got a novel therapy where there are no alternatives and you have any reproducible, meaningful benefit, that would be important to ascertain in a phase I trial or even a controlled trial.  But I think the hurdle gets higher and higher as you have more options and the science advances.  So, I think it's really a moving target.  Do you power a study based on a statistically significant change or a clinically relevant change or a change that addresses your scientific question is a separate issue based on where you are in the science and in the development of a new therapy.
		DR. DRACHMAN:  My favorite quote is Gertrude Stein who said, "For a difference to be a difference, it has to make a difference."  I think that's probably relevant here.  We've gone around and around the treatments for Alzheimer's and finally ended up with a CIBIC-plus, which means that someone other than a neuropsychologist can also tell there really is a difference, but it's very difficult. 
		DR. TROJANOWSKI:  I think just to follow up in the area of Alzheimer's disease, it's still a moving target.  I just came from the meeting of the WAC 2000, the World Alzheimer's Conference 2000, downtown within the beltway.  It was announced yesterday in the Washington Post a vaccine that may eliminate plaques.  That's really very compelling preclinical data.  But patients don't come in complaining of a head full of plaques.  They come in complaining of memory impairments.  There's still, I think, controversy in the field as to whether eliminating plaques will convert these patients into those that die of a "tauopathy" because their tangles continue to accumulate or not.  
		There are going to be imaging agents shortly I believe that will image plaques, maybe tangles as well, but I don't think we have our arms around all of the measurable important and relevant things that one might want to consider.  Certainly the patients complain of memory, and that's the most important thing.  
		I think an innovation that's just developing that I see in the Alzheimer's centers, the 27 centers around the country, is interrogating the caregivers for their views as to what an outcome might be that is beneficial.  Remember, these patients are cognitively impaired and cannot themselves always state whether they're feeling better reliably.  
		So, fortunately, the science is moving at such a pace that we hopefully will have all the measures in place when the vaccine -- they're actually going into people right now, but we won't know for certain right away.
		DR GAGE:  John brings up a really interesting point that be worth some further discussion about the ethics of it.  So, the tauists versus the amyloidists or Baptists.  
		(Laughter.)
		DR GAGE:  We don't know really whether or not either one of those pathological manifestations are involved in the behavioral deficits that are seen, which are really what's important.  But if you had a therapy which could eliminate the formation of the amyloid, even though you don't know whether or not elimination of the amyloid is going to change the nature of the disease, since there isn't an animal model that is excellent for the disease, is that justifiable?  You're asking a scientific question to some extent with the hope that there might be some therapeutic output.  But is that something that -- obviously, they're going to do it, but where does that fit on our --
		DR. SUGARMAN:  No.  This is the exact kind of conversation that ought to go on, and it's this conversation that often happens but in small groups and not among different groups of experts.  This is what should happen with each trial as it goes forward. 
		Now, it happens sometimes behind closed doors, but because of really legitimate reasons for those conversations to take place behind closed doors.  There are proprietary interests, but at the same time, they're very powerful in terms of what's going on in this trial and what's this trial about.  
		So, you have to say, what's this trial about?  If it's to look at plaques and then say, does that correlation between plaque formation correlate at all?  Do you want to know in that study even if that correlates with behavioral change?  Or is that a separate study?  Those could be discrete scientific questions involving different investigators and different outcome measures and how you power it.  So, this is the precise kind of conversation is what I would say.  
		Either one of those studies is probably okay to do.  You could measure plaques.  You're saying, okay, there's a way to measure plaques.  It's not invasive presumably to measure the plaque.  It may be, it may not be.  There may be more invasive ways.  What's the gold standard?  And you start to notch up.  You change the characteristics of the trial in each case and each of those calculi that you need to go through are related to that.
		But I would like to just encourage these kind of conversations to go forward and realize that each person coming to the table in that conversation is going to have a different reason for selecting an outcome measure.
		DR. SIEGEL:  I just wanted to put some of the last set of comments into a perspective in the context of clinical development from observations in many fields, which is that to the extent we're talking about first introduction into humans in early phase I trials, with some extraordinarily rare exceptions, there aren't treatments that you can measure efficacy in those trials.  You need to have control groups.  You need to have large numbers of patients.  There may be, as I said, a rare home-run treatment in a disease of entirely predictable course where you can tell that in a small number of patients.  
		We're talking about the distinction between what's measurable and what's meaningful.  It's compellingly important in this sort of therapy in particular, I would think, to have a measure of something that at least you hope and have reason to anticipate may predict efficacy because as these products develop, there are so many potential variables, many of which we've heard about.  Well, what happens if I give a few more cells, culture a few more passages, change the concentration of this differentiation factor, change the cell source, or whatever?  Am I going to have a better product or a worse product?  What happens when I up-scale to treat larger numbers of patients if I culture at a denser or a less dense -- and on down the line in many more ways than with a simple chemical molecule, many questions about variations. 
		The notion that each and every one of those questions can be answered by a multi-year controlled clinical trial comparing efficacy outcomes is, of course, ridiculous on its face.  So, if you don't have a measure that is measurable and you have some reason to hope will predict clinical efficacy, you're going to be doing a guessing game and you're going to be down the line trying to guess what's the right way to do it in a very expensive and a very large trial and probably have a lot of trouble finding people even willing to support or participate in such a trial.
		MS. SONTAG:  My name is Jordana Sontag.  I'm a patient advocate.  My son has a leukodystrophy called Canavan's disease.  He was part of a protocol at Yale University and successfully received gene therapy.  He's now part of a new IND that's being reviewed currently.
		When you started your presentation, you said that the general consensus or the general public trusts the oversight, and I'd like to say that is true as a parent of a patient.  
		But I'd also like to say that I feel that the oversight process perhaps has lost some trust in the patients and the families of patients.  I feel that sometimes we're perceived as hysterical, desperate people that are willing to do anything and put anything to try and save our families.  But the truth is we do want safety.  We don't want to harm.  I already have a fatally ill child.  I don't want to do worse to him.  So, I wanted to state that fact. 
		I also wanted to state that I feel that there needs to also be some definition in ethical lines as we move from gene therapy.  Now we're going into stem cells.  At what point do we as patients have the right to say we funded this research?  It has been worked on for two years.  We have all the safety data.  At what point does safety then turn into political pressures, media frenzies, where the FDA succumbs to that and loses site of the patients? 
		So, what I'd like to say is that I feel like the oversight committee should have more trust in us as patients.
		DR. SALOMON:  Can I ask you a question then?  We're talking about outcome parameters, measurable, meaningful.  So, in your own experience with a clinical trial on your child -- I mean, who would know their child better than the mother -- do you have an impression about what would be a meaningful outcome parameter that you would see as meaningful versus what the doctors who did the trial on your son thought were meaningful outcome parameters?
		MS. SONTAG:  I'm lucky enough to have both.  My son showed improvements through MRI.  He regenerated new myelin, which he couldn't do without the intervention of gene therapy.  So, I had the scientific proof, but then, on the other side, I have a child that is doing dramatically well.  He vocalizes.  He talks.  He's not on a feeding tube.  He's a happy child.  His quality of life is drastically improved.  He's gained the ability to use a communication device.  I took him to a symposium and a bunch of the researchers looked at my son and said, he has Canavan's disease?  If they're saying it without me even saying anything, then I know something is happening.  
		I'm not willing to just put snake oil in my son.  I'm funding research.  We have not received any federal funding.  This is all parent-run.  I don't want to put my money into something that I don't believe in or that I believe won't have a benefit.  I don't have that much money to spend.  So, I feel that we're objective and I think that we need to gain the respect of the oversight process as objective, passionate people that are looking to move science forward for everyone's benefit.
		DR. SALOMON:  Thank you.
		DR. CHAMPLIN:  As somebody who does phase I trials in cancer patients, I think there are sort of two levels here.  One is the patient's hopes for benefit, and every once in a while, a new drug does come along that actually is a major advantage and provides clear-cut, symptomatic benefit for those patients.  Unfortunately, it's the minority of the time rather than the majority. 
		But from a drug development standpoint, one is generally treating patients usually with far advanced disease where the prognosis is very poor.  In those situations, you're looking for some biologic effects that would then justify taking that trial into a better prognostic setting where, in fact, it may have more efficacy.  So, you need to have some endpoints, obviously, to measure that biology to determine is the drug promising to take forward into a more definitive population.
		DR. WALKER:  Dr. Sugarman introduced an important concept of "the gold standard," and then we didn't discuss it particularly any further.  It is critical that we have a gold standard of measurement that we think is important.  It started out years ago in cancer being solely death, and that's a pretty unsatisfactory kind of gold standard but sure is a standard.  It's easily measurable. 
		We then move into better and better ways of doing it.  I think here is the place we have a tremendous problem.  We do not do the research into the development of the better ways of measuring appropriate outcomes, be they biochemical, be they looking at plaques, or whatever it is. But we need to do that kind of thing.  It will lead to better trials, smaller sample size, greater efficiencies.
		DR. SALOMON:  Let me pose a question then to continue on this.  What I see happening whatever time in the future, but not the long future, is there's going to be a group of sponsors stepping up with a specific trial.  Today we have the chance to be generalists, but soon we'll be faced with a specific trial in "fill in the blank."  
		So, in that each disease process will have very different kinds of outcome parameters, if there were two trials considered at the same time, both of which are devastating diseases -- I don't want to compete the diseases against each other, but one which just happens to have very well-defined outcome parameters and measures and one that maybe doesn't.  The Alzheimer's disease is an interesting one already posed by this group.  What do you guys think of that?  Should you do both?  Or should you only do the one with the better outcome parameters given that this is a new field?
		DR. MACKLIS:  I had a question or a few naive questions that relate to that.  So, maybe I can throw my questions out on the table, and then everybody can discuss.
		Dr. Sugarman repeatedly used the baseball analogy of hitting a home run.  Sitting here listening, I thought that was a great way of telling me how to think about things.  As scientists, one likes to think one is hitting a home run. 
		But Mark Noble earlier raised a point about commercial pressures that you reinforced and we just heard about patient and disease driven pressures.  I wonder if there is a broader either societal or governmental or field set of ethics -- is it at odds or is it the same -- to deciding whether we might want to go for a double or a single or even a bunt.
		(Laughter.)
		DR. MACKLIS:  And do we have just a certain number of people to go at bat, to bring the analogy maybe four steps further than it ever should have been taken?  I apologize.  And do we limit that?  How many studies can we do, and are we willing to go for a single or a double more broadly than just a few home run hitters?
		DR. SUGARMAN:  I think one of the struggles with this set of conversations is what we're talking about.  I'll change the analogy slightly.  If I bring my ethics hammer to bear here, the problem we're talking about are all problems related to what we call justice, fairness in allocation, and fairness in access.  These are all justice problems and they've been a problem in ethical theory since Aristotle.  So, it's no surprise that we're still chewing on them. 
		In research, what you're seeing as a reflection here I think are multiple problems.  The whole regulatory scheme was built on the notion that patients were vulnerable and needed to be protected.  The reasons for that were a series of scandals in the '60s and early '70s that came out of disadvantaged subjects being used without consent for research.  We can go through each of those examples.  So, when the National Research Act was put into place, the notion there was to protect folks and to make sure that there were even more protections on anyone who might be perceived as vulnerable in any way. 
		Once the regulatory approach came into play -- and we have separate regulations for prisoners and kids and pregnant women, and we have this very restrictive approach.  With the onset of the AIDS epidemic and advances in cancer chemotherapeutics, we've seen a change in the calculus.  It's still a problem of justice, but the claim is in protect me as someone who is vulnerable but let me have access to something that could potentially help me.  There was sort of a corrective to what was an overly protective initial scheme. 
		So, what you've seen as a result of the lobbying efforts of folks and patients speaking loudly is a change in the regulatory structure.  You've seen different pathways for drug approval processes in FDA.  You've seen inclusion criteria in clinical trials to enhance generalizability in women, minorities, kids.  So, this is all a reflection of the same piece.
		Now, what has happened as a result of that is we're recognizing the multiple players in the field as we go from protecting folks to access and the right answer is somewhere in between.  We don't know how to divvy up all the resources the right way.  Sometimes those resources are the science itself for trials to move forward.  Sometimes it's which patients will be in a trial.  Sometimes it's which disease will be appropriate.  These are all criteria on justice that we are all really just learning how to talk about. 
		So, I think, yes, a bunt or whatever that would mean.  The home run may be a short question.  It may be that my home run is decreasing tangles, that my home run here isn't curing Alzheimer's disease or whatever the question is.  But that needs to be visualized.  If you share that goal with the same enthusiasm that we would have scientifically with patients and family members, this is where we are, this is the next question, this is the scientific question, and unfortunately the science isn't good enough for us to promise more, but do you want to help out?  Uniformly people will help out if that's where the science really is. 
		I think we just need to think through those again explicitly to try to answer those.  I think they're all home runs.  If we could achieve any of them, we could move forward.
		DR. NOBLE:  I want to make one concrete proposal to this often too abstract discussion.  If there is any way in a study to use imaging to determine whether or not you have had any kind of successful cell replacement, it appears to me that this has to be mandatory because we need a scientist to know whether or not we may be getting cell replacement in the absence of clinical improvement.  And if we simply use outcome measures that can be measured simply in the clinic, we don't even know whether the experiment worked because it might have failed because we didn't replace any cells at all, or it might have failed because we didn't replace enough, or it might have failed because we replaced them and it doesn't matter.  So, I think that's a critical component.
		The ethics questions -- I'll never forget what one of my friends with a glioblastoma told me.  He said, Dr. Noble, your ethics are a luxury that I don't have time for.  This is a pressure that we have for many of the patients.  We know a lot of spinal cord injury patients.  They're going to go and get shark embryo transplants in Mexico.  If we had some means of making sure that these patients were directed into therapies that might be more beneficial that maybe are not ready for the prime time that we would want, but they're better than shuffling off to Tijuana for a shark embryo, then maybe we could use this process to learn something also.
		DR. TROJANOWSKI:  With respect to whether there are measures that are adequate to indicate a response to a therapy or not, I think that is a very critical question.  I recall, Mark, the Motulsky Orkin report on gene therapy four or five years ago which said that too many of the clinical trials for gene therapy were not powered sufficiently, didn't have outcome measures to know whether the experiment would or would not benefit the patient.  And that's a horrible place to be, and I'm glad that they spoke out so eloquently and strongly about more basic science, more animal models before rushing to clinical trials.
		Although I mentioned that Alzheimer's disease has some perplexing conundrums about what the most toxic lesion is, I think we have many potentially informative measures for a response to therapy, both biological and cognitive.  So, I would argue that we should go for the clinical trials that have the potential to do something where the measures are in place. 
		I'm very much in favor of bunts and base hits because we in the Alzheimer's field, although I work in Parkinson's as well, would love to be faced with the dilemma of an L-dopa-like therapy that carried people for 10 or 15 years when the disease begins at age 75 to, let's say, age 85 or 90 so that they could enjoy their retirement or be cognitively intact enough to enjoy retirement years rather than being in a nursing home.  So, it doesn't have to be a cure for Alzheimer's.
		MS. WOLFSON:  I think one thing that has to be thought of, when we talk about the ethics of new therapies, is not only the quality of life that a patient might enjoy if this was successful, but the quality of the patient's death.  I think that in any informed consent procedure, the possibility of what will happen to the person without the therapy and the effect of the therapy on a person's death I think is equally as important. 
		DR. SUGARMAN:  I would like to underscore the points.  I don't want to send the false message that I want be a Luddite about the whole thing and stop science.  I'm doing science all the time, what I consider science, empirical work, and like to move forward and work in clinical trials.  I am the biggest fan of moving forward on clinical trials and advancing things, and if that wasn't clear from my comments, I'm sorry about that.  
		But I'm a fan of moving forward not for any of this right now.  When safe, when appropriate, when alternatives have been explained.  And I hope that the emphasis is when it's right, I'm all over it.  I sit on IRBs and DSMBs and love to see them move forward.
		I think it's critical to bring in these other points about what the alternatives are.  Just because we're not the folks with the alternatives when we are at major medical centers and the like doesn't mean that there aren't other alternatives out there that might be good for this patient or this patient's family.  It may be a death.  It may be going on vacation instead of spending their time in parking decks and waiting rooms.  It really depends on the state of the science, and I just think we need to be up front and forward about what we can to offer. 
		I think there's a lot of good to be had here and would like to see that we also focus on the outcomes of benefit as well to know that what we're doing isn't the snake oil.  Is it that we put something in a part of the brain that we think is going to be functional, it's not functional, but has a therapeutic effect?  We've heard this kind of conversation before.  We can fool ourselves too by the state of the science of what we think we're doing.  So, we need to have a little humility here about how good it is so that we can be honest and appropriate as we go forward.
		DR. SALOMON:  I know this is a great discussion.  I just want to do Dr. Mulligan in the back, Dr. Freeman and Dr. Kurtzberg, and then we'll be done for this and we'll move on.
		DR. MULLIGAN:  I'd like to come to the snake oil issue.  It seems that there's somewhat of an inconsistency between your principles of consent and the very low bar that I think you've represented to support going ahead for a clinical trial.  So, as I was looking at your bullet points, you're saying a possible benefit is desirable but not necessary.  The clinical equipoise is a sufficient metric.  Risk/benefit is not really an issue in the phase I.  
		If you're giving consent -- let's say we all know clinical trials that we think are not very interesting and let's say that there's one that manages to pass that very low bar, who is then appropriate to give consent to the patient?  One person, for instance, might say in his consent this is a pretty bad trial, do you want to do this?  Most people think this is not going to work.  It's really kind of ridiculous.  As opposed to the investigator who says, well, this has a chance, et cetera, et cetera.  
		I raise this because it forces you, I would think to have to deal with what's necessary in terms of preclinical information.  As you set the bar lower and lower, then the consent I would think becomes more and more an issue about to what extent you have to get across that there's great difference in thinking about whether there's enough science to support going ahead.
		DR. SUGARMAN:  I think one of the emphases is that in proposing these criteria for the first in humans kind of trials, bench to bedside kind of argument, is that it's safe.  Safety is the key as number one, that we have as much information as we have about safety preclinically that we know it's okay to try this or we think it's okay to try it.  We're not going to know until we try it in folks.   So, the bar is really high on not hurting people in the process that are willing. 
		The possibility of benefit is honest about where we are with science, and that starts to become part of the consent process. 
		You're right.  I think the consent process does play an important role here.  It's arguably hard to do.  We're collaborating with some folks at Hopkins about trying to enhance consent for phase I trials in oncology.  We recorded about 100 conversations with investigators talking to patient subjects about enrolling in phase I.  We're trying to enhance that process not necessarily changing the recruitment rates at all.  We saw no nudge.  But what we see is greater satisfaction, greater understanding, and greater knowledge about what's happening in the context of this trial.  
		So, I do think the consent process is critical, and whether it's appropriate for the investigator to give the consent or the nay-saying research nurse, I don't know who the right person is to do that in each case.  But it's something that a thorough look at the ethics of the research would say don't just outline your consent document, say this.  Who's going to get consent?  How are people going to be approached?  What kind of materials are they going to be given?  Just deliberate about what's the most balanced way to give that information that's fair. 
		I don't know if I evaded your question or answered it.
		DR. MULLIGAN:  You very effectively evaded it.
		(Laughter.)
		DR. SAUSVILLE:  I have to interject.  You created sort of a polar situation by presuming that the clinical investigator is basically going to talk it up and another person, whether a research nurse or the next investigator, is going to talk it down.  Again, I speak as someone who just yesterday participated in two informed consents for a phase I trial.  
		You've got to be able to say to the patient that, yes, as an investigator, I think this is a reasonable idea, but you also have to say to the patient that it's perfectly reasonable to consider no particular treatment and to use palliative efforts to relieve symptoms as potentially having no difference in outcome.  So, maybe that's part of an equipoise.  
		So, I don't think you have to have this -- what I read into this -- a polarization of points of view.  I think to make the consenting process totally informed, you have to open the possibility that supportive care and specifically not going on this trial is a perfectly reasonable thing. 
		DR. MULLIGAN:  Yes.  I'm attempting to really get a better definition of how much effort ought to be put into evaluating the science from the point of view of going ahead.  I guess I'm saying that it seems like reasonably the bar is set very low, but that there's a very arbitrary point of view put to the patient, almost by definition, let's say, because generally there are different points of view, and the physician that's carrying out the trial clearly believes in what they're doing and think that there is some hope.  But should the consent process somehow take into account, say, the prevailing scientific view?  Often there is a prevailing scientific view that something is not so hot. 
		DR. SAUSVILLE:  I would submit that both points of view need to be offered. 
		DR. SALOMON:  Okay, in the back.
		DR. FREED:  Curt Freed, University of Colorado.
		With the issue of outcome and if we're talking about cell therapy, there are two issues that always have to be addressed I think.  First of all is has the cell done what you think it was going to do, and second is has the patient had a response.  Because we're dealing with cells, not drugs, the cell has to go in, survive, and flourish.  In our neural transplants for Parkinson's that variable has been less variable than the patient outcome in which you have a whole different set of factors.  What is Parkinson's disease?  What is the range of actual underlying disorders that manifest itself as Parkinson's across age range and so forth?  So, we have two distinct parameters:  one, cell survival; second, patient response.  Both have to be considered as two endpoints, not a single endpoint I would say.
		DR. SALOMON:  Dr. Freeman and then Dr. Kurtzberg.
		DR. FREEMAN:  I think there is one set of special challenges for stem cells, in particular, when it comes to trials.  Although these are pharmaceuticals, they are pharmaceuticals that are delivered surgically, which brings up a whole set of special considerations which fall outside of the realm of standard pharmaceutical trial designs. 
		First of all, how do you adequately control for your outcome, and what are the ethics of placebo-controlled surgical trials?
		Secondly, the costs of a surgical trial are prohibitive in comparison to pharmaceutical trials which, first of all, will favor product-driven trials over, say, organ transplant, fetal tissue, or kidney transplants. Anything that's product-driven in a surgical trial will, therefore, be favored by that. 
		Secondly, it influences the size of the trial and therefore limits your ability to power adequately on primary clinical endpoints which is, of course, what is favored in a trial design and lends itself more to surrogate marker endpoints and radiologic outcomes. 
		Finally, from a trialism point of view, many surgeons are not adequately trained or prepared psychologically to deal with placebo-controlled trials.  Then from the surgeon's point of view, surgeons generally get paid to do surgical procedures which will limit their equipoise.
		(Laughter.)
		DR. FREEMAN:  And finally, if they do a placebo-controlled trial where you bill for the active arm by you don't for the placebo arm, of course, that's unblinding, and not many surgeons are willing to operate on people in either arm for free.  So, there's a whole constraint of issues which really are specific to surgical trials that really need to be thought out carefully.
		DR. SUGARMAN:  I appreciate that.  Also, any other comments people have along the way, I'm happy to revise and analyze and change, and throw things at me, as long as they're words.  I would love your thoughts and input on this as we think forward about this together.
		DR. KURTZBERG:  I don't think we've really addressed the topic of resource allocations.  I think there's really a societal disconnect between what we want to do as scientists and tests and what we have the resources to look at.  I think the FDA is going to be in a unique position of being able to control, in a way, how some resources might be allocated.  I think that responsibility has to be discussed and that systems and committees and whatever have to be put in place so that it's done fairly.  
		I don't think our society realizes they need to do research in certain illnesses until somebody they know gets sick, and then it's too late.  Then the resources get emotionally allocated instead of objectively allocated.  I think this agency has the power, if you will, to maybe change that and put direction on how we approach some of these diseases and how we allocate the resources.
		DR. SALOMON:  I think that was a very good discussion.  I think it's probably the first time since I've been associated with FDA advisory committees that we've had that much productive discussion on an ethics thing --
		(Laughter.)
		DR. SALOMON:  -- which may be a statement on previous committees or previous presenters.  But I mean that as a sincere form of compliment.
		So, what I came away with as sort of key issues that came out of this discussion from the group is that clearly this idea of outcome measures, measurability and meaningfulness, is really a big thing.  Picking up on Dr. Kurtzberg's last comment, I think bringing it around, these are issues for the FDA -- and for the NIH I might add -- in terms of where funding priorities are put in order to address which diseases we should start with, what resource allocation is going to be done and make sure that we do address the issue that it's not done emotionally. 
		The second thing is clearly I think everybody is sensitive to informed consent.  I didn't cut that conversation off even though it was a little more generic informed consent than neural stem cell informed consent.  If people want to get back to talking about informed consent, I would ask you only to integrate some specific issues related to neural stem cell transplantation if the topic should come back up again. 
		Jay, given your status --
		DR. SIEGEL:  Thank you.  I know you want to move on, but I just want to quickly say for the sake of Dr. Freeman, Sugarman, anyone else who might be interested, in 1994 and 1995, the FDA did have a two-day advisory committee discussion on issues in intraventricular therapies of neurological disorders, many of the same disorders, and many of the treatments involving placement of catheters, reservoirs, and pumps, and issues of sham surgery and issues of ethics in dementing conditions.  There was some very fruitful discussion there, and those interested in the issue may want to check back on that record.
		DR. SALOMON:  Well, given a technical glitch, maybe we've got a minute here.  Go ahead. 
		DR. TROJANOWSKI:  Could I ask for one more thing to be put on the future agenda of these discussions?  That is the role of media in reporting science.  
		I've been appalled at their reluctance to declare conflicts of interest, what their hidden agenda might be in some of their press reports.  The St. Jude's Hospital report of potential contamination of HIV fragments in a gene therapy trial that then were retracted five days later I think did enormous damage to the public's confidence in this.  
		Although at the University of Pennsylvania we're very sensitized to the conflicts of interest with Jim Wilson -- I'm not part of his institute at all.
		(Laughter.)
		DR. TROJANOWSKI:  I'm part of the institution, but not the IHGT.  I think there were accusations in the press that were completely unethical and, for reasons that I don't understand, they were allowed to go forward.  
		I don't know if members of the press are here, but I hope they do pay attention to what Dr. Sugarman said, and I would hope that -- this is a free country.  You can say whatever you want, of course, but I think you can say things in print that have an enormously damaging effect and there's no accountability. 
		DR. SALOMON:  I think one comment, though, that needs to be followed up on that is that, yes, the press has to be responsible and irresponsible reporting -- you gave a good example of it. 
		But the other side of it was, it was really a black eye.  There was not just Dr. Wilson, but there were several prominent gene therapists with ridiculous conflicts of interest that could have been dealt with in a proper way.  There's nothing wrong with a conflict of interest if it's up front.  So, I think it's definitely a two-edged sword here. 
		Our next speaker -- and then we'll take a break -- is Dr. Fred Gage, Rusty Gage, giving us an overview on animal models.
		DR GAGE:  So, I took the charge pretty literally, and when you sent me that list of questions, I took the questions out of the text and pasted them onto slides and put my answers onto them so we could keep it fairly focused. 
		I wanted to start by harping again on this concept of the definition of stem cells and the fact that there are no prospective markers for stem cells that would allow you to go in and look at a cell and say, that is a stem cell.  Rather, what we have are populations of cells.  At best we can say within that population we believe that there is a cell that's a stem cell. 
		The other point is that there really are two different definitions that we've been talking about here.  One is in vitro definitions which involve saying that the cell has self-renewing properties and is multipotent.  In most cases, at best what one will do is say that twice self-renewal -- you'll say that one population can be derived from a single cell, expanded, differentiated into multiple cells.  In that population, again you can take one cell out of it and give rise to that same property of self-renewal and multipotentiality again.  But all you've really demonstrated there is twice.  You've said self-renewal twice.  What we really invoke with self-renewal is perpetual self-renewal or for long periods of time.
		In terms of multipotency, certainly in the nervous system, most people are satisfied with looking at three markers:  some marker of glia, some marker of oligodendrocyte, and usually an early neural marker.  And then they say that they have a stem cell and maybe a complete stem cell population. 
		The other definition related here is the in vivo definition, and we really have no definition of an in vivo model of stem cells in the central nervous system because of the absence of ability for self-renewing properties. 
		So, one of the things that this gives rise to then in this little schematic is the idea that of totipotency, pluripotency, and the general multipotency.  As we go forward, rather than be pessimistic about this, I'd like to be optimistic that, in fact, while we don't know very much, we have the tools presently to begin answering these questions very systematically.  It's the answer to these questions which will help us progress further rather than throwing up our hands without knowledge. 
		So, I think one of the important questions that we have to address that has already been brought up is the relationship that exists between what we're calling stem cells and progenitor cells and whether or not there is a progressive unilinear progression of differentiation or whether or not there's a capacity for dedifferentiation or reversion back and forth between the cell types.  That I think will be a very important part for talking and discussing things. 
		One of the ways in which we've tried to address this in our lab by calling cells stem cells -- I'm going to give you demonstrations mostly from our lab, but also showing you the down side of what we're doing. 
		We take adult tissue.  We isolate the tissue on various isolation procedures, and we use retroviruses which have the capacity for single integration into individual cells as a marker.  We then take colonies and clone the individual cells manually.  But after we have what we believe to be a clonal population derived from presumably one cell, we do Southern Blots to make sure that that single integration -- it defines genetically that every cell in that culture that expanded from the retroviral integration is derived from a common cell.
		You can then use another retrovirus with another marker to then test for self-renewal, but then again, at the end of the day, all you've got is being able to say that within that population there are stem cells and within that population there is some evidence that at least some of them have self-renewing properties.  So, even under these very rigorous conditions, we do not have prospective markers that allow us to identify the individual cell.
		However, if you can then use these procedures to isolate repeatedly, using the same procedures, cells and ask about the potentially different cells, here's an example of what you can get.  So, here's the parent population and these are 6 different clones derived from the bulk population that are then differentiated.  While every one of the clones can give rise to a neuron -- a quote/unquote neuron -- an astrocyte and oligodendrocyte, the ratio that each one of the clones all derive from a single population have in differentiating down different lineages is quite variable.  But I think the good news is ‑‑ one has to look for the good news in the noise -- that each of the clonal populations is multipotent.
		Now, just to give you an idea of one of the things we're trying to think about in terms of these concepts of defining what is a stem cell, this is a film of an individual cell in a culture dish.  So, here's a cell moving around.  It's under conditions.  This cell can divide.  So, is that a self-renewing property or is this a committed cell?  
		Well, if we look carefully, you can see that first this cell rounds up and divides, and now this cell rounds up and divides.  These cells then can each individually divide and differentiate.  This is over a period of 2 or 3 days, and if you monitor these cells over longer periods of time, this population will grow up to be a multipotent stem cell population.  But even by tracking the cells individually in the culture, without prospective markers, we can't identify which one of these cells, other than the very first cell, was in fact a stem cell.
		So, the next question of the questions that were asked was production characteristics.  What are the objective, qualitative differences that distinguish stem cells derived from different sources, namely, embryonic, fetal, adult, and how might these distinctions impact safety and potential efficacy?
		Well, to date there has been no head-up comparison between cells of different origin, species, or age, in part because different methods have been used to isolate and expand and differentiate the cells.  But this I think is certainly a challenge and one that should be met.
		Examples of the differences that are existing in the culture dishes today are, we've heard, EGF versus FGF.
		Some people grow cells as monolayers.  Others grow them as spheres.  
		We don't know the difference between an embryonically defined cell and an adult cell.  
		How many passages does it take to change the cell and how important is that?  
		We hear about cells that are immortalized.  Actually rationally immortalized.  They're meant to immortalize the stem cells.  What's the difference between that cell and a non-immortalized cell?
		Then, of course, rat, mouse, and human.  Many of the discussions we have are interchangeably using the results that we obtain from one species and generalizing both in behavior experimental models and in the behavior of the cells. 
		Then again, the source of the cells. 
		So, the bottom line is, the answer to the question is it hasn't been done, but these are the head-up kinds of experiments that certainly could be done.
		So, we assume in some sense that there's a stem cell here that's dividing down into a progenitor cell, one of the other lineages and that these are some sort of linear track.
		I want to show you a recent observation by Sophia Calimari using a marker that you heard from Steve Goldman earlier.  This is a population of cells all derived from a single cell in a culture dish, and they're moving around.  It's very hard to tell from this who is the stem cell or who is the progenitor cell and who is undergoing cell division at any one particular time. 
		If we infect the same population with a marker that uses the alpha tubulin promoter driving GFP, you can see that in that culture, here's the cell with nice branches and it's a committed neural progenitor cell, as we've used these terminologies.  But if we look closely, follow that cell, it rounds up and undergoes cell division.  So, here we have a situation where we're using markers to define cells as committed down a neural lineage.  That same cell -- it's not fusing back together.  Don't worry.
		(Laughter.)
		DR GAGE:  That cell can actually undergo cell division and give rise to a cell at the same state of lineage, showing here that it's expressing the alpha tubulin promoter.  
		So, in addition to the propagation of cells in the most primitive states, it looks like at different stages along the commitment, cells can be expanded and propagated.  I think this is going to be a valuable tool and a piece of information for us to consider in this process of isolating functional cells. 
		Similarly, will the stem cell source influence the robustness, the durability, the longevity of an intended therapeutic response following transplantation?  Well, yes.  
		So, how do we compare, measure, and follow them?  Well, these are just a list of some of the things that we need to do with our cells when we consider -- and I think that the spirit of the question was how can we begin to characterize cells in some systematic way so that ultimately we can make comparisons between the different types of cells that people are bringing forward as being a potential cell source for a therapeutic intervention.  Certainly issues related to isolation efficiency, what factors are used to propagate the cells.  Do we need serum for this?  Most of these are fairly straightforward, but what again it comes back to is making comparisons between all of these variables between different cell types. 
		One of the methods that we've used to isolate these cells as a method of isolating the cells from the adult nervous system and enriched for certain cells has allowed us to look at cells directly isolated from the tissue in the absence of propagating them in conditions of mitogens.  One of the concerns that we all have in this field is when you propagate cells for indefinite periods of time or for any period of time with mitogens as potent as EGF and FGF, are you changing the potentiality of the cells. 
		So, we've developed a procedure where you can take adult tissue and isolate it on a Percoll gradient.  We have a pretty good idea of the density of the cells that we propagated.  Then when we isolate those cells, we can ask what percentage of those cells that are freshly isolated from the brain are able to give rise to neurons in a dish.  As Steve Goldman told you, the hippocampus has this potential for neurogenesis, as does the subventricular zone.  Here with 0 passages and induced differentiation, we do in fact get neurogenesis out of these cells. 
		But if we look at other areas of the brain, like the cortex where no neurogenesis normally occurs in the adult, or even the optic nerve, which is extended out into the rostral brain cavity, there is, we know, no neurogenesis occurring in those cells.  If we exposed them for merely 3 days to FGF in vitro, we can get those cells to begin to express neuronal markers.  So, here's a population of cells existing in a non-neurogenic site, when exposed to FGF, can in fact give rise to cells. 
		We believe that this, in fact, suggests that either there are quiescent populations of cells -- this is one interpretation -- that exists within the adult brain that can be activated by these mitogens or, alternatively, this idea of reprogramming emerges again.  Do mitogens reprogram the adult or any kind of stem cell to broaden its propagated capacity?
		So, here's the question again.  Please describe specific markers used in the isolation, characterization of stem cell preparations. 
		Well, if you read through the literature, some people will say if a cell is responsive to FGF or EGF, it's a stem cell.  Others will say that if it forms a sphere, a sphere-forming cell is a stem cell.  These are in the published literature.  The ability to passage a cell we've read may be used.  Again, I'm being a little bit tough here, but I think that the concept of using stem cells is being used in a much broader sense than original assumptions were, and I think we should return to those to some extent. 
		Nestin is another marker that has been used as a marker of stem cells. 
		Then in terms of differentiation, as I said, usually three markers are adequate.  
		I argue that in order for you to say that a single cell can give rise to a neuron, as several of the talks today have already documented, we need to be more rigorous in our definition of whether or not the cells are in fact neuronal.  So, markers needed for functional characterization would be the ability of the cells to actually myelinate in vitro.  Can they form myelin and myelinate axons?  Do they form dendrites, axons, and synapses?  Do they make neurotransmitters, and are they electrophysiologically active?  These are all methods that are presently available.  So, it's not that difficult.
		Here is a stem cell derived from a single cell infected with GFP so you can mark it.  This is a double labeling with synaptophysin, a marker for synapses.  MAP-2 is a dendritic marker.  What you can see here is that synapses are formed on the surface of these stem cells. 
		If you look at the action from this neuron derived from stem cells -- and this again are the synapses, and this is the dendrite from another cell, not this one -- we can say that the synapses from this cell are making synaptic contacts on the dendrite of another cell.  So, merely by using antibody markers and collocalization, we can begin to get the features which give us a little more confidence that the cells are differentiating down different lineages.
		Using really pretty standard electrophysiological characteristics now by taking those same cells, voltage clamping so you're looking only at current, you can see that a normal primary neuron in culture gives rise to EPSPs -- IPSPs and stem cells or cells derived from a single cell that differentiated down lineages also can give rise to similar kinds of IPSPs and EPSPs that are blocked by GABA and blocked by glutamatergic stimulation.
		As has been reported before by some of the other speakers, one of the things we'd like to know is can these neurons actually spontaneously fire an action potential as a neuron.  So, if you put them on a current clamp and just ask whether or not the cell has the capacity, in the absence of stimulating with potassium, but does it have the potential for generating an action potential, you can measure these properties in the cell and gain confidence that the cells have actually differentiated or can differentiate down fully functional neurons.
		Are there selected genetic or biochemical markers that accurately indicate the differentiation status of stem cell preparations or that assure the acquisition of correct functional therapeutic -- and this is a tough one.  Not from certainly any in vitro monitoring that is available.
		If in the standard cell preparation, all cells can give rise to a defined therapeutic cell phenotype in vitro and in vivo, then I think you're feeling a little bit more comfortable from your animal models that it might work in a patient.
		If the cell implant can reverse the functional deficits reliably in an animal model, I think that's another evidence of support. 
		In addition, if the functional recovery observed in the animal model can be causally linked to the phenotypic differentiation of the graft itself, then I think you're close to being able to say that that cell that you're grafting is differentiating into a phenotype that's responsible for the functional outcome that you're looking at. 
		If you use these sorts of criteria in the animal models, at least it begins to say that, well, we believe that it's not the cell secreting a factor or dying and it's that response that's causing the behavioral result.
		Alternatively, have specific markers been identified that could relatively predict transplant failure or indicate the likelihood of untoward events to occur, such as inappropriate -- yes.  I think that there are things we can look at in vitro and in vivo in animal models that are pretty bad predictors.  One would be that the cell that you're looking at can't differentiate down the appropriate lineages either in vitro of in vivo.  If you can't get your cell to differentiate to a large extent with some control down the appropriate lineages both in vitro and in vivo, that does not bode well for a cell.
		Cannot be induced for all the cells to stop dividing.  If in vitro or in vivo, there is any evidence that these cells are continuing to divide in culture, that all of your differentiation schemes -- you may have, well, just 1 or 2 cells are undergoing mitosis later on -- I think that that is a marker that we need to be absolutely aware of.  Cells need to be karyotyped to make sure that there are no abnormalities, and if there are abnormalities, then this needs to be evaluated.  
		And cells die following differentiation in vitro or in vivo.  I don't mean just that they can differentiate appropriately, but some will survive and then as soon as they differentiate, they die.
		Here's some of our own data of our passaged clones.  These are all clonal lines that you saw before.  After 30 passages, we karyotyped our cells, and while 7 of the 9 had basically normal karyotype, we had two abnormal cells that were out 40 or 50 passages.  When we grafted this cell, it actually acted just like a normal cell, but when we grafted this cell, we got tumors.  So, I think that's a beginning of at least a minimal amount of characterization that one needs to do for your cells and we should see that for every cell that is used.
		So, preclinical.  To what degree do particular experimental animal models mimic disease conditions in the human and to what extent are animal models predictive in terms of evaluating safety prior to initiating studies in humans?
		I'd just like to be a little pedantic here and say that there are three different kinds of animal models that many of us who work in animal models think about and what you can expect out of an animal model. 
		There are homologous animal models, and that is a model that has common etiology to that which exists in the human.  The pathology is the same and the behavioral outcome the same.  It's not even a model.  It's the animal has the same disease as humans.  Treating that will be a lot more informative than any other model.  But most of the models that we actually look at are isomorphic models.  While the pathology or the behavior may be the same, the actual cause of it, the initiating cause of it is unknown or is induced by the experimenter.  Most of our models, even our transgenic animals that we're generating now that have over-expression of certain genotypes, are really not homologous to the human form of these diseases.  Given that's the case, one needs to be aware of that whenever one is modeling these and looking at behavioral outcomes and anticipating moving on for a clinical application.
		There are obviously certain models that have a greater relationship or closer to the homologous sort of thing.  Having said that and being critical of isomorphic models, there are a lot of correlation models that are actually quite good.  There are a lot of in vitro models.  If you're modeling for one element of the human disease, then in some cases in vitro models are okay if the only thing you're looking for is to reverse that one thing.  So, being aware of the model that's being used.
		This is my list of all the diseases.  When I looked through the literature to see what people have begun modeling for and spoken about in terms of cell therapy -- this isn't something I would even think about -- there's an amazing number of diseases that people are attributing to as potential models for cell therapy at present.  They don't always say that they're going to be using stem cells, but it's striking. 
		So, then they asked me, so what are the relative strengths and weaknesses of any animal models described in your presentation?  I'll talk about a few things. 
		Most of the models we deal with are isomorphic.  They're not real homologous models.  The timing of implant is almost never taken into consideration relative to the disease.  The size of the target is minuscule relative to the clinical problem.  The number of cells that we're grafting have nothing to do anywhere near the cell number that we're going to be using in human.  Even when we go on to primates, the scale-up is quite big. 
		We work to reduce variability.  That's why we use animal models.  It allows us to make extrapolations a little bit better.  
		Unfortunately, little attention is given to in vivo measures to monitor the survival function.
		This relates to what Mark just said, which is we need to get working on and perhaps demanding the development of imaging techniques to monitor these cells if we're going to go forward in an aggressive way. 
		So, in the modeling, a few slides showing grafting or transplantation of cells.  This just relates to the idea that niche is important.  If we take a clonal population of cells and graft them into the hippocampus, what you'll see is the same cells, depending upon where they end up in the granule cell layer, will differentiate into different cell types.  So, if you get them just in the innergranular zone, you can get cells that differentiate into neurons, that are calbindin positive, they receive synapses, and by all criteria are indistinguishable from the other granule cells. 
		If, however, you damage the granule cells and you elicit a glial response, then those same cells would differentiate into astrocytes.  Tens of microns away or, say, 100 microns away, the exact same cells are going to respond differently depending upon the cues that are present in the local environment.  So, it's not just the cell type.  It is the microenvironment.
		I'm not going to talk about Parkinson's disease, but I hope that some of the people in the audience who have a lot of experience will and relate the successes that have been used with fetal tissue, where there's defined populations of cells, albeit not enough cells, and their hopes for stem cells or progenitor cells to be an expanded population and the reality of what really is expected out of a stem cell.  I feel confident in saying at present no propagated cell, CNS propagated cell, CNS-derived propagated cell, that can be even closely considered to be a stem cell gives anywhere near the functional effects that one sees with fetal tissue even in any of the most simple experimental models.  At this point, fetal tissue is the benchmark for both the experimental model in Parkinson's disease, as well as the clinical model, and the stem cell work is not even close.
		A quasi-homologous model might be -- I think eye disease has a lot of opportunities here because there are a lot of animal models with genetic disorders where there are selected gene mutations and retinal degenerations associated with this.  I wanted to show some pictures of some grafts that Mike Young has done with our cells and injecting in the Royal College of Surgeons rat which is a rat that has degeneration of the retina.
		If you infect the cells with GFP so they're fluorescent and you select a cell that stays on once it's grafted -- and this is really a selection process rather than knowing what we're doing in terms of where the virus inserts.  We can get cells that stay on after differentiation, months afterwards. 
		So, here's the degenerating eye and these green cells will target to the outer region.  They can send processes into the synaptic region.  So, this is the neuron in the middle layer, projecting processes into these synaptic regions.  If we look closely at that, we can see that in some cases synapses are being formed in the processes, and they line up in an appropriate way. 
		These are being examined for functional characteristics, but these are cells derived from the adult hippocampus that, when grafted into a degenerating eye, appear to target to these areas.  They don't take on the full, let's say, phenotypic characteristics of the same eye cells in their present state, and we don't know if that's a function of the fact that they're from the hippocampus or whether or not the adult brain or damaged brain doesn't have all the cues necessary to drive the cells down further.
		This is just an observation that is consistent with the idea that the cells are responding appropriately to the damaged cues.  In addition to seeing some of the cells fall into the ganglion cell area, we actually see axons coming out of the ganglia and into the optic nerve that are GFP positive, and you have growth counts that tip as they lead into the optic nerve head.  
		So, while the cells may have the will to differentiate and the degenerating target may provide some of the clues, the whole process of neural regeneration and recapitulation of normal circuitry down to a functional neuron is going to require an orchestration of things that are not just induced in vitro, but also requiring the host as well.
		A few cautionary notes about the in vivo effects.  So, when modeling diseases like epilepsy, which is thought of as a target for cell therapy, if you pulse a normal animal with bromodeoxyuridine, these are the dividing cells that normally exist.  If you give an animal pilocarpine, as an experimental animal of epilepsy, you get a massive proliferation of these progenitor cells.  Many of these cells migrate out into the granule cell layer and form ectopic, inappropriate granule cells.  So, anytime when thinking about transplanting cells into an epileptic host, one has to think about the fact that already in that damaged tissue the local circuits are recruiting these cells into aberrant locations.  This is a slide from Jack Parent and Dan Lowenstein in their earlier work.
		I put this in recently because I wanted to say what are some successes and what are some models that one could at least think about in terms of cell therapy and the rationales behind it.  The successes we'll hear about in the next couple of speakers.  
		We'd like to keep in mind defined populations of cells and defined phenotypes.  Here I'm not talking so much about the cell itself as the fact that the cells can be delivery vehicles for factors.  This for me is a real lesson that any cell that we put in there in and of itself may not be acting for its intrinsic property as a cell, but may be a vehicle for delivery.  What's important then is to understand the nature of those signals that are being secreted.  
		So, in the human and all mammals, there's a cholinergic system in the basal forebrain projecting into the hippocampus in the septal area, and we know that there are many trophins that are effective, but uniquely nerve growth factor will support the survival of these cholinergic neurons in aging animals and in damage situations.  It's a potent rescue factor but it also has a lot of side effects that have been revealed as this molecule would supposedly move forward into some sort of clinical application because of its diffuse effects throughout the system.
		But one of the lessons we learned was that if you take an aged animal and stained for markers which identify what the cells are, these cholinergic neurons, we concluded originally that there was a loss of cells in these regions.  When grafted cells that over-express nerve growth factor into these target areas, we ended up getting more cholinergic neurons in this region. 
		Now, one can conclude from that that if you were putting in progenitor cells, that in fact some of the grafted cells are differentiating into new cholinergic neurons.  In fact, that's not what's happening here.  We're up-regulating the expression of phenotypic markers on the surface of the cells that were basically blank before, and this could easily be interpreted as some sort of cellular replacement when, in fact, it's just genetic up-regulation of what's going on. 
		The other point that we face is this difference of size.  So, this is rat brain and this is a monkey brain. Depending upon the monkey brain, the human brain is going to be at least four times, if not more, and parametologists in the audience will discuss this with us.  But this difference of size is not one that I think is easily scaleable, especially when we're talking about cell therapy and cell replacement. 
		One of the things that we've done with Mark Tuszynski is to look in the primate and see how many cells do you really have to do to mimic the effects that you see in a rat in a monkey.  I think that those are the kinds of questions in the stem cell field which would be important. 
		The difficult thing, though, in cells like this where they form an aggregate or in fetal tissue where they form an aggregate and you can use various tracers like MRI or PET scanning to identify the graft, this is going to be significantly more difficult with progenitor cells and stem cells because unlike these cells, they migrate very, very far, and most of the human imaging techniques are not sensitive enough to really identify where those cells are going.  So, this I think is once again a real challenge for us.
		So, embedded in this also, as I restate this, is that we really have to consider this issue of immunology, if we're harvesting our cells from autologous tissues.  Whereas we can take some cells from the animals themselves and transplant them back, I believe it will be a more difficult situation to take and perhaps an ethical issue we could talk about of harvesting biopsy material from humans and then growing those cells and retransplanting those cells and the efficacy of doing something like that. 
		Getting to the last of these questions, to what extent do animal models facilitate the evaluation of cellular differentiation and integration following transplantation of stem cells? 
		Well, I come back to the point that rarely, if ever, are we really grafting stem cells.  In the central nervous system, these have not been the studies.  Most of the cells that have gone in are progenitor cells.  They're not self-renewing.  It's not known if the cells that do survive -- in fact, migrate -- are in fact stem cells of the population that go through, and many would argue that it really is a committed cell that's actually the one that's surviving and migrating. 
		An important question is how many species -- I think this is one of the questions that is related here to animal models -- and are monkey trials essential for moving forward in stem cell applications? 
		There are mouse models in some cases that could thought to be more homologous to the human disease than the monkey, obviously, in terms of transgenic animals, but the monkey provides many of the size issues that one would like to have addressed for some of these trials.  So, nevertheless, animal models are absolutely essential.
		And tracking.  So, describe techniques currently available. 
		Well, fluorodopa is being used.  Olle Lindvall came out with a very nice D-2 receptor occupancy, PET imaging study with fetal tissue grafts giving some functional index of the graft in Parkinson's patients.  I think we can begin to look.  There are studies now being looked at for BrdU PET to see whether or not there are dividing cells that exist within the grafts or in the brain normally, and then for some of these other grafts, if they don't migrate too much, one can begin to look at MRI.
		Highlight significant hurdles that need to be overcome. 
		We need markers.  We need markers for the transplant of cells.  We need markers that don't down-regulate when they differentiate, that are easy to assay in sections, and better, can be monitored in vivo and can be monitored in humans to follow the cells in vivo.  Develop some human markers that can be put inside these stem cells so that you can monitor where they are and where they go in vivo.
		You asked if there are any behavioral markers in animal models.  There is a ton of them.  You can measure every behavioral response that you want in an animal model to look at safety and efficacy, but I think what's missing in many cases are the appropriate controls.  For cell grafting, I think this is really important, especially in stem cells.  What is the control that you're using as a control for your stem cell to say that your cell is doing something different than damage or any other cell that would be used.  I think this issue of control and animal control is a very important one. 
		Define the extent to which animal model testing is useful for evaluating the impact of local environment on phenotypic expression. 
		Well, to the extent that one can make a homologous model of a human condition with the same pathology, then I think that's one of the considerations.  To the extent that you have an isomorphic model that mimics the local pathology of the human disease, then I think you're beginning to address these issues of microenvironment.  But if your models have no relationship to the anatomical microenvironment that's going on in the human pathology, then you really can't make any prediction of the fate of the cells in the human condition.  So, animal models, for stem cells in particular, should take into consideration trying to mimic either isomorphically or homologously the pathological changes. 
		To the extent that human cells behave the same in animal models as they do in human disease, it's obviously the gold standard.  This is what you want to do.
		I'm done.  I just want to remind, as did Steve, that there is neurogenesis and cell proliferation going on in the adult nervous system.  There's lots of it, and it's not just that there's neurogenesis going on in these regions of the hippocampus and the subventricular zone, there are cells dividing in the cortex, there are cells dividing in the spinal cord.  They're not necessarily giving rise to neurons, but they are dividing. 
		This is just a list.  You don't have to read it, but I'll just go through it.  This is a list of the recent studies that have shown either factors or environmental stimulation that can affect the endogenous rate of self-proliferation within adult animals.  So, EGF, FGF, estrogen, serotonin, glutamate, enriched environment, exercise, learning, stress, glucocorticoids, adrenalectomy,  stroke, epilepsy, and aging all have shown to have effects on the endogenous proliferation of certainly the hippocampal neurogenesis, but many cells throughout the brain.  So, in any consideration of grafting cells, we need to consider the fact that the environment can actually influence the fate of our cells once they are grafted into the host. 
		Thank you.
		(Applause.)
		DR. SALOMON:  Well, it's getting to that part of the afternoon where probably a break would be in order.  There was also a lot of stuff put on the table by Dr. Gage here that I think is worthy of some discussion.  The good news is that the next three talks are also on animal models.  So, can we maybe have specific comments to things that Dr. Gage brought up, and then we'll take a break.
		DR. DINSMORE:  Dr. Gage, you pointed out that there's no one marker for any one type of these cells.  There does not need to be one type of marker.  I think anytime you put in a population of cells, you just have to know repeatedly from time to time that you're putting in the same population of cells.  Therefore, your marker is for whatever characterizes a population, but I don't know of a single stem cell that doesn't have some marker that says you're putting in a population of liver-like cells or you're putting in a population of a certain type of neuronal cell that responds to EGF or responds to bFGF.  There's always somewhat of characterizing those, and I think the key factor is that you can do it repeatedly and have a repeatable population which you can use.  The only way you can develop a therapy is if you have something you can reproduce.
		DR GAGE:  Yes, I think the more completely you can characterize the cells the better, and I don't think you'd really argue that if you had a marker that could identify the cell that was the therapeutic cell, that you'd want to know what that marker is, and you'd be more satisfied with an identified cell than a population that contains perhaps the cell that you want.  That's where we'd like to go.  It's one thing to say where we'd like to go and where we are.  I agree where we are is we work with what we have.
		DR. BECK:  Tony Beck, Tissue Engineering Sciences. 
		One of the overriding themes all morning has been the fact that we have stem cells which are hard to define, but clearly you want to have a starting population that is consistent.  The other theme seems to be that the final residency of the cells often dictates their final disposition.  
		Now, there's a technology that hasn't really been addressed, and I address it to Dr. Gage and the panel in general.  The field of genomics has created a tremendous potential for defining the state of the stem cells you're starting with, and your slide on the karyotypic variation on those clonally expanded cells shows that certainly genomics or genetics plays a part.  
		Is it possible that some application of the EST, the expressed sequence tags, which really define the genes that are being expressed in those cells at that moment -- is that something that potentially could be applied to this quality control for stem cells?
		DR GAGE:  Yes, I think absolutely.  I think it's a very, very good point.  Ihor Lemischka has begun, as many other people have, to define the genetic profiles or the genetic fingerprint of cells that are defined either through FACS sorting -- I think that that's the starting point.  You have to have some reference point from which to make other comparisons.  So, I don't think that we all have to pick one cell.  I think that everybody should begin to genetically profile each of their cell types, bank that information from which we can begin to make comparisons.  So, I think that I couldn't agree with you more.  I think that's a terrific idea and certainly one that I hope the NIH picks up and does in some sort of formal way.
		DR. DINSMORE:  One has to be very careful about that.  This whole talk about stem cells -- we do get down to semantics of what a stem cell is, and we have to get down to talking about a symmetric division versus an asymmetric division.  In any population of stem cells, you have some cells that are potentially undergoing symmetric divisions and others that are undergoing asymmetric divisions.  It's hard to characterize a population like that as being uniform, and in many ways a true stem cell population is not going to be uniform because there may be some that are dividing symmetrically and others that are dividing asymmetrically.
		DR GAGE:  Sure, I agree entirely.  But I think that the technology is prepared to match that challenge at present, and by using strategies like laser capture where you can identify individual cells, you will begin to bank this information.  I don't think it really matters what cell you begin with, but we definitely need to begin to identify them.  
		I think what you're saying is an important point, and that is, are you going to do this in a population of cells, are you going to do it in somehow characterized cells maybe that are FACS sorted to be in a certain stage of the cell cycle or based on some marker on their surface?  In any case, the more discretely you can define the starting population, I think the more informative that technology will be.
		DR. SALOMON:  I just wanted to make a point before we keep going.  If you're coming from the audience, can you make sure that you identify yourself when you step to the mike, because it's very hard for our transcriptionist to do that.  
		DR. RAO:  Rusty, through your talk, you emphasized the fact that human cells may be different from mouse and from rat, and you also talked about isomorphic and autologous sort of transplants.  Do you think that there's some point that can be made about which would be a better model based on the fact -- what should we be using?  Would rat into rat be a better model in your mind than human into rat in that sense, given the differences?
		DR GAGE:  I think you've hit on a very important point, and I think probably some of the biological questions that are being asked are going to be better asked in mouse/mouse, mouse/rat, but as it moves forward to a clinical application where we're really asking whether or not the cell that you think you've characterized is going to be of clinical use, I can't see how you can avoid using the exact material that you think is going to be going into the human.  And it better be prepared exactly the same way that it was and expanded in the same number of expansions and characterized in exactly the same way.  
		That doesn't mean that there isn't a significant amount of information to be gained by using mouse for understanding the biology.  As you can see, we know there's lots to learn there. 
		DR. TROJANOWSKI:  Rusty, the adult stem cell work is enormously exciting, and I think it's clear that our concepts were wrong about neurogenesis.  But is there any evidence -- I don't think there's any published, but do you know of any evidence of functional benefit from adult progenitor cell differentiation into neurons or glia or what have you?  And if not, could one perhaps think of ways to ramp up that neurogenesis so that one wouldn't have to transplant but could exploit the patient's own stem cells as replacement therapy?  Is that inconceivable?  I guess nothing is inconceivable, but is that a plausible strategy?
		DR GAGE:  Well, I think several of the other speakers have already talked about it.  Jeff Macklis has a nice paper that just came out in Nature a week or ago or something like that evidencing the fact that there's some level of recruitment under selective damage situations where the populations of endogenous cells can target in some reasonable way.  
		I think there is a big theoretical question out there.  The brain has this capacity for generating new neurons.  Does this have some sort of functional consequence to it that's beneficial or negative?  All the evidence so far is correlative.  There's no causal link, but there's certainly disease models where aberrant type growth has been seen from these endogenous cells just like in, one could say, more therapeutic minded approaches, one gets an elevation or a function of function which is correlated with the increase in neurogenesis. 
		But having said that, I think that this is an area that's important, probably a little bit outside of the goals of this meeting.  On the other hand, we had to sort of consider that, that these are cells that are going to be activated or influenced by whatever procedure that we are imposing on them from the outside.  They are a responsive population.
		DR. TROJANOWSKI:  I was struck by the injection that was not saline but the other thing that really seemed to ramp up proliferation and could one exploit that in a positive way rather than having it be a confound --
		DR GAGE:  That would be the objective.  But we think of this whole process as a continuum.  There's proliferation.  There's migration.  There's differentiation.  There's function.  What that sort of demonstrates is that there are stimuli that can potentiate stimulation, but having the entire sequence for functional differentiation I think is not here.
		DR. SALOMON:  Well, then with that, we'll take a 10-minute break.
		(Recess.)
		DR. SALOMON:  The next speaker this afternoon is Dr. John Trojanowski from the University of Pennsylvania, from the university and from the Center for Neurodegenerative Disease Research and not from the Genetic Therapy Institute.  He's going to talk about animal models for evaluating cell therapy of neurodegenerative diseases.
		DR. TROJANOWSKI:  I realize that some of my slides are small, but I'll be able to read off some of the things that don't project to the back of the room. 
		The first slide has something up here that says "fatal protein attractions underlying neurodegenerative diseases," and it was my attempt in a cinematic way or an allusion to a film to capture what's wrong in many neurodegenerative diseases.  I'm going to focus on Alzheimer's disease.  The list of diseases here in abbreviations are some of the very common ones.  We know of Alzheimer's, Down's syndrome, Parkinson's disease; less common ones, the dementia with Lewy bodies, multiple system atrophy, Pick's disease, ALS.  
		All of these diseases have very different clinical phenotypes, very different pathological phenotypes, but they share one intriguing commonality that struck us and has stimulated our efforts to understand how the lesions form.  And that is, they're characterized by intracellular or extracellular protein aggregates.  These proteins don't start off in life doing bad things or they're not designed to be toxic, but because of a mutation, let us say, in PS1 or PS2 or APP, they give rise to toxic proteins, the A-beta peptide, tau aggregates, synuclein aggregates.  But they have a very intriguing symmetry and that is the wild type protein in the sporadic disease also shows the same predisposition, perhaps influenced by genetic risk factors, environmental factors, to also convert from a soluble protein that performs a normal beneficial function to a toxic, insoluble, and often filamentous derivative that then accumulates as lesions in one of the places that I just mentioned.
		So, that offers the hope that if we can find anti-fibrillogenic drugs, that we can take drugs that convert bad conformations into normal conformations or that are filamentous aggregate-busting or plaque-busting or tangle-busting, we may be able to translate an advance in a boutique disease like MSA or dementia with Lewy bodies to a very common disease like Alzheimer's disease.  In other words, you may be able to exploit that drug to bust not only Lewy bodies but plaques, tangles, and other bad accumulations of proteins.  
		So, I'll just start with Alzheimer's disease as the prototype of these diseases and the prototype, of course, of all late onset, adult onset neurodegenerative diseases.  These statistics are quite ominous and worrisome.  4 million Alzheimer's patients today.  By 2050, there may be 14 million.  So, this is something that will not only cause a lot of anguish and pain among families, but it threatens to break the bank as well because of the high cost of caring for patients for a very long time with Alzheimer's disease.
		So, the defining lesions of Alzheimer's disease -- there's are lots of way to become demented and there are many different neurodegenerative diseases that cause dementia.  Alzheimer's is about 60 percent of dementias over age 65, and the defining lesions are the neurofibrillary tangle and the amyloid plaque.  The tangle is inside neurons, the plaque is outside neurons.  The one inside is formed by twisted filaments largely.  They are the tau filaments.  The ones outside are the amyloid fibrils formed from short peptides called the beta amyloid peptide. 
		It's not known how they're related in sporadic disease mutations.  Of course, NF AD kindreds will produce plaques and tangles, but in most diseases, sporadic so far as we know -- and this is just a schematic from the work of Steve Arnold, who is at Penn, showing the distribution of plaques and tangles.  They pretty much collocalize but they're not always in the same place, and there's been no meaningful way to connect plaques as causative of tangles or vice versa as yet, except in the genetic diseases. 
		There are areas of the brain that are spared, however, motor cortex, sensory cortex, occipital cortex, offering the hope that if at least a few neurons are smart enough to escape this pathology, if we figured out how they got to be that smart, in molecular terms or genetic terms, we would be able to perhaps develop a therapy to help spare neurons, that otherwise would be affected, the fate of dying because of plaque and tangle accumulations.
		So, these pathways are two parallel pathways so far.  This is the hypothetical scheme of how tangles are bad for you and how plaques are bad for you.  I'm going to focus on the tangle pathology just as it seems more intractable at this moment.  I mentioned the report yesterday in the Washington Post extending the findings of Dale Schenk a year ago showing that you can somehow take a formed plaque and clear it by an A-beta peptide vaccine.  That was in July of last year.  
		In December at Penn, we began a clinical trial.  I'm the Director of the Alzheimer's Center at Penn.  I didn't personally.  I'm not taking A-beta myself --
		(Laughter.)
		DR. TROJANOWSKI:  -- although it looks really exciting and might want to consider it just to be sure.  But it is in clinical trial and it looks as though there are no deleterious consequences at all.  
		So, I think we'll rapidly have in patients confirmation, or the opposite thereof, of the amyloid hypothesis.  For the patient advocates in the group, we'll also know right away whether we have a therapy that may in fact be beneficial. 
		I have said that if you clear all the plaques, you're going to be left with another disease that is referred to as a "tauopathy", Pick's disease, CBD, because I don't know that this therapy directed at amyloid will necessarily clear tangles.  Those are inside cells.  They're formed from the protein called tau, which is a microtubule associated protein.  I'll say a little bit more about what its functions are, but one of the important things it does is bind to microtubules and stabilizes them in the polymerized state.  These are the guide wires, the train tracks upon which traffic moves from cell body to processes and back again.  If those ties fall off or there are too few of them, it's plausible -- and in fact we've shown it does happen -- the microtubules depolymerize, transport fails, and that severely compromises neuronal survival.  That's a loss of function when you pull off tau and sequester it in tangles.  
		The tangles also, which are shown here in red, and dystrophic neurites also in red in these little processes could have a -- the formation of the tangles could represent a toxic gain in function.  You'd have plugs of stuff occluding conduits that should be open for traffic flowing both directions.  By whatever mechanism, the ultimate consequence of these toxic gains of functions and losses of normal functions would be the death of a neuron and release of tau into the CSF, which is now one of the markers that can be used to follow responses to therapy potentially.  
		So, just without getting too technical, tau is a microtubule associated protein, one of many that binds to microtubules, and has some function related to the stability of microtubules, in this case keeping microtubules polymerized, and again microtubules are essential for axonal transport.  
		It's a phosphoprotein and phosphorylation negatively regulates tau binding.  The more phosphorylated tau is the less it binds.  That's one of the things that goes wrong in Alzheimer's disease.  
		All of us have as adults 6 tau isoforms that come from a single gene that's alternatively spliced.  This is the biochemical profile.  They have these either 3 or 4 microtubule binding repeats in the amino terminus of unknown function, and it's by this alternative splicing that one gets then what we call 4R2N all the way down to 3R0N.  As you will see, there may be some reason to believe that the ratio of 4R to 3R tau is somehow important.
		So, what is a tangle then?  A tangle was one of the two signature lesions that Alois Alzheimer discovered and associated with the disease that bears his name.  
		These are tangles that you see here.  He used a Gallyas or Bill Shofsky or some of the silver stain.  These are anti-tau antibodies.  This is an anti-tau antibody labeling of tangles.  
		I would say that what impresses me much more about the tau pathology in the Alzheimer brain is not what's in the cell bodies but out in the processes.  I just want to emphasize to everyone that you could conceivably preserve neuron viability.  You could keep all these neurons alive, but if their processes are caked with tau inside of them, they're not going to be talking to each other and there will not be any functional discourse that takes place between neurons that need to do so in order for cognition and other things to take place.  
		So, neuron rescue is a laudable goal, but it may not do the trick in all of the diseases that I'm thinking of and that were listed on that first table.  Accumulations of filamentous aggregates and processes can be the equivalent of a dead neuron even though you can still identify it in the section. 
		So, these are the paired helical filaments.  They're abnormal aggregates of tau proteins and they accumulate in the cell bodies and processes of neurons and sometimes glial cells.  After many years of controversy, it was determined that these proteins are, indeed, formed by tau proteins, derivatized though they are by being abnormally excessively phosphorylated, and that comes with a very serious functional loss.  
		These proteins used to be called A68, so just forgive the old slide.  It should be tau or PHF-tau.  
		This is dephosphorylated PHF-tau, adult tau, dephosphorylated adult tau.  You don't have to know much about microtubule biology or biochemistry to realize that the only guy that winds up on the left side in the supernatant unbound to microtubules is the hyperphosphorylated A68.  And that's bad.  That's not where it should be.  It should pellet with the microtubules where it binds to and stabilizes the microtubule.  So, this is the loss of function.  
		The good news is it's not an irreversible loss of function because if you can figure out a way to dephosphorylate tau, pull a few phosphates off, we don't know if you could convert it into a water soluble protein, but it will then bind to microtubules again.  So, that's one potential target of therapy.  
		So, PHF-tau is insoluble, cell bodies, aberrantly hyperphosphorylated, and unable to bind to microtubules. 
		Now, I have to mention that there are other diseases -- the tauopathies that I referred to that I worry about will emerge when you remove plaques -- are Pick's disease, corticobasal degeneration, the other frontotemporal dementias, including hereditary ones, frontotemporal dementia with Parkinson's disease linked to chromosome 17.  
		So, in 1998 Jerry Shellaberg called me up and said, I've got a mutation.  No longer will you suffer from mutation envy.  And knowing that our proteins were bad already, I was quite peeved.  I said, Jerry, I didn't need your mutations to tell me that.  I knew it already.  But I was pleased because the rest of the world began to take these proteins seriously as players in cell degeneration.
		What was already emerging when the mutations appeared was the fact that it was clear that Alzheimer tau, where most of the studies that have been done, has this abnormal banding pattern that you see here.  This is a cartoon, of course.  When you dephosphorylate, you can see all of the 6 isoforms that are seen in the normal brain.
		Other diseases had a more peculiar banding pattern, either the two lower bands here or the three upper bands, which, when dephosphorylated, revealed that you had a preponderance of either the 3 microtubule binding repeat tau or the 4 microtubule binding repeat, suggesting perhaps that an imbalance in the ratio of these isoforms could in fact be deleterious.  That is what rapidly emerged in studies of the frontotemporal dementia with Parkinsonism linked to chromosome 17 patients.  
		A flurry of papers in '98 demonstrated that there were intronic and exonic mutations in tau and that these caused disease.  This is sort of the cartoonist tau protein.  This is the segment of exon 10 and the intron following exon 10 where many of these mutations are.  We and others have shown that the mutations impair tau functions.  They reduce the ability of tau to bind to microtubules, reduce the ability of tau to promote microtubules polymerization, or they alter splicing that either introduces more exon 10 or less exon 10 into the tau transcripts that encode 3R/4R tau.  
		All of us in this room hopefully have a 3R/4R tau ratio of 1.  When it begins to deviate from that, for reasons that we don't understand completely, you get accumulations of the species in excess, and that can lead to disease, as is clearly evident in this group now.  There's about 20 mutations, about 30 families, and we don't know so far of any escapees.  Disease can begin in the 20s and the 30s and it runs a very malignant course, and patients are dead in 10-15 years. 
		So, because of this data showing that abnormalities in tau ratios could be an underlying mechanism of disease in "tauopathy" -- this is before the mutations -- we began to generate a tau transgenic mouse that doesn't have exon 2 or 3 and doesn't have exon 10.  So, this would be the 3R0N, the smallest tau isoform.  I just have to tell you that, although what I've just said applies to people, rats and mice only have the 4R, the 4 microtubule binding repeat harboring tau transcripts.  They don't have the 3R tau isoform.  
		So, we figured we would perturb their microtubule metabolism, if you will, and their tau metabolism by introducing the smallest tau isoform.  Here are the three lines, the copy abundance of the transgene shown here, driven by a prion promoter shown to be effective in previous transgenic mouse generation efforts.  So, we had these three lines that have increasing amounts of transgene and increasing amounts of protein.  We exploit an antibody called T14, which is human-specific here, the human recombinant tau proteins on the left.  The wild type doesn't have this because it's a wild type and the antibody, of course, doesn't dissect it.  
		The smear is due to the variable extent of phosphorylation.  It's not a crisp band like this.  But you'll see later on you can dephosphorylate it and get the same.  
		Line 7 was the one we used.  Line 27 died at 3 months.  And line 7 was the one that we selected for our studies, and I'm going to focus mostly on line 7.  You can see here, again with this T14 antibody, the polyclonal detects everything, but I'll just focus your attention on what is cortex, hippocampus, brainstem, cerebellum, and spinal cord.  Spinal cord has about 60 percent of that amount of tau found in the other regions.  
		So, we've got robust expression, and anyone who is involved in transgenic mouse production efforts knows that the 3 L's of mouse is levels, levels, levels, or higher, higher, higher.  The rapidity of disease onset and the robustness of the phenotype depends very much on the abundance of the transgene protein expression. 
		6-month wild type mouse.  T14, nothing.  6-month transgenic mouse.  You begin to see a few cortical tangles, in brainstem a few tangles, and here are higher power views of the 6-month tangles in, I guess, brainstem and cortex.  Well, I shouldn't say they're tangles until I show you the filaments, but they stain for Bodian methods.  So, here in spinal cord and in cortex are things that to somebody who does diagnostic neuropathology as well as basic neurobiology like myself is persuasive of being a tangle.  
		We now have older mice that I'm not going to discuss or present who have more cortical tangles that are Gallyas positive, Congo red, thioflavin.  For those interested, I can tell why -- basically it says it looks more and more like the tangles of human beings.
		Since the filamentous inclusions will be more abundant in spinal cord and the mice begin to develop those at 3 months and then developed a motor neuron disease phenotype by 6 months that progressed, we focused a lot of our attention on spinal cord.  
		This is a normal nerve, and you can see there's a filamentous aggregate in this normal nerve of a wild type mouse, transgenic mouse, higher power view of these filaments.  And these are labeled with antibodies to tau, as well as neurofilament and tubulin.  Some tangles in Alzheimer's disease brains, progressive supernuclear palsy, Guam amyotrophic lateral sclerosis, Parkinson's dementia complex, or Guam ALS PDC, diseases that are like their western counterparts, except taus, that I would have called pathology, also have some neurofilament proteins.  
		I don't want to confuse you.  Tau is the building block of the filaments in our mouse and in humans, but other filaments may co-precipitate.  In the older mice that we have now, 18 to 24 months of age, we're not seeing that, that is, the co-aggregation of other elements. 
		So, I'll just say that there are other signatures of tau pathology that may also be relevant for targets of therapy and so forth that you want to pay attention of and make sure in your transgenic mice.  And here they are.  
		There is a progressive accumulation of insoluble tau with time, and you see that not only in the brain but you see in the spinal cord.  In fact, it's more abundant in the spinal cord.  So, this is RAB buffer.  Water soluble proteins would be present.  This is a buffer that is a next level of solubilization ability, and formic acid is really harsh and will solubilize just about everything.  
		So, you can see that in the spinal cord, we had the most abundant material that required formic acid extraction.  And then if one thinks that phosphorylation is somehow involved in Alzheimer's disease, as we do, you would want to see that there is an accumulation of phosphates on these molecules, which is indeed the case, and that that is in the insoluble pool as well.  
		So, here's the ADPHF tau.  The mouse tau doesn't migrate at the same level because, remember, there's only one, the lowest, the smallest, the 3R0N tau isoform.  But the principle is that it's winding up in the insoluble fraction, and it is detected by antibodies, for example, PHF1, that don't see normal tau, see PHF tau, and then this increasingly phosphorylated mouse tau.
		This paper appeared in Neuron last year and we thought we had persuaded them for what we thought where the bar was set at that time on a phenotype due to the accumulation of tau proteins.  And they insisted that we do something of a functional nature, which we did, and that is axonal transport studies.  I want to draw your attention.  
		These are the fluorograms.  Basically you inject P35 into the spinal cord motor neuron pool, and this radioactive amino acid is incorporated into all proteins that move out by axonal transport.  This is the normal wild type mouse here and this is the transgenic mouse.  The delay in transport is represented by -- these are increasing millimeter distances from the spinal cord, 2, 4, 6, 8, 10 millimeters from spinal cord. 
		You can see the wild type mice move at a normal rate and those in the transgenic move at a slow rate.  They're still walking around and doing things.  So, these are the remaining residual neurons, and there are axons that are not yet dead but are not functioning at the level they should. 
		We also showed -- I don't have slide for that ‑‑ that the microtubules were depolymerized and lost while neurofilaments and actin filaments were not. 
		So, let me just say that there are many genetic factors, tau mutations, APP, PS1, PS2 mutations that can produce what we call a "tauopathy."  Alzheimer's disease can be called a "tauopathy."  Of course, there are other lesions, but there are diseases, FTDP-17, in which the only lesion is a tangle and that is associated with whatever clinical phenotype, dementia, Parkinsonism, and neuron loss.
		I haven't shown you the data, of course, but I have referred to the fact that these genetic lesions can perturb the ratio of normal tau, 3R to 4R tau, or cause a loss of function and/or a gain of toxic function.  Hyperphosphorylation is something that may be down stream of all this but would certainly contribute to the failure to bind to microtubules.  
		A big unknown is environmental factors, and we have a lot to learn about that.  But wouldn't it be nice if we could prevent Alzheimer's disease by determining something in the environment that was bad?  And we're trying to work on that. 
		Tau dysfunction then, in one way or another, leads to tau aggregation and neurodegeneration.  I think despite the controversies between tauists and Baptists -- and I belong to both camps.  I work on both tau and A-beta, but I think there's very little reason for even the most maniacal Baptist to say that tau doesn't play a role anymore in neurodegeneration.  So, we hopefully built bridges and resolved our religious wars and can get on with the real business of curing these diseases.
		So, Nancy Bonini allowed me to use this slide from some of her work on Drosophila models.  We like to think of mice and monkeys as being the way to go, but there's a great deal of appeal to mouse models.  Nancy talks about late onset disease at day 9 or 10, and I hear some transgenic people laugh.  I lust for a number like that because we have to wait for a year, 18 months.  It's not only expensive, but very nerve-racking to know whether your investment of $50,000 or $100,000 in a mouse line is going to be thrown into the trash because it didn't develop a phenotype or if it's going to work.
		So, Nancy I think revolutionized our thinking about models of disease when she reported -- Woek, et al. reported in Cell that she could create a mouse model of Sca-3, the Machado-Joseph disease, one of the trinucleotide repeat diseases, and do this in a most unconventional way by getting protein expression, the transgenic protein to be expressed in the eye, and could follow a disease phenotype in a very straightforward, easy way just by looking at the mice.  They do the sectioning and all that sort of stuff, but that is the real bottleneck, the rate-limiting stuff.
		DR. SALOMON:  Do you mean mice?
		DR. TROJANOWSKI:  These are flies.  Did I say mice?  Flies, Drosophila.  I'm sorry.  So, these are fly eyes.
		So, this is the normal eye, and then at 10 or 15 days, the flies -- this is the normal ataxin-3 protein with the normal length of polyglutamines.  These flies have an eye that's identical to the normal eye.  But flies that express the disease, ataxin-3 protein, develop a phenotype that's very, very evident even to someone unschooled in fly biology, like myself. 
		She went on to show that there were aggregates and she showed that hsp, heat shock protein, 70 accumulated in them.  And she began very creatively thinking that maybe hsp 70 is there for a purpose.  It's not just sucked into a vortex of a crashing cell but has some productive role. 
		Again, Woek, et al. in Nature Medicine showed that if you co-expressed the heat shock protein 70 with the expanded polyglutamine protein in the same cells of the fly eye, you could eliminate the phenotype.  So, here's hsp 70 alone in the fly eye.  Here is the diseased eye with the disease protein, and when you co-express both of them, you suppress the phenotype.  So, the abnormal confirmations that are assumed by this expanded polyglutamine stretch are not formed or are smoothed out or reversed when you have sufficient heat shock protein on board. 
		There's a lot more that we need to know about what goes wrong inside tangle-bearing neurons as well as how plaques kill neurons.  I will suggest that one way to go -- it was mentioned that one could use gene expression profiling methods.  This is work that Steve Ginsburg did recently in our laboratory before going to Baylor. 
		These are normal neurons in the normal brain of an elderly individual, and these are neurons, some of which contain tangles in the brain of an Alzheimer's patient.  Steve harvested single cells, labeled with an antibody so he knew what it was he was taking out of the brain.  And he wanted pure populations of neurons because, remember, the thing that one always has to appreciate about many of these neurodegenerative diseases is selective vulnerability.  Not every neuron is affected, and certainly many other types of cells in the brain, glial cells are not affected. 
		So, if you just homogenize these two areas and look for differences in gene expression, you probably see lots of differences, but you wouldn't know whether you should attribute them to dying neurons or something else going on in glial cells.  Remember, if you do a Western Blot, the most elevated protein you'll see in a very severely diseased Alzheimer's brain is glial fibrillary acidic protein because there's a lot of gliosis and astrocytes that proliferate it.  So, you've got to do this in a focused, intelligent way, and it's very appealing to be able to have single cells. 
		So, then Steve was able to pool these cells, the tangle-bearing neurons and the non-tangle-bearing neurons.  This was done in collaboration with Jim Eberwine and Virginia Lee, Scott Hemby at Penn, and it just came out in Annals of Neurology, if you're interested in the details.  
		But basically Jim Eberwine's aRNA amplification method allows one to amplify a million-fold.  Here are some controls that were done to show linearity of amplification.  We took two approaches.  One is the "gee whiz, what can we see that's different approach" and then what we called the "candidate gene approach" where we knew there were proteins already implicated in Alzheimer's disease and where there was even some data to indicate that their message levels changed.  We interrogated what we call custom arrays with the amplified transcripts that are labeled in the second round of amplification.  
		Here are the GDA arrays, and I forgot whether red is normal and green is tangle.  Yellow indicates where both levels are equivalent, but if you have more of one transcript, it will be red.  If it's down, it will be green.  Then you can plot and do statistics and group proteins together by their function, phosphotases, kinases, actin binding proteins, cell cycle proteins, cytoskeletal proteins, and look for those that deviate up or down.  This may be away to identify other candidate proteins for targeting for cell therapy or some other form of therapy.
		Obviously, this is the beginning of an approach to dissecting out the molecular mechanisms of neurodegenerative disease, but potentially a very powerful one.  I'll just say that the one that we followed out in the most detail was cathepsin D, which Randy Nixon and collaborators had shown was up-regulated to lysosomal protein, and we showed that, indeed, it was one of the most highly elevated transcripts in our array study relative to controls, and when we did in situ hybridization immunohistochemistry, we showed, as Randy had -- it was very pleasing to not have to struggle with data that went the other way.  I saw Randy recently and said, thanks for making our job easier because we were able to show what he had already shown so nicely earlier, that lysosomal proteases, in particular cathepsin D, are up-regulated.
		This is the second-to-the-last slide.  I'll come back and emphasize once again that the approaches one can use to correct a disease that may be a boutique condition, such as MSA or Hallervorden-Spatz or neurodegeneration with brain iron type 1, could possibly be exploited in other situations.  What could cell therapy do in these kinds of situations? 
		Well, one thing would be to make a heat shock-like protein that could be secreted and taken up.  So, if you were able, as Evan is able to do, to inject cells into the brain and get them to go everywhere you want them to be or everywhere you can get them to be, they obviously -- if you had more heat shock protein around, I don't think it would make good protein fold bad, but it could make the bad-folding proteins fold right.  That would be one strategy.
		Another alternative, and far less clear-cut to me at this point, would be to have the neurons go to the damaged area and perhaps rewire, if they were interneurons.  That's really a great leap of faith and beyond my imagination to work out in my own mind in detail.  But maybe they will function as these human neurons do in stroke trials right now where they seem to reinvigorate cells in the penumbra of a stroke cavity so that they function better.  I'm really just speculating about possibilities, for which there is at this time very little data. 
		So, I think that one could get a base hit in one of these diseases that could be a base hit over and over and over again in other diseases and have potential to cure all of these diseases that are caused by fatal attractions of brain proteins.  
		I'll just close by saying that these studies were done with my wife and colleague, Virginia Lee.  We've been working together on this tau and Alzheimer's disease problem for many years and have a wonderful group of collaborators in our Center for Neurodegenerative Disease Research that extends to many departments at Penn, psychiatrists, neurologists.  It takes the whole university and many universities, I think, to marshal the talents to make advances in this area, and there are many colleagues who are at other universities, upon whose expertise we've drawn.  Of course, we couldn't do any of this without support from the National Institute of Aging and NINDS and the Alzheimer's Association. 
		Thank you very much.
		(Applause.)
		DR. SALOMON:  So, I think one of the things that we need to do in these next couple talks is to take advantage of the fact now that these specific models here ‑‑ this is Alzheimer's.  This is a model for Alzheimer's as well as -- I think you made very eloquently -- a number of different degenerative diseases.  
		So, in the comments that I'd like to get from the expert panel, can we talk about then how this kind of a model now could be integrated into preclinical studies for a clinical trial in Alzheimer's, the pros, the cons, what kind of things you would want to see measured, et cetera?  We'll pick up on this more specifically again tomorrow, but just in the short time we're going to allot to conversation right now, I'd like to start in that direction.
		DR. TROJANOWSKI:  I would just add that at the meeting there were crosses of the APP, the presenile, and what have you.  It sounds like a word salad I know, but you can get all of these mice that make the different pathologies to cross breed and get plaques and tangles.  So, that's going to be there very shortly if it's not here already.
		DR. NOBLE:  It seems a fundamental distinction that needs to be made is that these kinds of diseases with the tremendous diffuse damage throughout the nervous system are very different than the focal problems of spinal cord injury.  So, a different order of problem, different biology entirely. 
		DR. TROJANOWSKI:  I was told to make it as tough on you guys as I could.
		DR. NOBLE:  You did a good job.
		(Laughter.)
		DR. NOBLE:  So, one question that comes to my mind is the extent to which one can delineate between being a cell therapy problem in the contemporary world and a gene therapy problem and whether one is simply using cells as an enzymatic delivery system.  So, for example, in the work that Evan and his colleagues have done in relation to storage disorders.  So, I'm wondering what your thoughts are.
		DR. TROJANOWSKI:  I think we needn't think in a compartmentalized way, of course.  You might want to try several different options, and one could even conceive in the same patient of more than one option.  One would want to start off, of course, in a very focused and meticulous way looking at one at a time, but maybe you could replace cells in hippocampus.  If, as Brad Hyman argues, that is the switch that does or does not enable memories to go -- remember, in Alzheimer's patients the short-term memory is okay.  It's the long-term memory.  They can't sock new memories away.  So, maybe you could repopulate the hippocampus and maybe local connections.  That's a very small circuit.  The hippocampus is as big as my thumb.  Is it going to be that hard to restore circuits?  It seems like an immense task to me even though the hippocampus is small.  But that would be one way to go, and then you could also do something more diffuse.
		DR. NOBLE:  Let's follow that along.  I'm trying to figure out how to get to a clinical trial.  What is the preclinical experiment that you would want to conduct?  If you showed that putting neuron-restricted precursor cells or neural stem cells into the hippocampus of these animals now led to a restoration of cell number or a decrease in apoptosis or some parameter like this, is that the point at which you would argue one should go forward?
		DR. TROJANOWSKI:  If you're a purist, you would say, well, it's not quite the model of Alzheimer's disease I want because there aren't enough tangles up north, in the brain.  They're most abundant in spinal cord.  But from a proof of concept point of view, it's much easier to follow motor dysfunction than cognitive dysfunction.  I don't know if you corrected a cognitive dysfunction in mouse, whether you're immediately assume that that's going to make a difference in people.  
		But from a proof of concept point of view, you could inject cells into the anterior horn and see if they would keep those that are alive functioning better.  If you could transfer from those cells a compound like a heat shock protein to eliminate, bust up, a tangle-busting drug, that might be efficacious.  I don't know if you would get elongation of processes to the muscle from the transplant.  No one has done that kind of thing, the equivalent of cutting a nerve -- actually the nerves aren't cut, so you'd have a better chance probably, but I don't know if the rewiring would be meaningful.  So, those would be the kinds of the things that I would think of as trial-like concepts.
		DR GAGE:  You mentioned clinical applications in stroke too.  I think that you've been involved initially in these studies with the N-Tera-2 cells.  I wonder if you could update us on that and the rationale behind utilization of these because this is really the first real application of human whatever cells in a --
		DR. TROJANOWSKI:  I'm a founding scientist of Layton BioScience, but I'm a full-time Penn faculty.  I'm not involved daily in discussions of how the trial goes on.  I'm certainly glad in retrospect, considering Jim Wilson's problems, that I was not closer to the clinical trial than Pittsburgh, which is where it takes place.
		But the underlying rationale was not that we were rewiring the brain, because it was clear from the animal studies that we weren't.  It seemed as though the cells that went into the perimeter or the vicinity of the ischemic damage survived and survived very well and somehow made those remaining neurons function better because we clearly were not reinvigorating.  We didn't do bromodeoxyuridine to see if more proliferated.  I guess that would be an option now that we didn't think of 4 years ago. 
		So, I think what the N-Tera-2 cells may be doing, the neurons may be doing, in people is having some sort of trophic effect on their neighbors.  That is completely unvalidated and unverified.  It's a subject of intense research at Layton, but not in my lab.  I don't do those studies in my lab.  That is, so far, I think all that we can say about what we know might be going on.  But it's not a rewiring.
		DR. DRACHMAN:  John, if tau is your target -- and certainly that was what Alzheimer described originally, not really plaques -- we've got to believe that tau is a very specific problem.  Yet, we know that one of the most nonspecific etiologies that you may think of produces huge amounts of tangles, namely dementia pugilistica.  A good knock on the head certainly is a far cry from a very specific biochemical abnormality.
		What are you thoughts about that and whether this is secondary, primary, or what?
		DR. TROJANOWSKI:  I think that this argument is rendered moot by the mutations.  I don't know that there's any way that one could argue -- at least in FTDP-17 that the cause -- we have prima facie evidence that the cause is a mutation in the tau gene.  It tracks with the tau gene.  If you have the mutation, you get the disease.  I don't know unless there's another mutation someplace else that no one has seen -- and that happens perversely to track with disease.  
		So, tau abnormalities absolutely cause disease, and it causes disease that -- the Jerry Shellaberg B337M family was first thought to be schizophrenic, then Alzheimer's disease, and then a "tauopathy."  So, these are diseases that can look very much like Alzheimer's disease.  I think the only difference is where the pathology falls, and we don't have a good understanding of that. 
		Dementia pugilistica is very interesting.  We are working ourselves very intently on head trauma in transgenic mice to see if we can dissect out what's going on.  Is it inflammatory?  It is up-regulation of a kinase, down-regulation of a phosphatase?  What is going on here?  Because most Alzheimer's disease is sporadic.  Many people sustain head trauma.  I fell off a horse when I was 16.  So, I've had the head trauma history, as probably many people in this room have, of some sort of a fall, hit on the head, or what have you.  If we could figure out what that's all about, perhaps we would put people on anti-inflammatories after they've had their head trauma or recommend that everyone wear bicycle helmets.  And we do that already, but may soccer helmets and football helmets and all the other things, particularly if they have the E‑4/E-4 genotype.
		Tangles are not nonspecific.  You can do a lot of things experimentally and fail, as many of us have for many years, to produce tangles in an animal.  It's a long list but it's a limited list of diseases that are caused by or have prominent tangle pathology.  So, it's not like gliosis in response to injury.  I just want to emphasize that.  It's real specific and linked to bad stuff.
		DR. MACKLIS:  I have two questions that will tie into this morning's discussion about the characteristics and appropriateness of animal models and then the characteristics and appropriateness of source cells.
		The first one would be, in the tau transgenic that you mentioned, or the mutant, there's anterior horn cell loss, as I understand.  To use Rusty's slide, homology versus isotypic, is that a model for us for AD or for part of ALS, for example?
		Then the second part would be we've heard a lot this morning and discussed whether neural stem cells or precursors or progenitors effect their potential recovery by trophin or growth factor secretion rather than by connectivity.  If that's the case, are we actually looking at them in all of these slide talks by the wrong axes?  Do we really care if they turn into oligos, astrocytes, or neurons, or do we really want them to stay round cells that pump a lot of factor X, Y, and Z and maybe migrate to wherever they go?  I think we may want to ask those questions in our models ahead of time so that we can answer them in our preclinical studies.
		DR. TROJANOWSKI:  I'd say yes to the second question.  My early training in neuroscience was in neuroanatomy, and maybe I'm just hung up with the minutia of wiring diagrams, but I think it's hard to imagine restoring all the complex circuits that all of us walk around with before disease strikes.  But maybe that's just limited imagination.  Maybe we'll be able to do it. 
		Your second part of putting out whatever molecules that will help cells function better is I think a laudable goal and a direction that we should think of.
		For those of us who struggled with animals for so long in Alzheimer's disease -- and it may seem like a blink of an eye to people who are still struggling to make animal models in other diseases, and there are people who are struggling -- we just wanted a proof of concept that this pathology is bad.  So, we have shown that tangles kill.  Amyloid transgenic mice don't kill neurons.  So, I believe that amyloid is bad, and I work on that too.  But it was most pleasing to have an animal accumulate tau, show a functional deficit, failure to bind microtubules, microtubule dissolution, many functional deficits actually, axonal transport, perturbation, disruption, attenuation, what have you, and that these mice got sick.  They were weak.  They had disease.  I would love to move all those tangles up north.  They're getting older now and they are getting more tangles in their cortex and hippocampus.
		And I know others who are making other transgenic mice with other promoters and other constructs.  It's going to happen.  A year from now, we'll be talking about tangles in hippocampus that look just like Alzheimer's, believe me. 
		DR. MACKLIS:  Just a quick follow-up on that, though.  There may be a dissociation where that same mouse may be an incredibly good and motivating model for biochemical and molecular therapies for "tauopathy."  It may or may not be as good a model for repopulation or cellular repair.  Do you agree with that?
		DR. TROJANOWSKI:  No.  I think you start where you can start.  These things don't have to be done in series.  They can be done in parallel.  I appreciate the personal insight or contribution of one of the patient advocates in the audience.  I think all of us know people, have relatives, what have you, who died of an untreatable disease.  I think we should do things in parallel.  I think we have the resources.  I think we have the intelligence.  We have the people and it should be full steam ahead.
		DR. KOLIATSOS:  Can I make a comment here?  I'm sorry.
		DR. McDONALD:  I just wanted to say that perhaps some of the neurodegenerative diseases like Alzheimer's that occur over decades are really a problem of turnover, more or less.  That is, you're accelerating the rate of death, and with the new concept of the nervous constantly turning over -- that is, neurons and glia are continually being replaced -- that we might do better just augmenting cell birth and survival.
		DR. TROJANOWSKI:  Hence my question to Rusty.  If we could up the rate of normal adult neurogenesis, might we be able to repopulate some of the hippocampal neurons that are maybe still alive but not functioning because their axons aren't talking to each other.  I would agree.  When I say full steam ahead, I just want to let you know that means clinical trials should be done with great oversight and contemplation, but I don't think we should fail to exploit the models at hand.  That's what I'm saying.
		DR. McDONALD:  For example, do we know, in those animal models where the disease is present, is there reduced proliferation and survival of progenitors?
		DR. TROJANOWSKI:  I don't know that.  We will have that answer in a year or less. 
		These are very important questions and I don't think we should hold back from addressing them.  There's only so much I can do in my lab, but if Rusty stops in Philadelphia on the way home, I'll give him a bunch of mice to take back to San Diego.  I'd love to know the answer to that question is what I'm saying, and I think it's important to the field.
		DR. KOLIATSOS:  I strongly feel that it is inappropriate to make any direct comparisons between neurodegenerative models and stem cell transplant approaches at this time, especially as far as molecular specificity of lesions.  I think it's not the job of this panel to go into the depths of neurodegenerative models, and it would probably be a very bad idea I would propose. 
		I think that this is just a very important input from John to let us know that there are powerful models out there which might be used when the time is appropriate for what we're talking about in this meeting not only in the sense of repopulating, but also in the sense of assessing some potential side effects of grafts.  For example, mice which have the genetic propensity to make plaques or tangles could be very well used as biological models to see if the cells we want to put in Alzheimer's disease are going to increase plaques and tangles by some side effect mechanism.  So, there is much more genetic value to the models that John described than looking at specific associations between circuitry and cells and so on and so forth.
		DR. TROJANOWSKI:  That's true.  If A-beta is as toxic as it's reported to be, it may kill all new grafted cells on contact.  In agreement with what you said about the grafted cells doing bad stuff, we thought of that and did do those kinds of studies on the N-Tera-2 cells five or six years ago.  They do make APP.  They're great little factories for studying amyloid precursor protein metabolism, but we showed that in over a year in nude mice, they didn't produce plaque or tangles.  So, that was something that we felt was important to look at.
		DR. SALOMON:  I would point out that one of the things that the FDA does want from us is some comment on what might be perceived as gold standard models for different neurologic diseases that, indeed, could be put forward as models in which preclinical studies should be focused.  Or equally important would be to identify where there are no models or where there are incomplete models and that's where perhaps resource allocation from the NIH or others should be put.  It's certainly not meant that anyone take apart John's model.  I think I was only using it as an example of --
		DR. TROJANOWSKI:  Just on that point, one controversy that arose at the meeting -- there is inflammation in many neurodegenerative diseases, including Alzheimer's disease.  Dale Schenk, who is from Elan and really conceived of the experiment of using A-beta as a vaccine, reported data saying that microglia were doing good things scooping up, gobbling up A-beta and eliminating it.  That elicited irritated comments by the neuroimmunomodulation group who thought that all the inflammation was really bad and what you want to do is suppress that.  Well, there's a lot of heat but no light here, and I think what you can do very quickly is transplant microglia into these animals and see if they will sweep through and scavenge up.  That could be a therapy.
		DR. KORDOWER:  John, if your clinical trial with the vaccine shows no clinical improvement and then when patients eventually come to post, you find that the vaccine did clear the beta amyloid, would that be enough data to suggest that we should go to non-beta amyloid models and abandon the beta amyloid models?
		DR. TROJANOWSKI:  I don't think you have to wait that long actually.  I think if they declined and the A-beta levels in CSF or blood -- I don't know that I believe this completely, but Steve Younkin said plasma levels of A-beta can monitor -- you're shaking your head the way I do, but I believe that the CSF. 
		So, just so that everyone knows, the best markers for Alzheimer's disease compared to normal controls -- it gets murky when you get into the other unusual, less common neurodegenerative diseases -- is diminished A-beta levels and elevated tau levels.  So, diminished A-beta is thought to be, because it's all retained in the brain for plaque formation, and tau goes up as neurons die and externalize or release their tau and it goes into the CSF.   So, if you followed patients and showed that the A-beta CSF levels rise to the normal level and they're still getting demented -- you would still want to do postmortem follow-up of course -- but that would --
		DR. KORDOWER:  Would that scenario be enough to abandon A-beta models?
		DR. TROJANOWSKI:  That's a provocative question.  I think it's maybe not meant to be provocative.  Many people tease Dale Schenk.  But, no, I think animals and people still are different in many ways and you'd want to look very, very closely I think and extensively at the A-beta levels and so forth to make sure you weren't throwing something out.
		DR. DRACHMAN:  No way.  You may remove a subdural hematoma from an elderly individual, remove the pathology and watch that person go downhill.  Now, that says that you've done some primary damage.  Neurons will not recover.  Then you've fallen below a threshold and normal aging will then cause further dementia.  So, even though I'm not a huge fan of giving beta amyloid -- I'm not sure that I believe that it will do a great deal of good -- the failure of that to reverse certain symptoms at a certain point may not be the proof that that's wrong. 
		DR. TROJANOWSKI:  There's a large Columbian kindred with FAD mutations, APP.  You know like clockwork when they're going to get demented. 
		There is this condition called mild cognitive impairments.  Individuals have a measurable decrement in cognition that does not equal dementia, and they convert from that state to Alzheimer's disease at a rate of 15 percent a year.  So, in five years you would know.
		DR. SIEGEL:  Just as an aside, I would hope that the measure of whether that therapy or other therapies in that or most other diseases worked clinically would not be observation as to whether patients continued to decline, but ultimately a comparison of two randomized groups, one treated and one not in a controlled manner.  And the predictability of decline in Alzheimer's and many other diseases -- there's so much variability that -- if what you really meant was if they continued to decline, that would be a failure --
		DR. TROJANOWSKI:  Relatively controlled.  Sure.  The clinical trial that's underway -- and I'm not involved.  The vaccine is vehicle and A-beta peptide.  They'll do imaging.
		DR. SALOMON:  So, I would summarize what I got out of this specifically with respect to the things on the table would be that even though these diseases like Alzheimer's are devastating diseases and the implications for this really path-breaking research is obvious, the complexity of these diseases, as they then translate into the practical realities of setting up an animal model where -- you picked looking at the tau, but that doesn't change the contribution of the fibrillogenesis that's occurring extracellularly.  Yet, to construct a true animal model of this, perhaps we're going to have to create them both and then cross breed the animals.  All of these are really amazingly daunting scientific tasks.  Just what you've accomplished is several years of work, as we both know. 
		So, it's just an interesting and sobering thing.  We have to be very careful then in the discussions tomorrow that if we take a position like, yes, you need this animal model and you've got to demonstrate specificity -- I think that was some of the concerns you were having -- and we get really into that, it may sound really good.  But then we've got to deal with the realities, what it really is like setting up these sort of animal models and doing studies in them.  
		A really interesting thing to spend the night thinking about is that there may be certain diseases that, if you feel compelled enough, based on whatever line of reasoning you're going to take from your lab, there may not be an animal model.  Or you may have to construct two or three animal models and pick bits of them in order to generate a rationale rather than this simplistic concept that you're going to have an animal model -- I guess, Rusty, you called it an homologous animal model.  I think that really comes out very clear here in these discussions.
		The next person who's going to speak is Evan Snyder.  He's from Harvard.  The migration and integration of transplanted stem cells within the recipient nervous system. 
		DR. SNYDER:  I was asked to talk about migration of neural stem cells.  I think it's very interesting that this is what I was asked to talk about because this is both probably one of the appeals of stem cells, at least neural stem cells, but also probably a nightmare for the FDA because this is exactly what they don't want is cells moving away in transplantation paradigms from their point of administration. 
		But to illustrate that what we who have been working in various models of stem cell-like biology -- and admittedly, we're all just working on models of this kind of biology -- have illustrated I think -- and I'm going to try to illustrate -- taps into what the brain may be doing anyway with its own endogenous supply of progenitors or precursors or stem cells, whatever we're going to choose to call it.  
		So, I want to start off talking about a non-transplantation paradigm, and that's illustrated over here by this well-known migratory pathway that Steve and Rusty and a number of the others have already spoken about where throughout life, cells in the subventricular zone are born and follow this stereotypical migratory pathway from their birth out over here into the olfactory bulb where they become neurons.
		Well, we've wondered what would happen to this stereotypical developmental migratory pattern if injury was imposed up here in the opposite direction.  The model that we decided to us what we had been doing in the lab because it actually emulated for the brain about as close as we thought we were going to be able to come to what hematologists can come to in terms of ablating the bone marrow.  Well, you can't really do that with the brain, we figured, but we can come fairly close.  
		If we actually take this particular model, which is actually, I should say, a model of a real pediatric disease -- it's hypoxic ischemic encephalopathy, which is a major cause of mental retardation, cerebral palsy, epilepsy in the pediatric population.  As the only pediatrician I think on the panel, I feel I need to be an advocate for the pediatric group. 
		Anyway, the way this model works -- and it's a very devastating model -- is you take about a week-old mouse, you ligate the common carotid artery, expose the animal to hypoxia, and you blow away a huge area of the hemisphere on this particular side, leaving this side as a pretty good, intact control.
		Well, Krucken Park in the lab wondered what would happen if he took an animal like that and at the exact same time as he imposed this kind of devastating injury, he started pulsing these animals with two markers of cells that would be proliferative at the time of this injury.  One way is to pulse the animals with BrdU.  Another way is to inject a retroviral vector into the ventricles which would then label subventricular zone cells going through S phase at the time of the injury.  The retrovirus would encode lacZ and you could follow that.  So, you could follow these cells either by their BrdU immunoreactivity or by their lacZ immunoreactivity, and the results are basically the same. 
		Let me just lead you through this very quickly.  Obviously, here's a parasaggital section of the animal.  These sections over here, right through here through the subventricular zone, are shown in coronal sections over here, and sections through the olfactory bulb are shown in coronal sections over here.  This side over here is the intact side.  This side over here is the infarcted side.
		If one looks at the cells that were labeled at the time of the injury on the intact side, they're actually not here in the subventricular zone.  They have appropriately moved out here into the olfactory bulb.  They've followed this migratory pathway as you might expect.  
		However, in the exact same animal, if you look at its opposite side, at the side ipsilateral to the injury, both here and here, it's very dramatically different.  The cells are not out here.  In fact, they're back over here around the subventricular zone and, as you can see over here, are starting migrate, or at least we think, they're starting to at least lean into the infarction cavity.  If you actually look to see what is going on in these non-neuronogenic regions, for instance, in the cortex, in the striatum, in the hippocampus, and you double label these lacZ positive cells for the mature neuronal marker NeuN, as you can see in this double labeling, they actually seem to have shifted and move up to this non-neuronogenic area giving rise to neurons where one would not expect. 
		Now, obviously, this is probably not enough to cure the animal.  However, it does suggest that there's a developmental mechanism of shifted and altered migration to areas of injury.  Here is a lacZ positive cell, double-stained for NeuN.  If you do double staining for c-fos, some of these neurons appear to at least be active in concert with the other cells, the other neurons in those particular regions. 
		So, this is my stem cell slide because I think that some of these plasticity is actually programmed, or at least the stem cell or what we'd like to call the stem cell is the repository of some of this plasticity.  The reason I'm using this is because this way I don't have to define what my vanilla ice cream is.  You can fill in your blank.  I don't have to even say what they're going to give rise to.  But the idea of some immature cell that's in small abundance that can be pulled out and expanded by the means that you wish to choose, by epigenetic means or genetic means, suggests that perhaps we can pull out and harness this kind of biology. 
		Now, one of the issues we have to figure out is how are we going to expand this cellular population.  We've heard about a number of different ways, and it's an open issue.  Are we going to use epigenetic growth stimulatory signals?  Are we going to try to intervene right in cell cycle?  Ideally we'd love some technique that either is constituitively or self-regulated in terms of propagating the cells or at least one that we can control.  We've used various models that have taught us a lot about stem cell biology that take advantage of a lot of these. 
		One of the early models that we used was actually a clone that was propagated or augmented by a mutated Myc, which was actually constituitively self-regulated.  In other words, it simply kept the cells propagating in a dish, but in the brain was constituitively and automatically self-regulated.  But it taught us a lot about stem cell biology or what we should expect.  
		I'll be initially talking a lot about that particular clone that seems to at least follow the operational definition of a stem cell.  It has also taught us a lot about what we should be looking for if we want to start anew looking at human stem cells or human neural stem cells.
		Now, what's interesting is that the biology at least of the neural stem cells seems to emulate a lot of the biology of stem cells from other organ systems.  This is some collaborative work that Richard Mulligan and Lou Kunkel at our lab are doing in which we actually will take these operationally defined clones of stem-like cells and put them through a FACS sorter looking for what's called the side population, what you've heard about before.  It's simply a population, actually defined over here, in which Hoechst red and blue dye are excluded.  It's a very small population.  It's a population to which hematopoietic cells seem to migrate to that reconstitute bone marrow.  Muscle stem cells seem to migrate there. 
		Well, it's kind of interesting that cells that fulfill the operational definition of a stem cell -- and you've heard a lot about that before -- actually seem to engorge that particular area and actually seem to amplify a population that even from primary brain seems to migrate there, or at least segregate there.  So, it actually engorges and amplifies a population in small abundance that segregates to this particular region. 
		Now, a few points can be brought out here.  First of all, it's interesting that this clone of mouse neural stem cells segregates to this area in exactly the same way as cells from the human nervous system that fulfill an operational definition of a stem cell also migrate to that particular area and augment it. 
		One can also see that if one actually buys that maybe this does recognize stem-like cells, it's certainly a small population and one will need to figure out a way to augment that, to expand it.  Hematologists, obviously, depend on expanding this population in vivo.  We in the nervous system are probably going to have to figure out a way to expand that ex vivo if we're envisioning transplantation paradigms. 
		Well, what do I mean by the operational definition of a stem cell?  Well, it should be self-renewing and we can discuss what that's about.  
		It certainly should be multipotent in a dish and also in vivo.  One way of showing that the cells are multipotent is to take this particular population that encodes lacZ and put it into a developing mouse embryo, let the animal go to adulthood, and see that the cells at maturity have integrated throughout the neural axis, and we know will respond to the particular temporal and anatomical cues present at that particular time.  
		They actually go to both sides, but I'm showing this particular section where they're segregated to one side to bring out the point that except for the fact that these areas are chimeric, the cytoarchitecture seems to be identical.  In other words, they seem to integrate in a fairly seamless fashion and what one sees there are the cells that you would expect based on your fundamental knowledge of neurogenesis in the developing nervous system.  So, if one does a transplant to allow cells to get into the embryonic cortex, they'll give rise to pyramidal neurons.  Here's a nonpyramidal neuron putting out a process onto a host pyramidal neuron.  
		Outside of that window of neuronogenesis, the exact same clones and the exact same region will no longer in a normal adult cortex give rise to these type of neurons, but will give rise to oligodendrocytes, for instance, or to astrocytes putting out foot processes onto blood vessels.
		Now, one of the operational definitions of a stem cell is this ability to do serial grafting.  It's one that hematologists do.  Well, we've tried to at least emulate that, also to show that the environment seems to mold these cells.  One can do, for instance, an embryonic transplant and particularly go to the forebrain, pull out cells, pull out a section, a section like that with engrafted cells from the adult in the forebrain, can pull them out, propagate them again, and then reimplant them at another time in another region, for instance, now in the newborn cerebellum, and in fact, find that those cells that have been retrieved now will give rise to granule neurons in the internal granular layer.  So, again, part of the operational definition of a stem cell to at least suggest that maybe we have a model that can teach us a little bit.
		Another major question is -- and this is unanswered I think satisfactorily in our field.  This experiment that I'm mentioning is not going to answer it either, but are these cells in any way functioning or are they simply there anatomically or is there any hope that they can start integrating into the circuitry.  Well, we really don't know because the experiments haven't really been done well.
		But one way of starting to get to that is to go to systems that -- at least this is one particular system I find very appealing, and that's a collaborative study that we've done with Bill Schwartz at U Mass who is a circadian rhythm biologist who focuses on this particular area, the suprachiasmatic nucleus.  Now, this is very appealing to us because these are cells, circadian rhythm cells, that need to respond to stimuli administered many way stations away, light to the retina, for example.  Yet, the cells here need to work in a concerted effort back here in the suprachiasmatic nucleus of the hypothalamus.
		Well, we know that these donor clone of neural stem cells can integrate into that region if you do a transplant at the right time embryonically.  These are these Xgal positive cells intermixed there. 
		Now, Bill Field uses c-fos to talk about -- it's somewhat nonspecific, but it talked about appropriate activations of the cells in response to a particular stimuli.  So, this particular area is stained for c-fos, so it will be a brown nucleus.  And then in an engrafted animal -- and you can see the cells integrated in there.  Now the lights are turned on, so to speak, and there's an up-regulation of c-fos activity.  You can see a burst of c‑fos activity.  And then a subpopulation of the donor cells over there.  It's a subpopulation that also expresses vasopressin and a lot of the other appropriate cytochemical markers.  A subpopulation of those cells also starts expressing c-fos appropriately and in the right percentage as the host cells.  
		If you don't buy the light microscopic view, this is confocal looking for c-fos among the host cells and then double staining for beta-gal and c-fos activity.  Again, the population of the donor cells in response to light, many synaptic connections away, will up-regulate c‑fos in the appropriate percentage.  
		So, what does this say?  Well, who knows what it says?  
		What is says, however, is that at least we have a model that can teach us a little bit about what stem cell biology would be if we buy certain operational definitions.
		What it also says is, in going through this, that whatever we're going to call them, there are cells that we can pull out, that we can propagate that can go into a brain, at least in a transplantation paradigm, might emulate proper developmental mechanisms, but you can grow them in a dish, put them back, and maybe have them become appropriate neural cells.  Certainly they can bring in foreign genes with them that, even as was brought up before, might be nothing more than glorified pumps, but this is a kind of pump that may be useful.  They can bring in these genes and they can do this in not only a local fashion, but in a widespread manner.  That's actually important, at least for a lot of the diseases that I as a pediatrician deal with.  
		They may also -- and this is a big "may" -- be able to repopulate.  So, just the idea of their being able to express therapeutic things throughout the brain we first tested, as was mentioned before, just by looking at a mouse mutant model of one of the lysosomal storage diseases.  We did this in collaboration with John Wolf down at Penn.  This particular mouse mutant was injected with these cells into the ventricles, allowing them access to the subventricular zone.  The cells distributed themselves all throughout the brain.  So, there was extensive migration of these cells, which is exactly what a model like this needs.  It needs the distribution of a missing gene all throughout the brain because it's missing.  This distribution of cells corresponded to the distribution of therapeutic enzymes all throughout the brain, almost like a bone marrow transplant to the brain.  Normally this mutant would have 0 levels, and that resulted in the elimination of this lysosomal storage, as you can see, in an untransplanted control animal in the cortex, but there the lysosomal storage in the host neurons and glia was virtually eliminated. 
		Well, this kind of paradigm of taking advantage of this migration can be applied to a lot of diseases where you need to replace not only genes throughout the brain, but maybe even replace cells.  So, some of the mouse mutants that require that are the white matter mutants that need dysfunctional oligodendrocytes replaced.  So, for instance, we're looking at a lot of these mutants that are white matter degeneration mutants just to test that.  
		So, we've looked at the twitcher mouse, which is a model of the leukodystrophies, the shiverer mouse, which is a model of myelin basic protein degeneration.  In those models we get that same distribution of cells.  
		In a twitcher model, for example, they will differentiate into oligodendrocytes that start myelinating host axons, and under EM you can appreciate this a little bit better.  Here's a donor-derived oligodendrocyte surrounded by 30 to 50 myelinated host axons.  The kind of myelin that one sees is actually fairly respectable.  As you can see over here, it has an appropriate thickness and compaction.  The Xgal precipitate indicates that it's donor-derived. 
		In the shiverer mouse, this kind of distribution of cells can even result in a decrement in some of the symptomatic shiver.
		So, taking advantage of this migratory potential can be used for certain diseases that need this perhaps.  So, one can use this perhaps to distribute cells and enzymes.  
		But there's another component to migration that I think is actually very appealing and very intriguing.  That's the idea that maybe these cells can migrate to areas of pathology, somewhat the way the initial experiments I mentioned illustrated that maybe the brain tries to also shift stereotypical migratory patterns to deposit cells where it at least perceives maybe they're needed.  Whether it does it sufficiently is another argument, but can we ride this crest?
		What I wanted to do was just very briefly talk about three different examples that somewhat demand alterations in migration of these cells.  That would be infarcts.  I'll get back to exactly the model with which I started, which is a very dramatic acute injury of multiple cell types, of selective cell death.  What I'm going to look at is motor neuron degeneration where just that cell dies but the other cells are pretty much intact, and then tumor, which is a slow, indolent kind of pathology, which in and of itself is also a very migratory pathology.
		So, just to remind you, this is the kind of injury that we're going to impose, and we're going to impose it on normal animals that simply have these reporter cells intermixed throughout and see what the cells do.  Krucken Park in the lab has taken animals like that and put them directly into the infarcted area or put them into the opposite side.  Some of you have seen these data before.
		But what I want to bring out is this migratory component.  So, the injury is imposed over there.  Cells are placed over here.  It doesn't really matter whether they're implanted first and then the injury is imposed or whether you impose the injury and then you do the transplantation of these reporter cells.  The phenomenon is the same.  In an intact animal, the cells are pretty much restricted to this particular side of the brain.  
		However, a different story is witnessed when there's damage done to this particular side.  Now, one sees that cells implanted over here will migrate across commissures or the corpus callosum, throughout the hemisphere, and move from their area of initial engraftment to home in, so to speak, into this infarcted or to this very damaged area.  The kind of cells that you would see there at higher power are what have the kind of profile that we recognize as being somewhat suggestive of a migratory cell with the leading process moving in the area of movement and then a cell, for instance, here on the corpus callosum, moving over here.  When it actually does get to the area of infarction, these cells under this EM will be migrating right into this very necrotic area as if there is a trophic response there.
		Interestingly, if the cells are placed directly into that area, they never move back in the opposite direction.  They always stay right there, which also suggests what one might want to ask.  But what happens when the cells are directly placed in there?
		Well, they seem to engraft very robustly into this very infarcted area.  The interesting thing would be, well, what happens to these cells?  They change their migration.  Do they change their differentiation?  Now, this is a time -- and if we look at the cortex -- neurons should not be born anymore.  Oligodendrocytes, maybe a low level of oligodendroglial genesis, but yet, these are the two cell types that take the biggest hit in this kind of injury.  Under EM, when we ask this question, we see something that electromicroscopists would like to say is a fortuitous EM section, one can see two donor-derived oligodendrocytes sitting next to a big, large donor-derived pyramidal neuron.  And the apical dendrite of this pyramidal neuron shown at higher power here -- here you can see the Xgal precipitate in there -- is receiving synaptic input from the host.  
		If one does immunocytochemistry not only to confirm this but to help with quantification, this top panel is anti-beta-gal antibody to recognize these donor reporter cells.  This is anti-cell type markers to clarify what they are.  This is under the merged image.  One, in fact, does see new neurons there, new oligodendrocytes, new neurons as much as a millimeter away on the side of the injury. 
		If one does the mathematics -- and I'm putting this up more to make a qualitative point than a quantitative point -- on the intact side, as you might expect, one sees no neurons at all, low level of oligodendrocytes.  But now there seems to be a shift, and now there seems to be new neuronogenesis in that particular area.  So, that as a developmental biologist is an interesting observation, and it suggests that maybe some of what we saw with the endogenous progenitors can be augmented with these exogenous progenitors or cells. 
		Now, you can look at this and say, well, 5 percent.  What's the big deal there?  Well, 5 percent of this large amount of cells may translate into tens of thousands of neurons, and we really don't know how much it takes to reconstruct circuitry.
		However, the fact that you get this robust lacZ expression suggests that perhaps one could actually engineer these cells to over-express a trophic factor.  Maybe if they're already expressing a trophic factor, perhaps they can over-express it and then respond to it.  
		In collaboration with Isack Fisher, these cells have been actually engineered to over-express NT-3.  It was first established that the cells actually have a TRK C receptor that appropriately becomes phosphorylated, that then signals to a MAP kinase intracellular signaling, suggesting that the cells should be able to respond in an autocrine or paracrine fashion to this NT-3.  In fact, they do give rise to many neurons.  
		Then one can do the exact same transplant, get the exact same kind of engraftment, find that these now engineered cells, these beta-gal positive cells, continue now to express NT-3 in vivo.  If one looks to see what the cells become, a beta-gal positive cell double-stained for MAP-2.  
		But the purpose of this slide is mostly to bring out the point that red fluorescence and green fluorescence, when superimposed will give you yellow or orange fluorescence because on the next slide, looking at the penumbra of the infarction where beta-gal is recognized in red, NeuN.  The mature neuronal marker is recognized in green.  Certainly one can see plain green cells and plain red cells, but now you see a fair number of orange and yellow cells, recognizing new neurons added to this penumbra as a result of this manipulation.  In the MAP, one can now see over 80 percent neurons in this penumbral area.
		But it also suggests that maybe if we understand some of the trophic factors we can use this perhaps to push cells down lineages that they're already pursuing but that are desirable to us.  Also, it's an interesting example of doing gene therapy and cell replacement with the same cells simultaneously.  The neurons that one sees are exactly what you would expect, donor-derived neurons that are cholinergic.  One sees glutamatergic and GABAergic. 
		So, this is a very dramatic kind of infarction, acute injury of many different cell types. 
		What about a selective loss of a particular kind of neurons?  This particular model is a well-known model in the ALS field in which if one first takes a newborn mouse, ligates its sciatic nerve, and then by the time the animal reaches maturity, these motor neurons have degenerated by apoptotic mechanisms.  
		Now, again, John Flax and Chou Xan Lo and Ted Tang in the lab have done a number of different kinds of manipulations here, but I want to show you one that brings out this idea of migration towards areas of injury.
		They would take the cells and put it not in the area where the ventral motor neurons have degenerated, but actually up here in the dorsal horn.  This is immediately after transplant, just to show that they've hit their target.  In an intact spinal cord, the cells certainly distribute themselves and engraft but pretty much restricted to the dorsal part of the cord in a normal animal.
		However, in a mature animal in which the motor neurons have been made to degenerate, particularly if you go towards the point of active degeneration of these cells, now something very different they've observed.  That is that the cells implanted up here have now migrated from the dorsal part of the cord down to the ventral part of the cord.  They've done it at multiple levels throughout the cord of this particular animal.  So, there not only is migration in this dorsal to ventral axis, but because these are semi-serial sections of the cord of one animal, they've done it also for about a millimeter in this rostral/caudal dimension which is the extent of motor neuron degeneration here.  
		If you look at the kinds of cells that are there at higher power, the kind of cells that you see are these large Xgal positive cells that certainly have the morphology reminiscent of a motor neuron, similar to one of the residual host motor neurons.
		They stain for NeuN, indicating that these donor-derived cells immunocytochemically are becoming neurons.  Here's a donor-derived NeuN positive cell sitting next to a residual host NeuN positive neuron.  
		Here's a MAP-2 positive cell double-stained for beta-gal sending its process out of the plane of focus.  So, it looks as if they're moving there and becoming neurons, but it looks as if they may also be trying to become spinal motor neurons.  If one does an in situ hybridization against ARIA, a neuregulin, called acetylcholine receptor inducing activity necessary for characterizing or determining that a cell is a spinal motor neuron, if you do an in situ on this section and engraft an axotomized section like that, you can see hybridization in this particular axotomized engrafted animal and a distribution that looks very similar to that of the engrafted cells and pretty respectable compared to the animal's own intact control, very different than an axotomized untransplanted animal.  The cells that seem to be hybridizing to the ARIA probe are these big beta-gal positive cells heavily overlaid by silver grains ARIA hybridization at a level much higher than background.  
		Interestingly, the beta-gal positive cells, which have stayed as small cells up in the dorsal horn, don't hybridize better than background.  And these kind of cells express ChAT.  They express ILa-2, suggesting that the cells have migrated there and are now trying to become perhaps motor neurons or at least responding to signals that may recapitulate some developmental signals. 
		It was mentioned that perhaps some of the efficacy of the cells is not only going to be whether they can replace cells or not.  
		These are the exact same cells -- actually this is a spinal cord slice done in collaboration with Jeff Rothstein.  The cells actually are placed out here of the spinal cord slice and one can see that the neurofilament fibers of the motor neurons are actually homing in right on some factor, almost looking as if they're making a ventral root, seeming to be expressed by this particular factor.
		I want to end with one last model and that's tumors, which is an indolent kind of slow growth that requires migration.  This is done by Karen Abuti in the lab.  She implanted cells directly into the adult cortex.  Now, one of the problems of treating brain tumors is the fact that they migrate and they insinuate into normal tissue.  Karen wondered what would happen if she first took the cells and implanted them directly into the tumor. 
		Here the tumor is seen in red.  Here the tumor is expressed to express green fluorescent protein so that it will fluoresce green.  The red are neural stem cells implanted in there for lacZ.  They're Xgal blue here. 
		The cells were implanted directly into the tumor and within 48 hours they migrate out to the very interface between tumor and normal tissue and come to a halt right there except, as you can see over here at higher power, where a tumor cell is infiltrating normal tissue.  There a neural stem cell seems to perhaps jump on top of it and move in direct juxtaposition to the cell, continuing to express its lacZ gene.  
		This is simply to show immunocytochemically that the exact same thing is happening, the cell migrating out, even in very virulent tumors, as there is rapid migration of tumor cells into the tissue, being followed by neural stem cells. 
		Now, how robust is this migratory capacity?  One would not necessarily want to treat tumors in this particular fashion, but Karen established the tumor over here but implanted the neural stem cells over here, and then after about a week followed to see what those cells were doing.  She could see, looking at the corpus callosum, that there were again cells moving from this area of implantation -- this cell shown at higher power here, this cell shown at higher power here -- classic migratory profile moving towards the tumor cell.  Then when she actually looked at the tumor, she could see that it was filled with blue cells.  The only source had to be from this particular implantation of neural stem cells over there.
		Now, this is the last point that I want to bring out.  This allows me to transition to whether this biology is preserved in human neural stem cells.  This is a human neural stem cell grown in growth factors that has been pulled out of a human fetus.  It's now an established clone that seems to emulate many of the qualities of mouse neural stem cells. 
		Well, Karen took a cell like that and did the exact same paradigm that I described before.  Here's the edge of a tumor over here.  Here's actually the edge of a human glioblastoma.  You can see it's filled with brown nuclei.  These are human neural stem cells recognized by a human-specific nuclear antibody, and it's completely engorging the tumor cell.  The interesting thing is that the cells were implanted over here on the opposite side and they migrated into there.
		The other interesting aspect is that this particular clone of human neural stem cells is engineered now to over-express a gene that would allow us to test bioactivity.  It's now been engineered to express the gene cytosine deaminase, which most of you know is a gene which has been used in prodrug paradigms against brain tumors.  If one exposes this benign drug, 5-FC, to cytosine deaminase, it converts it to the oncolytic drug 5-FU which then will kill tumor cells.  
		The neural stem cells over-expressing this particular gene now, first in a dish, are exposed to 5-FU to see whether this gene is bioactive.  Here they're intermixed with tumor cells.  One now overlays with 5-FC and the cells were now converted to the oncolytic gene product 5-FU and eliminates most of the tumor cells, as quantitated over here. 
		If one now takes this and does the exact same paradigm, taking the cells and putting it into the established tumor, and then does exactly the same thing, gives the animal 5-FU triggering the action, one can see in this image analysis of camera lucida, here is the tumor, our profiles of the tumor for image analysis, untreated, large, big like that.  If one now triggers this reaction mediated by these human neural stem cells, one can see a dramatic decrement in the size of the tumor, suggesting that the gene is bioactive, quantitated at an 80 percent decrement in the size of the tumor, suggesting in fact that one does have a bioactive gene delivered by this particular migratory cell.
		I'm going to skip this except simply to say that we see same type of phenomenon in motor neuron degeneration, also in stroke.
		And then simply to end with this last bit to say can this biology be translated to models that are closer to the human condition.  This is some work that Curt Freed in our lab did in which we would take these human neural stem cells and now see whether at least their biology will be recognized by a brain that's closer to that of a human. 
		So, we've gone actually to fetal monkeys.  This is a lot earlier than we've done it, but under ultrasonic guidance, taken these human neural stem cells, placed them into the ventricles of fetal monkeys -- except the animal is a lot older at this particular point -- let the animal develop, particularly the cortex.  The cells are labeled by anti-BrdU, so one will see a black nucleus.  The cells will migrate out from the ventricular zone, and one can see, recognizing the cells, migrating up radial glial fibers quite appropriately.  Those that jump off at the lower lamina of the cortex, where neurogenesis has stopped, will then differentiate into glial cells very appropriately.  Those that continue up this elevator, so to speak, and jump off at the higher lamina where neurogenesis is ongoing will now become neurons, intermixed right in the appropriate lamina with the monkey's own cortical neurons.  And that these are neurons is indicated by double staining for NeuN, doubling staining with BrdU for calbindin, but will also give rise to oligodendrocytes and to astrocytes, suggesting that they're responding to the appropriate developmental cues.
		Interestingly, a subpopulation that does not migrate out to the cortex will hang as a quiescent population around the ventricle, conceivably to become this population that later on in life may be activated.  So, the hope would be that human neural stem cells, because they're reflecting a biology, may be able to also recapitulate this biology in the human brain perhaps as they're doing it in non-human primate brain.  
		I think this is probably the touchstone of the entire meeting that I think we have to question all of our previous precepts, and that life's answers being questioned over here, but I think by the end of the two-day session, we're going to find out that all of our answers that we thought we had are certainly being questioned as we explore more of the stem cell biology.
		Sorry to have gone over.
		(Applause.)
		DR. SALOMON:  I think, just because of the time constraints here, what I'd like to do is go on to the last talk of the day, and then if there's some discussion, we can have that after both the talks.  So, I invite John McDonald to come and talk about animal models of spinal cord injury. 
		Comments?  Go ahead. 
		DR. DRACHMAN:  Evan, you're at Children's Hospital.  So is Judah Folkman.  You surely must have looked at VEGF, angiopoietin, basic FGF, and so forth to see whether that's what's leading your cells around.  Did you?
		DR. SNYDER:  We're actually in the midst of looking at all of that.  As you would imagine, we're doing a lot of chip technology.
		The thing that I think is going to be clear is that I think is going to be clear is that it's not going to be one factor.  It's probably going to be a cocktail of factors not only some diffusible factors, not only the factors that you mentioned, but also changes in extracellular matrix.  There's probably also going to be alterations in not only attractants but also disinhibition of repellants and things of that sort.  
		So, yes, we're starting to look at that.  We're trying to go at it, not in a fishing expedition kind of way, though you could say, well, that's what chip technology is.  But those are certainly candidates.
		One of the interesting things that we're seeing that's a difference between acute and chronic injury, which is where we see a lot of these phenomena going on, is alterations in various cytokine receptors, alterations in LIF receptors, various G proteins.  So, some of the candidates that seem to be very important in fundamental stem cell biology also seem to be changed in these milieu where we see these alterations in migration.
		DR. SALOMON:  John, you're on.
		DR. McDONALD:  So, what I'm going to focus on today is really embryonic stem cells.  What I'd like to do is go over some of the models in spinal cord injury and then use an example of a couple different models as an approach to potential clinical trials. 
		So, we'll go through a single example and really define what the problem is, what's doable, what's doable in regeneration, what are we modeling.  Appropriate age of the models is incredibly important.  What models are available?  What are ES cells?  It's still very early days and what to do to move forward. 
		So, this is the real problem with spinal cord injury.  You get breaks in the bony spine, compress the soft spinal cord.  The most common cause is motor vehicle accidents.  And you're left with an injury oftentimes that looks like this where you get a cyst in the middle of the cord.  In fact, here we're looking at a cross section of that same cord.  In between the dotted line and the closed circle line is the spared tissue.  So, the spinal cord is kind of built opposite of the brain, where the white matter containing the long tracks in the brain and going to the brain are on the outside.  So, death typically occurs in the middle.  You are always left with a small rim or donut of intact connections.  
		The key thing is that this is a person that has run triathelons.  So, you don't need to cure the spinal cord to regain function.  You just need to get minimal anatomic improvement.  The estimates are that you need less than 10 percent of the functional connections in order to walk.
		This is really to point out that the choices are really immense.  When you look at the choices between time of treatment after the injury, what cell type do you use and what disease to choose.  So, in this case we're going to choose spinal cord injury, a subchronic treatment at 9 days using ES cells at a very specific neuroprogenitor stage and focus on myelination.
		So, the thing is the critical balance in moving forward with potential preclinical studies or any studies in this.  I think that you'll see the studies that we look at have many of these features. 
		Of course, it's important that there's very few alternative treatments, making it more reasonable to use interventions such as cell transplantation.  Also, the potential risks of the treatment are reduced.  I think as I'll point out, transplanting oligodendrocytes really pose less of a risk than transplantation of neurons.  Neurons have a much higher propensity to do abnormal things such as pain, abnormal transmission, seizures. 
		Doability of treatment, of course, is very important.  And myelination I think fits this.  That is, after an injury and even in demyelination, many of the circuits remain but are dysfunctional, and you simply need to remyelinate.  That's going to be an easier task than recapitulate the circuits.
		And then severity of disease.  I think Evan pointed this out very well, that it's going to be much harder to fix a hole in the spinal cord than it is to replace single, individual lost cells.
		Then proof of principle.  What do we know in preclinical studies as far as mechanisms, anatomical and behavioral effects and the appropriateness of those animal models?
		Then most importantly, we need to have measurable and interpretable endpoints for a clinical trial, both anatomical behavior and physiologic.
		So, far as doability, of course, preventing the injury is much easier than regeneration.  In the spinal cord in animals, there are multiple new treatments that have been designed to be given immediately after the injury that are able to prevent some of the secondary injury.  Despite substantial progress in the last 10 years on regeneration, overall it's still very limited.  In humans, we only have one acute medical treatment, methylprednisolone and no regenerative treatments.
		So, if you really look at level of difficulty ‑‑ this is my personal view of this as far as regeneration strategies.  I think if you look at some of the easiest things, cell birth, such as glial cell birth, in many cases we want to limit this, in the case of astrocytosis.  Remyelination and birth of oligodendrocytes are just remyelination of surviving oligodendrocytes.  Axonal sprouting is doable, particularly in the local fashion.  Cellular expression of molecules through gene expression is becoming very doable.  Neuronal cell birth, as Rusty pointed out, is becoming something that we're just at the beginning of getting handles on.  
		The most difficult things, of course, are axonal sprouting, particularly over long distances, with appropriate reconnectivity.
		So, in spinal cord injury, just to put things in perspective, here are the different events.  Trauma.  Of course, you want to reduce any compression that's occurring immediately, then limit the secondary injury, limit secondary complications, and that's the main focus of rehabilitation, and then of course, interact with mobility and environment, which again is ergonomics of rehabilitation.  Then what we want to focus on today is just replacing lost cells, myelin and axonal connections.
		So, the first important question is what should we be modeling in spinal cord injury.  There are just a zillion different choices.  It really depends on what condition you want to model because there are many different causes of spinal cord injury.  The most common is trauma which is mixed from vehicular accidents, violence, falls, sports.  But then there's a myriad number of medical causes including demyelinating disorders, orthopedic tumors, infectious degenerative disorders.  So, we have to decide on which one of those we want to treat, some of which potentially are going to be easier than others.  But in most cases, most of those disorders are a one-time hit and it's not a recurrent event.
		Now, this is just a schematic that I put together today just to really look at the age appropriateness of models.  I think Evan pointed this out incredibly well, that if you transplant progenitor cells earlier in development, they really migrate a much greater distance and probably have a better chance of survival as opposed to the same transplantation in the adult.  Then as Evan also showed, injury also supports additional length of migration.  So, it's very important to assess the appropriate age for the model that you want to look at.
		Then most importantly, it's a constantly changing problem.  For example, if you want to look at spinal cord trauma, it's a very different problem if you want to have an intervention that's to be instituted immediately after the injury blocking, say, for example, necrosis, or one a little bit later blocking, for example, programmed cell death, or a regenerative type event.  Even the regenerative events are going to be dramatically different depending on the timing, whether it's 1 week, 2, weeks, 2 months.  So, the problem you're facing is constantly changing.
		So, what models are out there right now that people are focusing on?  I think it's useful to divide them up into these mixed cell death models where you get death of everything, basically infarcts, neuronal, glial, and axonal injury, and then more selective cell loss.  
		So, the weight drop contusion model is probably one of the best held models for traumatic spinal cord injury.  You recapitulate the syrinx that occurs in the middle of the spinal cord and many of the aspects of the physiology as well.  
		Compression injury is a bit of an older type model, but it's still being use.
		Ischemic injury, replicating what happens with triple A surgeries. 
		And then transections, both partial, complete, and suction methods.  
		A lot of these have come out mainly because these are very difficult models to care for the animals.
		But then the more selective cell loss injury models, such as just focal myelin loss, like Evan pointed out with the myelin mutants, chemical demyelination, which you heard about earlier today, and then focal neuronal loss, including neuronal mutants and neurotoxins.  These can be subdivided into axon sparing and non-axon sparing.  That really changes the model and what you want to accomplish.
		So, what is a stem cell or an embryonic stem cell?  Well, basically it's the earliest form of stem cell capable of making, in theory, every subsequent stem cell.  It's self-renewing.  It's truly pluripotent.  In most cases for new ES cells, we proved this by producing teratomas in SCID mice with cells from all three germ layers.
		Probably one of the most important aspects of an ES cell is you have the best proof of principle that it's genetically normal.  That is, you can produce a chimeric animal, which you really can't do with almost any other stem cell. 
		It's highly genetically manipulable.  Of course, this is one of the reasons for the transgenic error that's occurred, and this is going to have a lot of advantages.  I think in the long run embryonic stem cells are going to have most of its input as a tool rather than as a therapy, although a therapy will be important as well.
		And they have the same advantages as other stem cells.
		So, from the public's standpoint, I think these types of videos are very useful.  What is an ES cell?  Well, basically you just take an egg and a sperm and do in vitro fertilization is one of the most common ways it is done.  It can also be done in an animal and then flush the fallopian tubes.  But if you let that grow to a blastocyst stage -- and at this stage, it's still pre-implantation.  If it were in the animal, it would be in the fallopian tube before it got to the uterus.  
		If you split that in half, it's the inner cell mass that you're interested in.  If you take out these cells, these are embryonic stem cells.  More typically it's done through immunodissection.
		This is very easy to take these cells and culture them really indefinitely.  So, it really provides an unlimited source of cells.  You begin the week with 1 of these, you end the week with 256 flasks.  So, already in the last couple of years, there has been over 50 knock-ins and knock-outs in ES cells that are frozen cell lines that you can just obtain from other people around the world. 
		So, let's move on to transplantation.  So, we use a differentiation protocol worked out by David Gottlieb at Washington University called the 4 minus/4 plus stage embryoid body where you take the ES cells and you grow them floating in a dish so they don't stick for the first 4 days, and then expose them in the last 4 days to retinoic acid.  Retinoic acid has been shown to be a very strong stimulus to push them down and commit them to a neuroglial lineage.  
		So, what is one of these 4 minus/4 plus EBs?  Well, if you cut these in half, you can see that there are very few differentiated cells.  The majority are nestin positive or neural-restricted precursors.
		If you take these and culture them at that point, you can show that you can make, of course, neurons, type 1 astrocytes, type 2 astrocytes, and oligodendrocytes.  Really now, applying just basic tissue culture methodologies, you can obtain almost every type of culture.  This is a very typical mixed neuronal/astrocyte culture where you see ganglia of neuronal cell bodies with axons over astrocytes.
		This is the model that we use for transplantation.  It's a contusion model injury where we drop a 10 gram weight over a laminectomy site over the exposed spinal cord.  They have a moderate/severe injury such that when the animal recovers over the next 2 or 3 weeks, it plateaus to the point where it's not able to bear any weight on its hind limbs.  It can move its legs, but not in a coordinated fashion, and it can't place its paws.  So, it's one of the most sensitive points on the BBB Scale, which is an open field assessment of hind limb function following spinal cord injury.
		In this study, this is the experimental time line we used for the transplantation.  We did the contusion injury and then we waited 9 days.  We waited 9 days for two purposes.  One, we wanted to wait till the inflammation went down, and two, we wanted to get out past the period where most cell death occurs.  If you really want to look at regeneration, you should make sure you exclude potentially preventing cell death.  Many regenerative studies are done with co-transplantation.  Then we assessed behavior over this whole time period and then 2 and 5 weeks later looked at the histology.
		In these studies -- these were very early studies -- we used multiple labels because no one label is perfect.  So, the cells were genetically labeled with lacZ or GFP.  We could also use species-specific antibodies.  We transplanted mouse male ES cells into female rats so we could also use Y chromosome in situ hybridization.  It turns out that the mouse-specific antibodies were very effective because you were able to see processes very well.
		We always did triple labeling.  It's not enough just to see lacZ or GFP, but we want to see the appropriate phenotype specificity, as well as a nucleus that's appropriate for that cell type because it's very difficult.  There are so many macrophages that occur after any injury that it impairs the assessment of this. 
		We moved on now to label these cells with paramagnetic markers so that we can follow them with MRI, and there are other cell tracking methods as well. 
		So, if we look 2 weeks after transplantation, this is a longitudinal cord from one of those rats.  This is the formed cyst that occurs within 2 weeks.  This is the middle of the transplant.  We actually transplanted a million cells in 5 microliters right into the forming cyst.  2 weeks later we found that the cells would migrate up to a centimeter down.  In fact, cells would be a centimeter down within 12 hours after transplantation, and they're not doing this by migrating through the tissue.  This syrinx connects with the central canal, and they would walk down the central canal in both directions, and then migrate out across the radial glial.
		We can quantitate this.  I'm not going to go through this in detail, but basically you can demonstrate that there are all three lineages, oligodendrocytes, astrocytes, and neurons, and they have the appropriate phenotype.  NeuN doesn't stain the nucleolus.  It labels the nucleus more than the cytoplasm.  There's a nucleus that fits in that, and that fits in this label for M2 which is a mouse-specific antibody, larger astrocyte, larger nucleus, co-labels with M2, which is a surface label marker.  There's an oligodendrocyte.  APC is a cytoplasmic label, and there's the nucleus that fits in.
		What we were very surprised at, because what we were hoping to do is just see cells survive and then use those cells as genetic vectors, as Evan mentioned, was that we did see a very consistent behavioral reproducible improvement in function.  For those of you who have worked with the BBB Scale, you know that this is not a linear scale, and this 2-point difference is a big functional difference.  2 points down here between 6 and 4 or 6 and 8 means almost nothing.  2 points between 18 and 16 means almost nothing.  But I'll show you what this does.  
		We've repeated this and added an additional control.  We added another control where we looked at adult mouse cortical cells and transplanted them to control for a rat versus mouse immune response.  Certain groups have shown that pro-inflammatory cytokines can improve some anatomical function after injury.  But we saw the consistent same 2-point difference. 
		What I'll show you now are little video segments that are from animals that are exactly these scores, so they're the average scores.  It's not the best or the worst or one group.  It's exactly the average.  You'll see it's a dramatic difference. 
		This is a control animal that just received the control media.  You'll see he walks.  He can't lift his tail.  He can move his hind limbs.  He doesn't bear weight and he can't place his paws.
		This is the average transplant.  You can see he bears weight.  You can see he lifts his butt off the ground and his tail, and he can stand on his hind limbs.  All the animals would do this, so this is a very average response.
		What we found in that study is that the majority of the cells went on to become oligodendrocytes, over 60 percent, and less than 10 percent were neurons.
		In the next phase, we looked at how well can these ES-derived oligodendrocytes myelinate in culture.  This is a culture that's derived from 4 minus/4 plus EBs and were stained for O1 here which is a myelin product.  This is an oligodendrocyte with its multiple branches, and it's overlaid over a phase image.  The white arrows indicate axons, and here this axon continues and then becomes myelinated.  Here's an axon.  It then becomes myelinated.  You can actually follow each branch out.  So, you can see that a single oligodendrocyte will wrap multiple axons or multiple segments of a single axon.
		Now, of course, to prove that this is myelination, you can do standard transmission electron microscopy to demonstrate early wrapping.  
		This is an amazing amount of wrapping.  This is an average amount that we saw in the culture already occurring by 9 days.  So, not only did the cells have to differentiate from a neural progenitor, but they had to form both cell types and interact within 9 days.  So, it's occurring more rapidly than primary derived oligodendrocytes.
		We went on to assess this in a more focal model.  Rather than doing a contusion injury, now we said let's just do a chemical demyelination and irradiate the animals.  So, here you primarily lose astrocytes and oligodendrocytes and some axons, but you spare the majority of axons. 
		So, we did the demyelination and then transplanted 3 days later and then assessed anatomy 10 and 30 days later.  It was a very similar transplantation paradigm, although we've transplanted half as many cells. 
		What this really depicts is that basically the majority of the cells that are transplanted turn into oligodendrocytes, not astrocytes, not neurons.  
		So, what's shown here is a panel stained with M2, which is the anti-mouse antibody.  It just really depicts the transplant site.  These are three different animals.  This is a control that received just vehicle medium, so it's a very good antibody.  This is a small growth of a transplant, a much larger growth of the transplant.  APC labels oligodendrocytes, and at this low level you don't see APC, but you see it mainly in the transplant site.  But interestingly, the GFAP is almost excluded from the transplant site.  It was only on the edge.  
		You can actually see at this early time point, which is 10 days after transplantation, there's actually increased nuclear density.  If you were to look at these same animals 30 days later, they would achieve close to a normal nuclear density.  So, cells would either die or migrate. 
		We can immunolabel the myelin to demonstrate that oligodendrocytes derived from ES cells myelinated.  You can see the immunoprecipitant.  This is 30 days after transplantation.  It's a similar picture, now at a much higher power, showing the immunoprecipitant in the multi layers of myelin and yet next to axons that don't have labels.
		We then went on to develop a pretty simple method for enriching for oligodendrocytes, really following methods developed by McKay and Ian Duncan, but a little bit different.  
		We took the 4 minus/4 plus EBs and ground them up and then made oligospheres and grew them in a specialized, defined media so that we could highly enrich for oligodendrocytes.  This procedure also enriches for neurons.  So, basically if you look at this, now the majority of the cells are beyond nestin positive, and if you culture these cells in oligosphere condition media, you can get cultures that are over 92 percent oligodendrocyte pure.  But there are virtually no astrocytes under these methods.
		If we take those cells and then transplant, like Evan discussed with the shiverer mouse, you can see that they integrate.  This is a CTO-labeled cell that lines up with the other intrafascicular oligodendrocytes.  This is an animal that doesn't express any myelin basic protein.  So, if you use myelin basic protein and see here's the cell body, here's the nucleus that fits in there, and you can see its processes.  It's a transplanted cell.  
		This is a sham transplant and a controlled ES cell transplant.  You would see myelination for a couple millimeters, and at high power, you can actually see individual axons with the myelin wrapping them in the intrafascicular white matter.
		At a much lower power, you can see that the transplant rapidly falls off.  So, we don't see nearly the migration that you see in the immature animals.  You initially see a higher nuclear density and then it decreases over time, in this case, to below normal.
		You can use transmission electron microscopy to demonstrate that there's over 10 layers of more highly compact myelin achieved within a month in these animals, so this has to come from an embryonic stem cell.
		We've gone on now to utilize fluorescently labeled cell lines, ones that are just on a CMV promoter.  This is 2 weeks after transplantation in the middle of the transplant.  Then we also have ones that are labeled selectively for tau and then also for myelin basic protein so that we can track individual cell types. 
		The other advantage is people like Austin Smith have developed a genetic selection.  So, they've linked a resistance gene to the SOX-2 so that they can select for neural restricted precursors just by adding an antibiotic.  He's been giving that out.  So, that's a very effective selection method to get purified cultures very rapidly and cheaply.
		So, just to follow up what we started with, transplantation and myelination.  I don't think this is necessarily the best thing to move forward from a clinical standpoint, but it's a good example of something that's doable.  
		There are not very many alternatives for a spinal cord injury.  There are very few treatment options.  There are rehabilitation treatment options.  There's functional electrical stimulation, but otherwise not much to offer in these people that are very debilitated and have a long life span.
		The potential risks for transplantation of oligodendrocytes is much lower than neurons potentially.
		The doability of treatment.  Multiple laboratories have shown that they can reconstitute myelination around circuits.
		The severity of the disease is important.  It might not be the best thing to start off with the most severe injury.  A more selective injury, transverse myelitis, where you don't see an infarct might be a good place to start. 
		Then proof of principle.  I think that myelination is one of the areas that has some of the best anatomical behavioral correlates.
		Then in humans, this is a very measurable and interpretable endpoint.  Just with MRI alone, now with the higher magnets, you can follow myelination in the spinal cord.  Now with the paramagnetic molecules, you can really follow it very easily.  Of course, you can't use this quantitatively.  It really lights up the spinal cord, but this is a very effective way of following these cells.  Then, of course, you can have both positive and negative controls.  You can see tracks that have been remyelinated and tracks that are not myelinated, and you know exactly the function of that track.  You can use motor volt potentials and somatosensory volt potentials.  So, it's a very well-outlined anatomical system to look for physiologic correlates.
		I'd just like to thank my collaborators at Washington University and the people in my lab.  If you ever get out there, you can come visit us. 
		(Laughter.)
		DR. McDONALD:  Junior faculty move around a lot.  But this is where we're moving now.  We're really marrying the idea of having inpatient rehabilitation for spinal cord with the basic science.  The basic science for spinal cord will all be on this fourth floor and all the rehabilitation will be on the lower floors. 
		Anyway, thanks.
		(Applause.)
		DR. SALOMON:  Well, we did it.  Thank you very much.
		Well, guys, it's 10 after 6:00.  We're 40 minutes late, not too bad given the fact that we went into lunch 40 minutes late.  So, we've stayed basically on track I think.
		We did get through a lot of material.  I for one feel privileged to have been allowed to hear all these excellent talks.  I certainly got a lot out of it.  I had a lot to get out of it.
		Do we want to have any discussion at all at this point?  We're going to return to this whole thing in depth tomorrow.  I think the time is late.  Everybody is a little bit tired.  So, I'm going to make an executive decision.  I hope that the last two speakers don't take it personally.  But I think that clearly these last two talks and a couple that followed it all starting with Rusty with the animal stuff is really going to be front and center tomorrow as we talk about what kind of animal models are appropriate for clinical trials.  I think we'll get plenty of time to discuss these.  
		So, again, I'd like to thank everybody for the energy and the time all the speakers took to put these talks together and for your attention.  
		See you tomorrow morning at 8:30.
		(Whereupon, at 6:15 p.m., the committee was recessed, to reconvene at 8:30 a.m., Friday, July 14, 2000.)