NATIONAL HEART, LUNG, AND BLOOD
INSTITUTE DIVISION OF BLOOD DISEASES AND
RESOURCES
WORKING GROUP ON STEM CELL
PLASTICITY
March 21,
2000
The Workshop on Stem Cell Plasticity was held on March
21, 2000 in the Rockledge ll Building in Bethesda, Maryland. The Working Group
was constituted to assess the state-of-science in the stem cell plasticity
research area and identify future research directions to be explored in this
area. Brief highlights from the scientific presentations, group discussions,
and suggestions of potential research directions in this area are presented
below.
Dr. Makio Ogawa presented
murine data which clearly demonstrated that CD34 expression of adult mouse stem
cells is driven by the activation state of the stem cells. Various
manipulations were used to "tip" stem cells into cycle such as, 5-fluorouracil
injection, culture with c-kit ligand and interleukin-11 and G-CSF mobilization.
In all of the above circumstances, stem cells in a "resting state" were shown
to be predominately CD34- whereas upon activation, the stem cells transitioned
to become CD34+. As to developmental changes of CD34 expression, stem cells
from 5 week old and younger mice are all CD34+; 7 week old mice showed both
CD34- and CD34+ stem cells. The majority of stem cells (~80%) in 10 week
old mice are CD34-. Thus, CD34 expression by adult stem cells appears to be a
mirror of the activation state of the stem cell.
In the ensuing discussion, it was pointed out that
AA4.1 appeared to be another marker associated with activated but not quiescent
reconstituting cells. AA4.1 is present on stem cells in murine fetal liver,
where stem cells actively cycle, and on marrow stem cells cycling during
regeneration following exposure to 5-FU, but not on stem cells in normal
marrow. The Working Group agreed that it is important to make the link between
the presence or absence of the CD34 marker and global change or biological
potential (e.g., trancriptional, phoshphorylation) of processes within the
cell. It was also noted that correlation between phenotype and biological
potential is present but less than absolute. The Working Group acknowledged
that the molecules that are tracked in phenotypic studies are not likely to be
closely related to the mechanisms that specify lineage or self-renewal.
Dr. Sharkis gave an excellent review of the
current literature and his own work regarding CD34expression in murine
transplant models. Transplant studies were conducted primarily with mouse bone
marrow cells collected at a flow rate of 25ml/minute by elutriation (FR25
cells) This cell population has been further enriched by selecting cells that
are negative for lineage antigen expression (Lin- cells). An intracellular
enzyme was used to positively select FR25 cells which were further separated
cells into 34- and 34+ cells. The 34+ cells represented approximately 30- 40%
of the above FR25 cell population. FR25 34+ but not 34- cells from this
population reconstituted animals. CD34 negative and positive cell separation
data was presented using a rat anti-mouse CD34 monoclonal and polyclonal
antibodies. Transplant results indicated that the frequency of CD34- stem cells
is significantly lower than CD34+.
To extend the previous studies and to diminish the
concern of CD34 contamination in the cell population, CD34 knockout mice were
obtained from individuals in Toronto (Mak) and Genetech (Lasky). Studies were
conducted which include stem cell isolation, transplantation, long-term marrow
culture and homing. Transplant studies conducted with (Mak) mice (previously
reported to have no stem cell phenotype defect) marrow and normal mice marrow
indicate no difference in reconstituting capability in a normal recipient.
However, transplant studies with the Lasky mice marrow (reported phenotype -
significant reduction in progenitor cell number) showed a reduction in
reconstituting capability at each dose of cells.
These studies and others have suggested that CD34 may
be an important adhesion molecule and important in stem cell homing. CD34- FR25
lin- cells (Mak mice) were isolated, PKH labeled, and injected into irradiated
wild type mice. Forty-eight hours later marrow and spleen cells were harvested
and analyzed for the frequency of PKH-positive cells. Using PKH bright cells
from normal mice transplanted into secondary recipients, 80% of mice (using 100
cells) are protected with cells from the marrow whereas cells recovered from
spleen fail at 100,000 cells to short-term reconstitute lethally irradiated
animals. Thus, 2 days following transplant with normal cells it appears that
long-term reconstituting cells have a selective advantage for going to the bone
marrow compared to the spleen. However, using the homing assay with the Mak
mice, the recovery is predominately in spleen cells and not in marrow as seen
with normal cells. These data indicate that cells from the knockout mice may
have a difference in homing capability. Thus, a primary function of CD34 may be
the regulation of the localization and differentiation of hematopoietic stem
cells within the hematopoietic microenvironment.
Dr. Lemischka presented the utility of his Stem
Cell Database (SCDb), an interactive World Wide Web resource designed to
provide insights into the molecular nature of stem cells and their
microenvironment. An important objective is to precisely correlate global gene
expression profiles with quantitatively measured biological properties such as
multipotentiality, self-renewal ability, and the potential for in vivo
engraftment. An important goal of these studies is to construct a comprehensive
genetic program primarily for long-term repopulating cells and secondarily for
various, putatively distinct stages of the hematopoietic developmental
hierarchy. The characterization of such comprehensive genetic programs will
facilitate the elucidation of potentially complex regulatory pathways and
networks. The SCDb (a joint project between the Lemischka laboratory and the
laboratory of Dr. G. Christian Overton, the director of the Center for
Computational Biology and Informatics at Penn) contains the results of numerous
global computational analyses which in many cases have assigned a protein
family membership and/or a candidate biological function to the novel
gene-products. Most of the analyzed gene-products in SCDb are from the mouse.
There are also gene-products from a smaller human project in which a high
quality, representative cDNA library from bone marrow CD34+Lin-CD38- cells was
subtractively depleted of sequences expressed in the closely related but more
committed CD34+Lin-CD38+ cell population. Collectively, SCDb has approximately
34,000 individual sequences. The sequence data have been analyzed using
sophisticated bioinformatics. omparisons to the public databases and
peptide-motif analyses have identified numerous known and novel gene-products
with structural features that are consistent with regulatory functions. These
include transcription factors, cell surface molecules, and intracellular
signaling proteins. These molecules have been identified by performing
subtractive hybridization, high-throughput sequencing, sophisticated
bioinformatic analyses, and high-density array hybridization.
The SCDb will eventually be made available to the
public. It will contain information from functional assays, sequences,
transplant competitive repopulation results down to molecular biology
(tutorials). The Working Group was pleased to hear that the database would be
made public and felt strongly that this would significantly facilitate and
encourage new work. It is anticipated that other investigators will take
advantage of SCDb to deposit and analyze sequences from their own molecular
efforts. In this way, the SCDb will become a central resource for the entire
stem cell research community.
Discussion arose regarding the subtraction
hybridization strategy used in the above studies. Fractions that were being
subtracted were not homogeneous and early stem cells might constitute only a
small percentage of the fractions. There was uncertainty expressed whether stem
cell-specific genes would be accessible by subtraction if stem cells were only
a small subset. Dr. Iscove described how this issue drove the conception of his
approach, in which cDNA is obtained from single cells whose exact biological
potential is known. The approach resolves transcript expression to specific
stages and is able to discriminate transcripts expressed specifically in very
early stages. Prospective work is directed at capturing cDNA from long-term
reconstituting cells directly confirmed by in vivo assay of sibling cells. The
work has identified transcriptional "previewing" of lineage maturation
transcripts in multipotent cells. Previewed transcripts are subsequently
down-regulated downstream and then eventually up-regulated again as typical
transcripts in maturing cells (Ig, Ly6A/Sca-1, lysozyme). He is attempting to
define the breadth of the previewed repertoire. Significantly, only
hematopoietic transcripts have been detected in multipotent cells, while
transcripts that characterize a spectrum of non-hematopoietic lineages
including neural, connective and epithelial tissues have not thus far been
detected.
Currently, the issue of stem cell homogeneity is a
complication in all enrichment strategies, and is very difficult to rigorously
address; given that in vivo transplantation is the only reliable assay for the
most primitive stem cell population.
Dr. Mulligan summarized several of his lab's
recent studies which have focused on further characterizing the surface
phenotype of murine bone marrow-derived cell populations capable of the
reconstitution of lethally irradiated recipients, and determining the potential
of bone marrow and organ tissue or organ-derived populations to give rise to
specific non-hematopoietic cell types after bone marrow transplantation (BMT).
He first reviewed studies which indicate that, using a FACS-based procedure
involving dual-wavelength analysis of Hoechst-33342 stained bone marrow or
muscle cells, it is possible to isolate 'stem cell' populations (termed SP
cells) which are capable of giving rise to both hematopoietic cells and muscle
cells after BMT in a mouse model of muscular dystrophy (mdx mice). Although the
proportion of muscle fibers showing evidence of donor cell derived engraftment
was variable, in one animal approximately 10% of all muscle fibers showed
contribution from donor cells. Interestingly, in the case of animals engrafted
with bone marrow SP cells, it was not possible to detect the presence of donor
derived 'satellite' cells, the stem cell population in muscle utilized for the
normal regeneration of muscle, while in the case of animals engrafted with
muscle SP cells, there appeared to be some engraftment of this 'compartment'.
Dr. Mulligan indicated that these results may suggest that the engraftment of
muscle via SP cells may involve a novel mechanism distinct from that utilized
by satellite cells.
Dr. Mulligan also presented data that bone marrow
derived cells can give rise to endothelial cells after BMT.
Interestingly, although in unperturbed BMT recipients, no evidence for
endothelial contribution was observed, donor derived endothelial cells were
readily detected in BMT recipients which subsequently received organ
transplants, or in recipients in which a myocardial infarction was induced.
These results suggest that the ability of transplanted stem cells to give rise
to non-hematopoietic stem cells may be dependent upon local environmental
stimuli, induced by organ or tissue injury.
Dr. Mulligan then reviewed the results from his
laboratory which suggest that, using the FACS methods his laboratory had used
to purify bone marrow hematopoietic stem cells, it may be also be possible to
isolate stem cell populations from a variety of adult tissues, including
muscle, brain, heart, kidney, and liver. Specifically, experiments with muscle
show that muscle SP cells exist, and give rise to both hematopoietic and muscle
cells after BMT. Preliminary experiments with CNS derived SP cells
suggest that they are enriched for engraftment of the brain. Dr. Mulligan
indicated that current efforts are being directed towards further evaluating
the developmental potentials of both bone marrow and other putative adult stem
cell populations, and the biological circumstances necessary to reveal those
potentials. Dr. Mulligan summarized his studies by suggesting that a variety of
adult stem cell populations may exist and have common functional properties,
and that the ability of the different stem cell populations to give rise to
particular cell types may be dependent upon local environmental stimuli.
Accordingly, it may be critically important to understand the nature of those
biological cues for differentiation, and how to manipulate the cells and the
host in order to facilitate production of desired cell types after stem cell
transplantation. Lastly, Dr. Mulligan presented data which suggests that only
CD34- SP cells are capable of the complete and long term reconstitution of
lethally irradiated murine recipients. These results was discussed in detail,
in light of the results presented by Drs Ogawa and Sharkis, and the possibility
that different transplantation models may reveal the ability of different cell
types to provide for hematopoietic cell engraftment was raised.
Dr. Verfaillie described the isolation,
cultivation, and differentiation of adult bone marrow derived mesodermal stem
cells (MSC). Mesodermal progenitor cells were isolated by depletion of CD45 and
GlyA positive cells from mononuclear bone marrow cells. The differentiation of
MSC into three types of muscle (skeletal, heart, and smooth muscle) was
discussed. Regarding cultivation of MSC, after plating approximately 5000
cells, single cells grow out in about every third well. It is almost 3 weeks
before anything can be seen growing. Currently, the phenotype of the cells is
unknown and will have to wait until enough cells can be isolated. The MSC cells
are essentially negative for most markers except cytokine and adhesion
receptors. If cultured "correctly," (very low density/confluency) cells do not
change phenotype after numerous cell divisions. It takes 14-21 days get them
going and they divide every 40 hours. If the cells become confluent, they stop
growing, start to differentiate, and express CD44, HLA antigens and other
markers. The cells have been taken out to 65-70 doubles. Telomere length (in
cells from adults and children) does not change much before and after culture
and no major cytogenetic abnormalities have been seen.
The frequency of the MSC with massive
multi-potentially is approximately 1 in 10,000,000 whole bone marrow
mononuclear cells. Cells are plated with limited dilution. One cell per well
does not grow well at all and it maybe that cells need to talk to each other.
It is not clear at this time whether a single cell can generate multiple cell
types. Specific culture conditions (developed by trial and error) to
differentiate MSC into endothelial cells, adipocytes, skeletal muscle, and
neural cells were discussed. These cells also differentiate into osteoblasts
and chondroblasts.
Dr. Verfaillie has attempted to coax the
undifferentiated MSC to hematopoiesis by changing culture conditions (using a
combination of hematopoietic cytokines) and culture the cells on the AFT024
feeder layer. No blood cells have been generated at this time although the
cells that emerged were GATA-2 and c-kit positive. Three types of CNS cells
were generated in this culture system; astrocytes, oligodendrocytes, and
neurons. Testing is now ongoing using clonal analysis of retrovirally marked
MSC to demonstrate that undifferentiated MSC generate a neural stem cell that
then gives rise to the three types of CNS cells. These cells have been assessed
in an infarcted rat model. Undifferentiated MSC can differentiate in situ
into cells that express early and more mature neuronal marker including
astrocyte and oligodendritic markers. After implantation, the infarcted rat
model appears to be functionally better. Characterizing the molecular
mechanisms underlying neural stem cell differentiation will be extremely
important for the development of therapies for traumatic or degenerative neural
defects.
The challenge of doing these experiments on single
cell level was discussed in detail. Being able to clone these cells and
recreate in vitro results in vivo are among the next major challenges facing
this research.
Dr. Iscove's presentation outlined a framework
in which findings that have been interpreted as "plasticity" could be
explainable alternatively in terms of our current state of knowledge. The long
prevailing view was that stem cells resident within each tissue (e.g.
hematopoietic, epithelial, liver, skeletal muscle, neural, testis) were
restricted in their repertoire to maintaining only that kind of tissue. Recent
findings have been interpreted as challenging that view. His comments
focused on repertoire, site, and origin of individual nuclei in multinucleate
cells. Regarding repertoire, the definitive way to determine the potential of a
cell is to address it "clonally." The nature of a cell can not be established
definitively simply on the basis of origin from a particular location, a
defined population or possession of a particular phenotype. Proof of
"plasticity" is likely to require some attempt (cloning, limiting dilution,
insertion marking) to ask "what can one cell do?" Clonal demonstrations have
been lacking in most of the recent findings suggestive of plasticity. Location
of a cell may not be a reliable indicator of its origin. Marrow stem cells can
circulate and we know how to enhance their frequency of doing so. Throughout
the body a large number of sites are potentially hematopoietic. For example,
0.5-1% of the normal thymus is hematopoietic, and most of the organs including
skeletal muscle can become infiltrated with actively hematopoietic cells in
various circumstances. These observations suggest that hematopoietic stem cells
may be widely distributed in non-medullary tissues, although normally
quiescent.
With interest in potential plasticity recently
kindled, some non-hematopoietic tissues are being measured for hematopoietic
potential perhaps for the first time. Like the bone marrow, other systems may
also occasionally export stem cells to the circulation and to diverse tissues.
Reservoirs throughout the body could contain mixed populations of stem cells
individually restricted to their own kind of repertoire, but mixed together in
heterotopic sites and possible sharing very similar phenotypic features.
Additionally, macrophages of marrow origin extensively populate most tissues
including the musculature. Experiments designed to probe hematopoietic cells
for potential to differentiate into non-hematopoietic lineages will have to
prove quite conclusively that cells, or even fused nuclei, within a
non-hematopoietic tissue do not simply represent or derive from differentiated
hematopoietic bystanders. Clearly such alternative explanations would have to
be ruled out in future studies, ideally by clonal methods, for the concept of
"stem cell plasticity" to acquire a firmer footing.
Dr. Darwin Prockop was unable to present his
data due to time constraints but called our attention to a PNAS article that
would shortly be published. I have attached the reference and abstract of the
article.
AUTHORS: Colter DC; Class R; DiGirolamo CM; Prockop
DJ; Center for Gene Therapy, MCP Hahnemann University, 10118 New College
Building, 245 North 15 Street, Philadelphia, PA 19102-1192, USA. Proc Natl Acad
Sci U S A 2000 Mar 28;97(7):3213-8.
ABSTRACT: Cultures of plastic-adherent cells
from bone marrow have attracted interest because of their ability to support
growth of hematopoietic stem cells, their multipotentiality for
differentiation, and their possible use for cell and gene therapy. Here we
found that the cells grew most rapidly when they were initially plated at low
densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies.
The cultures displayed a lag phase of about 5 days, a log phase of rapid growth
of about 5 days, and then a stationary phase. FACS analysis demonstrated that
stationary cultures contained a major population of large and moderately
granular cells and a minor population of small and agranular cells here
referred to as recycling stem cells or RS-1 cells. During the lag phase, the
RS-1 cells gave rise to a new population of small and densely granular cells
(RS-2 cells). During the late log phase, the RS-2 cells decreased in number and
regenerated the pool of RS-1 cells found in stationary cultures. In repeated
passages in which the cells were plated at low density, they were amplified
about 10(9)-fold in 6 wk. The cells retained their ability to generate
single-cell derived colonies and therefore apparently retained their
ultipotentiality for differentiation.
Recommendations
The Working Group discussed a number of important
research needs and several areas of focus are identified as follows: (1)
substantiate the stem cell plasticity concept (e.g., defining the repertoire of
stem cells in various anatomical locations); (2) determine whether cells with
hematopoietic potential can express other potentials; (3) whether cells
with other potentials (muscle, CNS, etc.,) in other sites of the body also have
hematopoietic potentials; (4) definition of relevant cell populations and
develop approaches to address the issue of clonality; and (5) develop
clinically relevant models to make use of the concept of stem cell
'plasticity.'
The Working Group acknowledged that much work is
needed to substantiate the stem cell 'plasticity' concept which clearly has
important therapeutic implications. Collaboration among research groups was
identified as the key element in our ability to better substantiate this
concept and to characterize the biological mechanisms that might drive the
process. Thus, the Working Group strongly recommended that research in this
area should be supported by using a collaborative/interactive research grant
mechanism which allows investigators from various locations to share
information and resources. They strongly emphasized a 'no walls concept' was
needed to foster efforts in this important research area.
PARTICIPANTS Richard Mulligan, Ph.D.
Chair Childrens Hospital, Enders, Room 861 320 Longwood
Avenue Boston, MA 02115
Norman Iscove, M.D., Ph.D. Senior Staff
Scientist The Ontario Cancer Institute 610 University Avenue, MSG 2M9
Toronto, Canada
Ihor Lemischka, Ph.D. Professor, Molecular
Biology Department Lewis Thomas Laboratory Room 210LTL Washington
Road Princeton, N.J. 08544
Darwin Prockop, M.D., Ph.D. Director, Center
for Gene Therapy MCP Hahnemann University 10118 New College Building
245 North 15th Street, Mail Stop 421 Philadelphia, PA
19102-1192 |
Saul Sharkis, Ph.D. Johns Hopkins
Oncology Center CRB Room 244 1650 Orleans Street Baltimore,
Maryland 21287
Catherine Verfaillie, M.D. Division of
Hematology 420 Delaware Street, SE,, Box 806 University of Minnesota
Health Center Minneapolis, MN 55455
NHLBI STAFF Helena O. Mishoe, Ph.D.
Health Scientist administrator National Institutes of Health
National Heart, Lung, and Blood Institute
Rockledge II, Room 10156 6701 Rockledge Drive, MSC 7950 Bethesda,
Maryland 20892
|
|