activity
of these cells.
In vivo, you need to look at a model
where these cells will survive. Can they
survive the delivery? I think it was
mentioned in Jonathan's talk. And, also,
are there going to be immune responses to these products?
I can tell you that this is not
necessarily an easy feat for our programs.
I think it took us about a year to a year and a half to establish a
robust system where we could get long-term graft survival, and
long-term
meaning greater than nine months and to a year. So I think it's important to selecting a model
so that if you're looking at these cells, that you can see that the cells, in
fact, survive.
An important feature, also, is
looking at the phenotype, looking at the phenotype in vitro and looking how
does that translate in vivo. Did they
maintain the same phenotype? Do they
mature over time? Are they proliferative
when they're in vivo in their environment?
Also, then, looking at the activity
in disease models, looking for clinical efficacy, looking for their
histology. Can you find evidence of
these cells and are they having any particular effect?
For instance, in this particular case, we're looking at a
spinal cord-injured animal and we're looking in brown for a human nuclear
antibody in EC4, area chrome cyanine, which is Stan's myelin. You can see in animals that are either
injected with vehicle or no injection for that matter, you can see these large
cavities that tend to develop in the spine cord-injured animals.
But that in animals injected with
GRNOPC1, the oligodendroglial progenitor cells, you can see brown, human cells
that are filling the cavity, and that if you look very, very closely, and
here's a high magnification of the center of that cavity, you can now see that
there are myelinated fibers traversing that lesion site.
So it's important to look at
clinical efficacy, histologically what cells are doing. What are the doses and how does that
translate into the human equivalent dose?
What is your delivery site in volume?
What kind of volume of cells are you going to have to inject in order to
meet your targeted efficacy? What kind
of implications does that have for the number of injections that you might need
to give? And then finally the timing of
the treatment in relationship to the disease process or the injury process.
Next question is where do the cells
go? That has both safety and efficacy
implications. You need to look at the
sensitivity of your assays to detect particular cells. Do the cells go to the site for intended activity? Are there sufficient numbers of cells at the
site in order to elicit efficacy? Is
there distribution outside of the target site?
And is there migration at the local site? And looking at extended periods over time.
This is just some examples from one
of our biodistribution studies where we looked by PCR for looking at tissues
throughout the body and also by immunohistochemistry to look at cells within
the spinal cord. And here we looked at,
in this case, the distance from the most proximal, the most distal cells in the
spinal cord as a function of time and dose.
So in this particular case we gave
animals either 2.4 x 105 or 2.4 x 106 cells, in
alternating 2 x 75, 2 x 76. And we looked at two days, 14 days, and 180
days post transplant. And you can see
there that these cells do migrate, and by six months post transplant we can see
that there is about a 16 to 17 millimeter distance between the most caudal and
the most rostral cells injected into the spinal cord.
For the toxicology studies, one of
the big considerations is where do the cells go so that you can start looking
for toxic activities. Again, some
considerations are looking at the dose of the product, looking at what your tox
model is. Again, we've looked at, in our
particular case we looked at a spinal cord-injured rat for many of our
toxilogical considerations just because we want to see what the cells do in
that scenario that probably is the closest model where we can get good
long-term cell survival to see whether these cells have any toxilogical
effects.
You really need to look at the
feasibility of the model. Can you get
long term survival of your whatever model you're choosing? The duration of the studies and looking at
the duration of the human cell survival, obviously, it's really important to
demonstrate that your product, if it's required to be there for a long term,
that it is there for a long term.
For our particular program we looked
at the toxicity of the delivery. We
looked at animal survival, clinical observations, any evidence for systemic
toxicity, hematological coagulation parameters, clinical chemistries in detail
in both macropathology and micropathology to look to see are there any adverse
events associated with the injection of the cells. And then also on some very, very, very --
activities that are specific to our product, in this case the evidence for
allodynia.
In the four tumorigenicity studies,
again, I think we've heard a lot about that this morning already. But, obviously, you're looking for not only
teratomas, but also evidence of ectopic tissue.
Obviously looking at the local injection site and distal sites to see,
again, where the migration patterns are of the cells. Do you find any evidence of teratomas?
There are some challenges to these
studies because in an ideal world you would love to be able to inject the human
clinical dose in these models. And if
you're looking at, again, as I think Melissa said, that if you're putting these
cells in any animal model, it's a xenograft and you want to look where you're
going to look at the most boast out of the cell survival is optimized. So, again, looking at long-term cell
survival.
You'd like to look at large numbers
of animals, not just one or two. You'd
like to look at large numbers of animals so you can boost your statistics.
You want to mimic the human clinical
setting as best as possible. It's not
always feasible, but you want to take that into consideration at least when
designing the tumorigenicity studies.
There are large animal models. For my perspective at this point, the large
animal models for looking at tumorigenicity studies can be quite a challenge
due to, in most cases, the absence of immunocompromised animals, large animals. It requires very long and very laborious xenografted
immunosuppression, which I think can be
a challenge for looking at the tumorigenicity of any embryonic stem cell-based
therapy.
There's also some suggestions, that
have been suggested to me anyways, about looking at homologous embryonic stem cell
systems. Looking at, for instance,
taking your protocol, apply it to a mouse embryonic stem cell, put that into a
mouse and see, in fact, do you get teratomas, do you get tumors?
Unfortunately, the protocols that we
use for differentiating human embryonic stem cells just aren't applicable to
the fine detail that we need for our human embryonic stem cells. And so what you would see with a mouse embryonic stem cells are just not
necessarily representative of what you would see with humans.
There are some lessons learned, that
-- through our studies -- that we have learned through, again, through the
execution of the studies, and that there are many important factors in teratoma
formation. For instance, we're looking
at embryonic stem cell number. In this
case, the more cells you put in, the more changes of having a teratoma.
In this case, we've looked at, we've
taken our product and deliberately spiked it with undifferentiated embryonic
stem cells. We held the total number of
cells constant at 2 x 106 and injected them into the spinal cord, in
this case of immunocompromised mice, and
assessed for teratoma formation 12 months after.
So what we did was a graded number
ranging from 50 percent undifferentiated cells all the way down to actually
zero percent undifferentiated cells.
What you can see here is that there is this curve that anywhere below
one percent we actually did not see any evidence for teratoma formation in the
spinal cord and in the spinal cord of these animals. Actually, in this particular experiment, we
also did not see any at five percent.
Five percent seems to be about the
threshold at which we start seeing teratomas.
So this is the undifferentiated cell number in this context of -- our
product is something that is important for establishing the formation of
teratomas in these animals.
We also know that the site of
implantation is important. We find, for
instance, in the spinal cord, that there's over a order of magnitude grave
sensitivity for teratoma formation than when looking at an intramuscular
injection. So the site of delivery of
your product is very important.
And, also, the cell aggregation
state so that clump cells tend to form teratomas at least more quickly and with
a slightly higher frequency than cells that are single cells.
Other studies to take into
consideration are allogenicity studies.
Is immunosuppression required?
And, if so, what is that duration of immunosuppression? I think that most common practice or common
logic would suggest that these cells are going to be rejected. But there's snippets of information out there
that suggest that they might not be rigorously rejected and that they might in
fact reduce the required reduced immunosuppression or reduced irration duration
of immunosuppression.
Some of the challenges, again, are
that this is very hard to measure, very hard to measure in an animal model
because, again, the embryonic stem cell-based products are xenografts in all
animals requiring extra rigor to get their survival. There's also, it's very difficult to look at
the allogenicity of maturing cells in vivo in these animal models, again,
because they are, in this case by definition, a xenograft model.
Humanized models are challenging,
and challenging to get the tissue, and challenging with respect to determining
allogenicity because it's not necessarily clear that all elements of the immune
system are in fact recapitulated in these models with the type of activity that
you might expect to see in a human. And
so in many cases we're left with in vitro analyses.
Some of the approaches is to look
at, again, the allogenicity in vitro; looking at trying to recapitulate in an
inflammatory site in in vitro culture; looking at removing immunosuppression in
your xenograft model, see if cells survive; and looking for ways to see if you
can monitor in the clinical setting engraftment, monitor injection. And, more importantly, what are the effects
of rejection? Are there any adverse
events in case these cells do, in fact, reject?
A couple of other things I'd like to
discuss, and that's the cells for the delivery.
Obviously, you have to decide whether it's going to be a local delivery
or a systemic delivery. What is going to
be the site? Is it going to be an
intraoperative delivery? What is going
to be the rate of delivery? Is it a
one-time administration? Is it multiple
times of administration? What's the
skill set required for the delivery? And
a delivery device, do you need a delivery device?
For our application, again, we, in
fact, did design a, what we call a syringe positioning device, because we're
looking at injecting these cells over a period of a minute or two in the spinal
cord, and one of the things we wanted to do was to make sure that we could
stabilize the syringe for neurosurgeons during the actual delivery of this
particular product. And, obviously,
there's training that's required.
So the last thing I'd like to talk
about are the design of the clinical trials.
And the design of the clinical trials, again, there's the most important
consideration is patient safety. And
it's really important to have consideration and interactions with a plethora of
different experts in order to design these clinical trials. We really need to, in this case we've looked
at, having a steering committee, a data monitoring committee, an outcomes
committee so that we can define how these trials and what are we going to be
considering for safety and efficacy, in this case, looking with our
neurosurgeons, who are the group that are going to be delivering these cells;
the radiologists who will be, in fact, monitoring these cells and the
progression of these cells, and, hopefully, looking for and not seeing, but
looking to see if there are any adverse events at the local sites.
Obviously, with the investigators
and the ESCRO committees and various committees to look at -- and IRBs who are
going to be reviewing the protocol. So
the idea here, again, is to look at the protocol, the logistics of the trial,
minimize potential risks. How can this
kind of therapy actually be established and maintained in the normal standard
of care for your particular patient?
Our proposed phase one clinical
trial would be an open label trial that
would be looking at treating patients with subacute, functionally complete T3
to T12 thoracic lesions, injuries, transplanting anywhere from seven to 14 days
with a temporary immunosuppression.
Again, the primary endpoints are safety.
Secondary endpoints are looking at
the feasibility of the multiple outcome measures that we're anticipating in the
trial. We're proposing frequent
short-term and
long-term
MRIs, again, to look at the site to see if we're seeing any adverse or positive
effects at the site, and looking at a variety of different immunological
monitoring.
So I think I will conclude there
just to say that there are numerous considerations in developing an embryonic
stem cell-based therapy. Some of the
questions are actually common to all cell therapies. Some are more specific than others to human
embryonic stem cells. That the specific
non-clinical
study designs really need to be designed with their clinical application in
mind. That the clinical trials designs
do require intradisciplinary input and they need to be designed with frequent
monitoring involved.
Thank you.
(Applause.)
CHAIR URBA: Thank you.
Any questions for Dr. Lebkowski?
Dr. Allen?
DR. ALLEN: You mentioned that in the context of
long-term safety testing and particularly large animals that it was potentially
possible to generate ES lines from these animals, but that your primary concern
were that they're manufacture essentially differently, the process
differently. I mean I think that this is
something that we struggle with with any cell-based therapy.
So I guess my question is, other
than that fact, are there any concerns?
I mean, if you can demonstrate that your differentiated ES cell, large
animal let's say, but essentially animal ES cell functionally behaves
similarly, has phenotypic features similar to those of the natural cell, is
there any reason not to test that in a large animal model?
DR. LEBKOWSKI: I think there's no reason not to test it in
an animal model. But I would add one
more criteria, that we have to show that the composition is equivalent, and we
need to have the right markers to show that the composition is equivalent.
DR. ALLEN: But, essentially, if the things like, for
example, spiking experiments, you could potentially do spiking experiments and
confirm that what you see in a mouse with a human ES cell you do or do not see
with, for example, a dog-to-dog or primate-to-primate.
DR. LEBKOWSKI: Yes.
That's feasible.
DR. ALLEN: Okay.
Thank you.
DR. LEBKOWSKI: As long as, you know, the question is is how
well do the undifferentiated human embryonic stem cells survive in your large
animal model because that could be a question too.
CHAIR URBA: Dr. Calos?
DR. CALOS: Jane, on the teratoma issue, you're scoring
teratomas and are you using that strictly to mean benign, or are you also scoring
malignant tumors?
DR. LEBKOWSKI: When we have looked at these studies, they're
benign teratomas, okay, so they're well differentiated.
DR. CALOS: So what is considered the issue with ES cells
if they're always benign teratomas? Do
you also get malignant tumors?
DR. LEBKOWSKI: In our experience, if you take enough
undifferentiated cells and you put them into a very vulnerable site, okay, you
can see cells that I wouldn't necessarily call them malignant, okay, but they
will look undifferentiated, okay. So we
haven't characterized them as to whether they are malignant or not, just
haven't looked at that. But the question
is, you can still see cells that look undifferentiated. Again, that's putting lots of
undifferentiated cells into a very vulnerable site, okay, for instance, the
brain.
The second things is the issue with
benign teratomas are, you know, what are the consequences of that? Do they continue to grow uncontrollably? And can you remove it? If you can't remove it, what happens? Can you remove it? You know, what are the consequences of
that? So those are the issues that we're
dealing with.
DR. GERSON: You showed us a variety of data of cell dose,
but I didn't see data on dose effect or dose range. Could you give us some sense about how you
approached that?
DR. LEBKOWSKI: Dose range for teratoma formation or dose
range --
DR. GERSON: No, you showed us percentage of cells in a
fixed dose. And in the migration study,
you showed us essentially no dose effect.
You showed us a time effect.
DR. LEBKOWSKI: Yes.
DR. GERSON: Is there a dose effect and are you
anticipating studying dose effect?
DR. LEBKOWSKI: Yes.
We can see dose effects on teratoma formation even when we look at not
spiking but using just plain, undifferentiated cells and putting them into
animals. We can see a dose effect on
teratoma formation.
With regards to any toxicological
parameter, for instances, even allodynia or anything like that, we have not
seen. We've looked at two doses in many
of our studies and have not seen any effect of that.
DR. GERSON: Is there a therapeutic dose effect?
DR. LEBKOWSKI: The answer is no. We have not seen any therapeutic dose effects
yet, but we might need to actually go down in dose in order to see that.
DR.
TAYLOR: Along those lines, you just
talked about if you put lots of cells in a vulnerable site you're likely to
see, potentially, a tumor, be it benign or otherwise. And there are data in the literature that
suggests small numbers of undifferentiated cells can form malignant tumors
depending on the site.
We've also heard from you and a
number of other presenters that we don't have a good sense of where cells
migrate, how cells migrate. You have
data that cells do migrate. So how do
you account for and begin to control whether or not cells are reaching "a
vulnerable site" and how many cells are?
And then, finally, in terms of the homologous cell studies you were
mentioning, there is continually a mixture of differentiated and
undifferentiated cells present, so how do you begin to account for that in this
broader context of tumorigenicity?
DR. LEBKOWSKI: Okay.
So let me be a little bit more specific.
For instance, if we take and try to inject two million undifferentiated
cells into a rodent spinal cord, intact rodent spinal cord, you will see cells
that not only have benign teratomas, well differentiated teratomas, but you
will see some cells that look still undifferentiated, and that's the target
site that we're looking at.
I think the question is, from your
biodistribution studies, you need to look at where, for instance, the cells go
and to see whether there are vulnerable sites that can, in fact, be -- that are
populated by cells or in potentially undifferentiated cells. So, for instance, in the biodistribution
sites, I didn't show you all the data, but we've looked at not only where the
cells go upon injection into animal that has a spinal cord injury, not only
looked at the spinal cord, but we've looked at essentially every tissue in the
body by quantitative PCR to look for, are there cells present, and they aren't.
DR. TAYLOR: Let me ask a followup on that. In terms of a location like spinal cord,
which is geometrically constrained, and brain, which is geometrically
constrained, and heart, likewise, have you noticed adverse effects by injecting
even small numbers of undifferentiated cells when there is the potential for
compression being an issue, for example,
even with a benign tumor?
DR. LEBKOWSKI: Yes, yes, I mean if you have enough
undifferentiated cells in your population.
So like, for instance, in the data that I showed, if you have 100,000
undifferentiated cells in the population that we deliberately spiked into, for
instance, our candidate population, OPC1.
We can see teratomas in there, okay.
And if we've titrated down, we don't see those.
DR. TAYLOR: The argument is that teratomas are
benign. I guess my question is, in some
situations a tumor is a geometrically unfavorable condition, be it benign or
otherwise, have you accounted for that, how do you account for that, and --
DR. LEBKOWSKI: Well, we're looking for any kind of adverse
events. And the question is, do we see a
benign or any other kinds of tissue in there that would be a problem?
CHAIR URBA: Dr. Chamberlain?
DR. CHAMBERLAIN: I'm curious.
In a human clinical trial where you're delivering to a relatively
inaccessible site like the spinal cord, how do you monitor cell survival? And is there any consideration given to
tracer studies or putting in marker genes like luciferase or anything like
that?
DR. LEBKOWSKI: We have not looked at putting in any marker
genes in there, but your question is quite relevant. It is going to be difficult. We are going to be proposing to do some
experimental assays to look for whether the cells are there or not, but it is,
actually, a challenge, and looking for the appropriate marker or the
appropriate tracer that can be used to monitor those cells, that can be used
clinically.
DR. GOLDMAN: It's a fault, but to the point with regards
to benign teratoma versus malignant teratocarcinoma, that's essentially a
histologic definition based on degree of anaplasia. But within the nervous system, any
neurologist or neuroncologist or neurosurgeon will tell you there's no such
thing as benign tumor within the brain or spinal cord. Anyway, that's a comment.
But I wanted to ask essentially the
same question that I did of Dr. Dinsmore.
Are you sorting up front or is the assumption that the population you're
starting with is essentially purified with regards to aligoprogenitors?
DR. LEBKOWSKI: We do not do any sorting by any flow
cytometry events or anything. It's
strictly driven by the differentiation protocol.
DR. GOLDMAN: So this thing segues into the spiking experiment. I'm just wondering about that. If I understood that graft correctly, when
you get to roughly five percent spiking, you start to see approximately ten
percent teratoma generation?
DR. LEBKOWSKI: Yes.
DR. GOLDMAN: That's out of two million. So that means that your threshold for
teratoma generation is effective 100,000 undifferentiated ES?
DR. LEBKOWSKI: In this model system, that's correct.
DR. GOLDMAN: So that's what I'm getting at. The model systems, in the typical -- in an uninjured model, the cell number's
much lower than that, well, of course, because of teratoma?
DR. LEBKOWSKI: Yes, that was a uninjured model.
DR. GOLDMAN: That wasn't an uninjured model.
DR. LEBKOWSKI: On an injured model, yes.
DR. GOLDMAN: So then I'm wondering then, is it --
DR. LEBKOWSKI: We've done both.
DR. GOLDMAN: Is the cell survival less than you think it
is or are there paracrine effects within the population such that the
undifferentiated are being suppressed by differentiated? Because I'd be very surprised that it takes
100,000 cells to get a teratoma or at least any incidence of teratoma.
DR. LEBKOWSKI: You know, all I can say is what we
deliver. We have monitored for human
cell survival in all of the animal models for all of the tumorigenicity and for
all the toxicology studies, and we see them there. We see them in good numbers.
Can I say that, you know, 100
percent of the cells that I have delivered are there? I can't say that, you know. There's just not the tools to be able to
answer that question.
CHAIR URBA: Two last questions.
DR. CHAPPELL: I'd like to follow up on the previous
discussion. You originally presented the
results that we've been discussing in terms of percent.
DR. LEBKOWSKI: Yes.
DR. CHAPPELL: So five percent or more undifferentiated cells
give rise to detectable teratoma levels.
But we've converted those to 100,000, which is five percent of two
million.
DR. LEBKOWSKI: Yes.
DR. CHAPPELL: So I'm wondering whether it's the absolute
number that you think is more relevant.
So would it be 100,000 with 1,900,000?
When your experiment, would that be equal to 100,000 alone, or 100,000
plus five million? Or do you think it's
the percent that's more relevant? Or do
you have any evidence to answer that?
DR. LEBKOWSKI: We have evidence to say that it's probably
the cell number. We've done some
studies, but not as many as in -- not as many animals. Looking at just undifferentiated cells alone
injected, graded numbers of undifferentiated cells in the spinal cord versus
spiking them into, for instance, the product, and we see about the same
thresholds. So that is data suggested
that it's probably not necessarily what's in there with it's buddies, okay.
On the other hand, I am a little
cautious about making that conclusive remark yet because we do know that there
are states of aggregation. Other things
can influence how these cells and how they form teratomas.
CHAIR URBA: Last question.
DR. FRIEDLANDER: So in assessing risk benefit, one likes to
think there is always some benefit to balance the risk here and we didn't talk
a lot about that. So I'm just wondering,
when you were injecting cells like these oligodendroglial progenitor cells, I
presume you're hoping these are going to differentiate into glial cells or
oligodendra sites, myeline damaged nerves.
But presumably there's a secondary or paracrine effect of these cells
also. How do you -- I mean it's fine in
the tissue culture dish to look at this, but how do you do this in vivo? How do you assess these sorts of issues?
DR. LEBKOWSKI: I mean, one of the things that we've looked
at extensively is looking at their effects on behavioral activities, for
instance, the BBB score, but also looking at histological. I just showed one little picture. But looking histologically at what these
spinal cord injury sites look like, okay, and seeing sparing of tissue and
looking at reconstitution or myelinated fibers going through these sites, so
it's a combination of factors.
CHAIR URBA: Okay.
We've reached the time for the morning break. I'd like to thank this morning's speakers for
their great presentations, for staying on time.
I'd like to remind the committee members that during the break we ought
to not discuss the topics that will be held this afternoon because we all want
to hear everyone's comments. And we will
reconvene here at 11:20 a.m. Thank you.
(Whereupon, the foregoing matter
went off the record at 11:08 a.m.
and went back on the record at
11:25 a.m.)
CHAIR URBA: So we're ready to begin the second part of
the morning session. I'd like to start
with our next speaker, Dr. Isacson, Director, Center for Neuroregeneration
Research, McLean Hospital/Harvard Medical School.
DR. ISACSON: Don't want to deprive the member of coffee.
I want to thank as an academic and
invited guest, the FDA and CBER and CGT for this terrific exploratory meeting
which I think is unique to us as a country and gives us an opportunity to
really air the issues. And the briefing
document that was described by Dr. Bauer to begin with I think is a very good
start.
So what I would describe to you
today is essentially a set of experiments and experiences over the last 15
years in which the desired cell type is actually one for potential use in
Parkinson's disease. And I will speak to
you mainly as a scientist, but also as someone who has participated in clinical
trials.
I wanted to start by showing my
opinion from current and previous work about this field, in particular because
I saw in a previous slide the word pharmacology show up, and I do think that
when we are discussing stem cell therapy we really are discussing live cell
therapy and they are really, specifically issues when you use investigational
new cells as opposed to the standard IND.
And, obviously, we already talked about what is the reasonable way to
evaluate stem cell derived cell therapy compared to the risks inherent in such
therapies.
And, finally, or almost finally, I
want to discuss with you the ability in each of these models, be they muscle or
diabetes or neurological disorders, how we generate a desired cell type just
like we would do in pharmacology for a desired substance, and how we define
that stability. And then to reiterate
that the animal model or models that we use, how are they going to tell us both
about benefit and safety data as the previous speakers also brought up.
Now, in Parkinson's disease cell
therapy has been considered for a number of years. The reason being that in the mid brain, the
base of the brain of a Parkinson patient, the cell that's most vulnerable and
actually produces the signs and symptoms of Parkinsonism died. They are known as dopaminergic neurons.
And in a paradigm in which the basic
idea is that since drug therapy fails over time, the replacement of such
neurons producing dopamine, here you see a patient from our trial in Canada
that participated in, which here you have the patient prior to transplantation
with a PET signal for fluorodopa and here you have three years after transplantation
to one side that restoration of signal indicates that the cells produced
dopamine.
And they do so and they grow so in a
very normal phenotypic way. In these
transplants here are actually four or five tracts and they grow very
specifically to create this dark substance, which are dopamine terminals, in
the unplanted side. There are very few
fibers left and the threshold for diseased is when the patient had lost about
75 percent of their normal terminals.
And these cells grow in what we call
phenotypic manner and restore a terminal network. This is very exploratory therapy, but it has
some up side. In particular we found
that patients typically recover over a period of two to three to four years
when the terminals grow into place that the desired cell type, here in red, is
the cell type that dies in the diseased.
So as of early this week a paper came out, in fact three papers, out of
the four groups in the world that are actively pursuing this work, three in
nature one and one is ongoing, in which, in our case, these transplants
survived for up to 14 years without any signs of Parkinson's disease
pathology. Two other studies showing
signs of some very minor protein aggregation.
But the overall message I believe from the four studies are that the
majority of patients who get these cells, very phenotypically specific cells,
have them active for a long period of time which gives you a rationale for
pursuing a better cell source than fetal cells.
So I hope you saw the title of my
slide here, which is that these are non-stem cell derived fetal neurons. But, in fact, those are the very one cells we
desire to get from the stem cells.
So the next question then is, what
kind of animals would you use here to gain recent information about safety and
benefits? And in the use of animal
models, as we heard previously, the combination of both your desired clinical
method and safety is the best choice I believe.
Certainly models that inform us that are disease relevant, and I will
show you examples of this, and also models that provide safety data at the same
time. And even though it's more
difficult I believe to present data for clinical trials, the combinations of
such models frequently give you a more comprehensive view.
As we discussed previously, in all
our cases we've had to choose animal models both of allo- and xenografted
combinations, and the immunosuppression methods have been applied I would say
actually with some reasonable data outcome.
I feel that those experiments have given us a lot of information that
weren't too influenced by the host donor immunology. So as we heard from one previous speaker, I
think these are issues that may be manageable.
However, the last point there, for
those of you in the back, it says engraftment, implantation and transplantation
procedures. Typically, in my field,
which is an academic field not driven by industry yet, a cell concentration
dose, delivery and implantation sites don't sound very exciting to a scientific
paper, but they are extremely important to the outcome for the patients and the
rodent models and primate models we view.
So I hope to give you some examples of those.
Now, what about an overall
understanding of function? The key
problem in our field of Parkinson's disease was that people basically didn't
believe you could use cell therapy when we started this work in the early
'80s. And it took a long time before the
field realized that, in fact, it was a dopamine neuron specific behavioral effect. And I've used a summary slide here in which
the behavioral syndrome is on the left side of this y-axis, which your graph
actually rotates because had lost dopamine neurons on one side of the brain.
And then you have here weeks
post-transplantation,
and I believe these experiments illustrate an important point. If you do a xenogeneic combination of putting
human fetal dopamine neurons, these are
post-mitotic,
very young, but they will not divide after this point, our desired cell type
will grow in the brain, takes about 20 weeks, maybe sometimes 16, in which a
rat receiving the same cell dose essentially as a allogeneic rat-to-rat combination recover. If you put in, say, a primate fetal VM which
has a faster gestational growth, the same dopamine neuron post-mitotic will
make the animal recover faster. A pig
has 115 days gestation, also does it over eight weeks. And if you take a mouse dopamine neuron, it
takes about four to six weeks.
If you put in, and this is work we
originally used to show the principle that you could get the desired phenotype
from ES cell using what's known as a default pathway, we implanted ES cells at
very low concentration where ectoderm dominates and we got dopamine neurons,
and now we face shifted this by 14 days because that's the number of days it
takes for dopamine neurons to be produced from embryonic stem cells. So if you follow the reasoning here, you can
predict the behavior recovery depending on your donor cell age and growth
patterns. So that's sort of proof of
principle.
Another example I would like to
share with you is that it's very different than pharmacology, and I like that
it's different, meaning cells have -- the reason we use cells many times is
because they have feedback control of releasing the desired substance, here
dopamine. So the dose response curve
with dopamine cells here from survival of 100 cells, where you see survival in
the rat, up to 10,000 cells, the behavior response plateaus even though you
increase your dose from 100 to 10,000 surviving cells.
The reason being, and you can find
this in articles, that the cells will shut down their own release of dopamine
as an adaptation to the host brain. So
that's why we are pursuing this particular cell therapy rather than more drug
design.
Now, what about stem cells? So the first work we worked with actually
involved Dr. Dinsmore, who was then collaborating with us, was control
experimenting, which we were actually producing another cell type. But in the control experiment, which was scary
to most post docs in the group, we actually implanted pure ES cells.
And the remarkable finding was that
not only did we find teratoma, which is in the center here, but also large
number of neurons of the desired cell type that we have been looking for
previously for about 15 years. So, obviously,
we were very attuned to this finding, and over the next couple of years we used
the finding to define a way to obtain a better outcome that did not include
teratomas.
But, actually, very pertinent to the
discussion this morning, we did the cell dose experiments that we had questions
about. When we injected, and this is
using the same concentration but actually increasing cell dose, 50,000 mouse ES
cells into a rat brain generate smooth muscles, a little bit of neurectoderm,
but also endothelial cells and skin. If
you reduce the dose somewhat, you get towards a more neurectodermal neuronal
graft, and, finally, at very low levels, this was actually based on early work
that was done many, many years ago based on neuronal induction paradigm, if you
don't tell an ES cell what to do, it becomes a neuron. We could in some cases obtain pure neuronal
grafts that were of this desired dopamine neuron.
And, moreover, we used this finding
to test the functional hypothesis. Could
these dopamine neurons function? Indeed,
they did. They had very, exactly the
desired cell type and they could grow exactly into the phenotype with the
markers we had. And implanting them into
rats and later into primates we could show by PET scanning, post mortem that
these dopamine neurons functioned exactly the way we wanted to. Using FMRI and PET, they activate blood flow
the same way that the fetal cells had in early clinical trials that we had done
on patients. So the functional issues,
which obviously is my interest, had been accomplished.
But we also had to pursue very
fundamental questions as had the previous speakers. For example, one example which goes against
the common notion that ES cell depends on implantation site, and perhaps
suggests that some of it depends on survival in the implantation site, was that
we performed in those early experiments implantation of this very low dose of
ES cell either into the brain or the kidney capsule and they are virtually
impossible to distinguish, meaning certainly for teratoma formation, that was
dependent on cell dose not location. So
the old experiments using kidney capsule as a location was a fairly good way to
test these tumors.
Now, obviously, the next step was to
use embryonic stem cells, and we pursued this, and many times in collaboration,
but the obvious problem for us was actually two-fold, that human cells grow
into bigger tissues, as we heard previously.
I think we need to be plain about this.
Tumors are not unicells grow into larger bodies than do mouse cells and
that is a problem.
Normally ES cells, and I want to
quote Mahendra Rao is in the room, unless you have a teratoma, you don't have
an ES cell. And I think he put it best
of all, namely, teratoma is a desired outcome to prove that you have a real ES
cell, namely, just all the tissues are produced in this benign, I should say at
least in teratology, as a childhood tumor where you find all the cell
types. So this is the normal outcome of
a normal ES cell.
But more interestingly in a recent
paper by Steve Goldman, who is on the committee and in this room, showed that
when you use human tissue, if you have a cell that proliferates, say you know
that our brains are very large and that's primarily because our cortex grows
much larger than other species, and if
you have such cell in your cell population it will grow to the size or try to
grow to the size of a human cortex, which makes it very problematic for animal
models. And so one needs to consider
these issues I think very plainly and biologically.
A
more challenging problem that we have already discussed is when an ES cell
becomes transformed. And in my view and
my experience it has to do primarily with passaging of cells. So the more a cell is allowed to or will
divide, the higher the likelihood obviously that some kind of event will occur
that will perhaps favor division.
Obviously, for those of you who are
not biologists in the room, it's all the in vitro cell culture favor cells that
divide. So there is the selection
pressure to have cells transform. So
passaging needs to be known and, obviously, studied very carefully.
And, as mentioned previously, subtle
effects may occur. And
teratocarcinoma is a very thing to me
than a teratoma consequently. That would
be uncontrollable growth, meaning the cells will not stop dividing even though
you differentiate them. So that's a
different type of cell.
Another issue there I think the
biologists can help you understand is the cancer biologist's concept of
de-differentiation. During passaging sometimes
a cell will lose it's control of division and proliferation and basically
become like an ES cell. In fact, that is
the concept used for induced pluripotency, which is the current work on making
skin cells into ES-like cells.
But for today's discussion let's
focus on the human ES cell. And we
performed early, this is now seven years ago, the equivalent study that we did
in the mouse using uninduced, the pure embryonic stems as implantation human
cells into rats. And, as expected, with
a very low dose of cells here we ranged, again, on the order of a few hundred
to a thousand.
We typically, here's a rat brain,
right side/left side, and here's the red outline is of graft. These grafts contain not only sometimes
teratoma, another tissue type, but frequently these neural tissues. These are called neurectoderms. Here we have human antigens, we have in green
here, and they form these structures that are the beginning of the nervous
system.
But they also at nine weeks produced
the desired cell type, the dopamine
neuron. So we knew then that it
was worthwhile to try to work with these cells to get out of this mass of cells
the desired type.
How did we do this? Well, actually, by this time other groups had
developed new techniques using standard cell tissue procedures, and, in
particular, Ron McKie and Lauren Studer's groups developed ways in which they
try to simulate the process of the Day 0 cell, which is the embryonic stem
cell, this induction of the rosette that I just showed you, the green rosettes,
and then as the cell matures, we have to expand those precursors into large
numbers called neuroprecursors, and finally, in the last few days, we hope that
they turn into the desired cell type.
They look typically like these
balls. But the reality is, and that's
why I don't like to show the schematic, and this is a population of cells. It contains all this beautiful green dopamine
neurons, but also in the center of the ball other cells that may have escaped
pattern.
This became very obvious to us. And even though a number of groups, and I
apologize for the details of this scheme here, but essentially, again, Day 0 to
45 using all sorts of factors that scientists believe are necessary to grow the
cell, sometimes it actually spontaneously differentiates, we can stimulate
these rosettes, shown as gray spots here, we actually have to cut them out of
the dish. This is how crude this
technology is.
And then propagate them again until
we get large masses of those, and eventually we get some dopamine neurons that,
thankfully when we transplanted them, and this is now also worked on by my
colleagues in other universities, they restore function in the
dopamine-depleted rat model at the predicted rate of recovery. So as we showed in the first slide of
function, they don't immediately create benefit to the animal, but at the rate
in which they would grow into the host brain.
But the reality, which I'd like to
share with you, is that this is a fairly big graft in a rat host. So in a human brain it wouldn't be so
detrimental. But these cells were
relatively rare, and even though many of them had the right cell type, there
are also cell types that we didn't recognize immediately but later found were
derivatives of dopamine neurons that were not in the mid brain but in other
places of the brain, not necessarily harmful, but not exactly the functional
cell type.
And I think this is typical for the
stage of research of current embryonic stem cell work. We are trying to get a cell type. But, in fact, as pointed out wonderfully by a
previous speaker, we actually have to use in vitro in the dish methods to do
what nature does normally.
So these in vitro cell culture
models then became our work for the next five years. And to bullet some of those discoveries, the
questions that we always ask now, are the changes produced by cell culture and
passaging monitored and understood? Do
we understand what happens to the cell over time? And also, obviously, if you grow the cells
for 40 days or sometimes for six months, do you really know whether those
functional cells you have are the typical ones that you want for heart islets
or brain? Are they as optimal as say a
drug design would be? What criteria do
we use?
And finally on the safety, to what
extent can we remove the unpatterned cells?
It's a very pragmatic question.
If you know you have the desired cell type, how do you get rid of the
other ones? And that's the last point.
And I will show you some
experiments, but I wanted to share with you how it looks like in the dish. If you look into the microscope at we called
Passage Zero, Day 9 of a human embryonic stem cell culture this is how it looks
like. It's not well ordered lines of
crop cells, but rather a series of aggregated cells that, depending on
concentration when you plate them, some of those will form into these rosette
structures that we know will turn into dopamine neurons.
Over time we can grow them, and we
actually have to manually cut around those and passage them further. And we do so using methods that are very
standard in the field and we start at zero and around 42, sometimes 47 days.
But I wanted to share with you a
discovery or an observation we have in a paper published by Jan Pruszak last
year that at Day 35, when you think you have patterned the cell, actually in
some cases you have these SSEA-4, which is the unpatterned ES cells in green,
the precursors to dopamine in red, and also some of the neurons desired here in
blue. This is a population of cells.
And the obvious idea that follows,
perhaps I should show you the explanation I believe in first, is that at that
time, 1, 2, 3, 4, 5, et cetera in your dish, some cells will differentiate
according to their normal, natural schedule, others will be remain on pattern because
stem cells tend to talk to each other at certain densities. And I know there was interesting question
from the committee earlier. These things
also matter.
If a cell is very close, if an ES
cell is very close to its neighbor, it may exchange what we call cytokines and
other patterning factors to tell the other cells what to do so that's why I
showed you the dish before. A cell C can
remain in this dish for 35 days without really seeing the differentiation factor
you want.
So if this is the reality, what do
you do about it? Well, there are
examples of how to reduce this risk, and I believe that science eventually will
solve this problem and I'll give you a few examples. That actually was illustrated previously.
When you look at the embryonic
antigen 4 and you grow the cells over time, in certain instances you can grow,
here you see days in vitro 42, that this green antigen, here studied by flow
cytometry, is decreased as the cell population ages. So less than 0.5 percent in our best cases, and
here's another example of a marker that disappears in the population.
Now, as we heard previously, well,
is that sufficient? Is 50,000 human
cells or 100,000 going to be too big a risk in these trials? So obviously, as suggested, one can perhaps limit
the procedure to a more differentiated proliferating cell as a precursor of
dopamine neuron.
Here is an example where we labeled
the precursor using a marker and then connecting it to a fluorescent
protein. So when the cell expressed this
marker, it was also green, fluorescent green.
And in that way, when we implanted the cells, instead of getting these
big tumors here, we managed to eliminate virtually all the tumors. So this is technically and scientifically
quite possible.
Now, also, as my last data slide, I
want to show you something that we actually published last Friday in stem cells
by the author Hedlund et al. And where
we actually labeled by a green fluorescent protein the very dopamine when it expressed
a factor that's unique to this cell that dyes in Parkinson's disease, and using
flow cytometry we transplanted only neurons and, quite surprisingly, they
didn't apparently need any neighbors because we had surviving cells that then
had eliminated completely or virtually completely all other cells types. So even though these are very challenging
issues, I believe there are technical and scientific ways around this.
So, in my experience, the factors
that come into play are the animals models, and there are reasonable ways of
choosing those including species and cross species. The animal species in new suppression methods
are known and can be addressed in an effective way. If I may add one thing is that you actually,
if you have a teratoma that's not growing any more, it will not have mitotic
cells in it. So there is actually a very
good way of addressing, I think some member of the committee said this, you can
actually look at earlier time points, and, if your graft is not growing, that
means that all the stem cells are depleted, meaning they had gone down their
natural developmental path. So, I think,
again, this can be understood and dealt with.
However, the engraftment implantation transplantation procedures I do
think belong not only in the laboratory but in industry. The number of variables you have to test here
are demanding, but I think they perhaps should be the same way as we deal with
pharmacology.
Potential tumorigenicity we have
talked about. Stem cell derived cell
identity and stability to me means that you really want to decide cell type
there for the patient you are treating.
I think that's an absolutely given and that there should be sufficient
criteria from the proposing clinical group or company to prove that and that
they have the desired cell type because the benefit must be present to the
patient.
And, finally, in summary then, my
point is that I think these models can provide both benefit and safety data and
should be carried out before clinical investigations.
Thank you very much.
(Applause.)
CHAIR URBA: Dr. Taylor?
DR. TAYLOR: Hi.
That was some elegant work. I
have a couple of questions.
So most of the abilities to sort
that you used were based on labeling with something like GFP. And, clearly, that's not clinically
applicable.
DR. ISACSON: That was just an example. In fact, we have other methods including cell
surface antigens and so I didn't have time to go through them. But I would say that there are at least two
or three antigens, again, depending on the specificity of the antibody for each
stage. So we actually have a paper
that's in preparation showing what we call a code, so each cell type, like in
hematology. As for hematology, there is
a way for obtaining a cell surface marker code that is useful.
DR. TAYLOR: Sure.
DR. ISACSON: So I don't think that's a necessary
requirement in the future, but an example that you can identify and isolate the
cell type.
DR. TAYLOR: And so are you saying you have codes for
various stages of differentiation then?
DR. ISACSON: Exactly.
DR. FRIEDLANDER: I have three very short questions for you.
In your cultures where you saw
SSEA-4 positive cells, did you see them associated with any other specific cell
type that might help you predict the future?
DR. ISACSON: I don't think so, but I would defer to
someone who looked at more cultures than I do, but I have not seen any
association. Except when you saw that
neural structure, the rosette formation, that actually does have a biology in
it. So cells are born in the center of
that crater and then migrate through and out.
DR. FRIEDLANDER: The second question is, did you ever look for
SSEA-4 in your tumors?
DR. ISACSON: Yes.
That's the one thing I tried to say at the end there. We started doing this actually in the mouse
teratoma, and by 21 days, I remember this very specifically, by 21 days in
mouse teratoma, we no longer see any growth, so that the teratology, the
embryological childhood tumor is formed.
Whereas with human cells, the SSEA-4 typically will go beyond several
months. And, obviously, that actually
sometimes leads that the animal is lost as is stated in the briefing
document. The animal dies from a
tumor. But you can use that marker
effectively to say whether there is still undifferentiating cells because they
will self renew.
DR. FRIEDLANDER: Correct.
DR. ISACSON: But the point biologically is that the cell
will tend to try to fully differentiate.
There's nothing that says that an SSEA-4 cell necessarily will maintain
it's proliferation because I've seen that in other animal species stem cells.
DR. FRIEDLANDER: So that leads me into the third question,
which is, in your primary cultures that you talked about first, how did you
measure the viability of those cells and do those cells actually have to be viable
or proliferating to actually have a positive effect in the graft?
DR. ISACSON: Absolutely.
I hope that that was a premise that went through. If you transplant dead cells, you get
absolutely nothing. Otherwise, we
wouldn't be doing this work quite frankly.
But let's see if I can answer the
first part of your question. Yes, so
it's quite easy to do that. You have
live viability and dye exclusion tests, so typically 80, 90 percent of the
cells are alive at the implantation.
But I want to point out something
that was mentioned previously. Somebody
called cataclysmic death. When you
implant cells into the adult brain, which I think is the desired host, there's
a lot of cell death in the first few days.
In fact, in the developing brain you
have something called developmental cell death.
Fifty percent of your brain has been sculptured from a larger number of
cells. But I would say in most cases up
to 90 percent of the cells will die in the first few days. So that's an important thing will live cell
therapy that the scientists and the clinicians and the companies understand
that they need to go through those paradigms and simulate it in a reasonable
way.
DR. SNYDER: I just had a brief, two-part question. In addition to looking for the gross evidence
of teratoma, did you ever see any evidence of non-neural cell types in the
brain that may not have been deforming but would have only been picked up by
immunocytochemistry, like smooth muscle or early evidence of cartilage, cell
types that are inappropriate to the brain?
DR. ISACSON: Well, I showed you a slide, right? Did you see that?
DR. SNYDER: I did at the low dose. You did not.
DR. ISACSON: At 50,000 cells.
DR. SNYDER: Okay.
So you never saw inappropriate cell types, not in teratoma formation,
but just forming there like vascular endothelium, things of that sort.
DR. ISACSON: No, that was what that slide showed. If you have 50,000 cells, you get all the
tissues in a teratoma. Wasn't that
clear?
Okay. But let me ask you a question another
way. So, yes, teratoma is exactly what
teratomas are. And you make me think of
another point which is that some neural cells, including glial cells, will migrate
away from the site of implantation.
So if you have a glial cell, and
this pertains to some of the previous questions, a glial cell is a
microglia. It has to be. It is on the non-neuronal tissue. Neurons will remain within gray matter and
never move if it's a fetal cell.
Progenitor cells in glials that will migrate along white matter bundles
in the brain quite extensively, and we have published this and others have, so
there is actually an importance in understanding what they do and where they
may end up in the brain.
Fortunately, I have never seen any
case of demyelination. In fact, some
people will argue that maybe they are beneficial to the brain. But they are not the functional cell that we
have studied for Parkinson's disease.
DR. CHIEN: I was going to ask you about the one
component I guess we haven't talked a lot about is let's say you have the
correct cell of interest, getting it to survive in the organ system or tissue
you're interested in is a challenge, for example. In most of the heart stem cell therapy or
cell-based
therapy that's been done, it's become fairly clear that very few of the cells
delivered in any approach and almost any cell survives long term. It's a very small percentage.
And I was wondering in the Nature
Medicine issue where your paper just appeared, there were two others that
also suggested that the transplanted cells actually either don't survive or
they acquire the disease, the graft actually gets the disease. And I was wondering two independent groups,
and so that wouldn't necessarily be a vote of confidence that this is going to
work. It's a no-brainer let me put it
that way.
DR. ISACSON: No, but let's --
DR. CHIEN: I'm trying to liven it up a little bit.
DR. ISACSON: I thought I described the facts more
clearly. So all three papers show
survival of cell for up to 15 years.
That's indisputable. All papers
show that most cells, in fact probably 99 percent, because I have looked at the
other transplants. We have this as a
shared collaborative effort.
When the transplants show what's
called protein inclusions, which doesn't mean that the cells don't work by the
way, this is also a fact, they do so at a fraction of less than one
percent. So my interpretation is
actually different than yours then, which is that it actually lends a lot of
optimists to this field.
And the fact that -- I mean one of
the papers speculates that maybe the transplant gets Parkinson's disease, but
there absolutely no evidence that's the same type of reaction as in the
patients because the transplant that we have looked at from other groups have
have a lot of microglia around them. So
it actually could be something that pertains to today's discussion is something
that the host may generate as a response to the transplant not unlike what you
may get in your heart transplants if you put in the wrong cells in the wrong
place.
But I actually think that you said
two papers, but they have the same basic tenet.
There's a fourth paper coming out that has exactly the same outcome as
our paper. I think it basically shows in
fact that there are variable outcomes and that the variable outcomes depend on
surgery, technique, cell dose, cells that don't belong in the transplant were
transferred including angiogenesis.
The paper that had most of these
cells had donor tissue that wasn't dissociated as a primary cell culture. So the whole tissue chunk was put in the
brain. And we know from science that
that contains donor blood vessels which have very high component of antigens
such as MHC1, which are, of course, T-cell targets and microglial triggers. So I believe that will be signs and our
observations will be explaining these findings.
But the bottom line is that cell
therapy, which was stated by all papers if I may add, have functional effect in
the patients for at least ten years, which is as well as perhaps heart surgery
can do currently, and this is a very prototypical and, as I hope I said, very
crude method at present, which will not be comparable I hope, I think to future
work on pure cell neural transplants.
DR. CHIEN: Okay.
That was transient though, I think, in one of the papers. I just read it.
DR. ISACSON: It's quite different than heart.
DR. GERSON: You, if I could, the preceding presenters
have suggested that there is cell loss at the point of the insertion of the cells
by whatever method into a tissue. Is
that a non event or are there cellular events or host response events due to
this cell death that is purported to take place? Is this something that we should give
attention to or something that we should ignore?
DR.
ISACSON: That's an interesting question
and I don't think there is an easy answer to that one. I mean fact of the matter is when you implant
cell, 80 percent die. The remaining 20
percent will grow quite well over a 15 year period. Those are the facts.
But I mean it may be important to
study that specifically. So if you
implant cells that may contain fibroblasts or endothelial precursors, they will
have a very massive -- I mean shouldn't exaggerate, but the immune system will
look at those very differently than nerve cells. So, again, you know, specificity in
experiment is required. Generalizations
are somewhat weak.
But I would say that if you as an
experimenter know yourself have composition, you can make reasonable
predictions. If somebody comes to the
FDA and makes a suggestion, I want to transplant these cell types, and they
contain a population that you know is going to be highly immunogenic, even
though most of the cells may die, they need to specifically show that there
isn't an adverse effect. But I wouldn't
say generally that it's a huge problem.
But in most of the work that we do currently, we spend a lot of time
designing the experiment and the therapy to avoid inflammation and try to avoid
all of the immune reactions.
DR. SALOMON: I think that I just want to pick up on that
too. What bothers me is this idea that
we just accept the way you put it that there's going to be 90 percent, 80
percent, different percentages thrown out of cells dying with a transplant.
Number one, I don't think that
that's something that you want to accept.
I think if you get the efficacy and you set up the protocols in such a
way that that's there in the early stages of a study, then that's what you
find, and I'm not saying that, per se, I'm biased against that, but as a
principle I don't think that's a good principle. You know, we do kidney transplants. We don't expect 90 percent of the kidney to
die after the kidney transplant.
I also want to say that I think that
there is an easy answer to the question.
That if you put a cell transplant in any tissue site and 90 percent of
the cells die within a few hours or a few days, that will have immunological
consequences both for innate and adaptive immunity.
DR. ISACSON: Yes.
So I want to differ with you on one point though, which is important,
just like pharmacology. Tissue organ
transplants, even though we are part of the same society, American Society of
Transplantation you and I, I would say cell therapy, and we have Gordon Weir
here, is a very different principle than organ transplants.
Your analogy I don't feel applies
because I'm not talking at all about that the transplant itself died. If you put in another organ and the organ
dies, that's a complete failure. But in
those patients that we have that have been off and on dopa for a decade, I
would say maybe 75 percent of initial dopamine population died, but that was
sufficient survival that these patients improved.
Of course I agree with you on the
principle, that in the future we would like to have 100 percent survival and it
may be possible to do that in various ways.
I just want to point out that, you know, it's an early young field and
we need to know the differences.
DR. TOMFORD: You mentioned the idea of the cells acting
individually versus sort of together.
What stimulates the transformation of the cells into a tissue? In other words, we're transplanting
individual cells. Eventually they're
going to form a tissue. Could you
comment on what happens there or what your thinking is?
DR. ISACSON: I wish I was able to. No, if I understand your question correctly,
for example, brain tissue is formed from the neuroectoderm. So at some point these cells talk to each
other and send signals that are translated into transcription factors that then
eventually tells the cell what to become, and they are spatially dependent and
time dependent. It's more like
initiating programs that run for a certain period of time.
To some extent, we can force these
programs. And experimentally the lab,
because we are not so good a simulating the environment, many scientists in my
field have now resumed what they call forced expression of a transcription
factor. So you express the factor that's
dominant and forcefully drives the program to which cell type you want. So you can do it either by intrinsic genetic
mechanism or try to simulate in the environment the time, the dose of those
factors that are typically in surrounding cells.
CHAIR URBA: One last question.
DR. FRIEDLANDER: I'd like to come back to that concept of the
effects of the transplanted cells on endogenous cells. And I recognize there's a difference between
putting a cell which you expect to become a dopaminergic -- producing neuron
and cell like a vascular endothelial cell, which may have some sort of
paracrine effect on tissue around it.
But should we be thinking a lot more
about the staging at which we put these in and what effect they could have on
endogenous cells in terms of resuscitating or maintaining diseased cells, which
ordinarily would die but perhaps not? So
how do you look, in fact, endogenous cells be maintained as opposed to newly
differentiated cells with tissue you put in?
DR. ISACSON: I think your question falls in the range of,
you know, can you implant a cell that stimulates the process in the host? The answer is probably yes. Can you put in a cell that does something
specific to heart and muscle or brain that you know you have lost? And the former yes means that it's almost --
the second time I've used the word, it's more challenging than the latter,
meaning when we put in nerve cells in the brain, we get the cells making the
rats less Parkinsonian because if we kill the cells, they are Parkinsonian
again. So they depend on the factor you
put in the cell.
However, over time you see, we
cannot today really measure that. When
the cell starts communicating -- and this could be paracrine by the way; I'm
not saying for example in ALS, which is a different type of pathology of the Parkinson's disease, even
though they're related -- there may be a different way of using glial cells to
substitute for cell that participate more actively there in the disease process
than I believe they do in Parkinson's disease.
So I'm open. I think we should all be open to innovation
and keep a very open mind of the choice the scientists and the clinicians for what reason and how they prove
they have efficacy. The most frustrating
thing in the field if you're in it is actually when somebody, we had that
opportunity, right. You put in a cell,
it dies, and the group says, well, the animal got better.
So you ask, well, how did the dying
cell provide the fact of that? And now
scientifically you're in such a broad scenario, universe of possibilities, that
you simply can't move because there is no hypothesis to test. But you would presume there was a substance
then, right, released by the cell that you perhaps could develop a drug
for. And I think that's perhaps beyond
the FDA/CBER premise of what stem cells do.
But since you asked the question, I
think a lot of people think about those issues with live cells, and I think it
will be a major scientific field in the future.
Homeostasis of cells, they talk to each other all the time.
CHAIR URBA: Okay.
We've heard all morning about where the cells go and how do we track
them. And that leads to our last talk of
the morning with Dr. Bulte, who is the Director of Cellular Imaging, Institute
for Cell Engineering at Johns Hopkins.
DR. BULTE: Good morning.
There are three things that I will talk about, and I've heard some of
the issues before that are very, very relevant for clinical translations. The three things: are the cells delivered
correctly; where do they go; and what do they become, assuming that they survive?
So during the last 50 years we have
made tremendous progress in organ transplantation, be it the pancreas, kidney,
together with the other development pioneered by Howard Hughes, the rise of the
modern airline industry so that the organs arrive alive. So the purpose of the meeting today is sort
of I think looking into a crystal ball what the future will bring, and we don't
know.
I do think that one of the things
that has not been addressed is perhaps the administration of cell
cocktails. As we know, transplanted
cells interact with the host, and they work in teams and perhaps they work
better with friends they already know, let's say, from the same background.
The majority of examples that I will
show do not involve the use of human embryonic stem cells for a variety of
reasons. However, the examples I will
show, and I will really show a variety of imaging techniques that can be used,
they're all applicable to human embryonic stem cells.
I'll start right off. There is no perfect imaging technique. They all have their limitations. So that's kind of unfortunate that we cannot
just pick one for all applications.
Now, I'm showing this example
here worked on by Steven Goldman and
worked on earlier by Evan Snyder, how we can transplant in the brain of animals
if it is myelinated disease, and we see here on the right that they shiver
less, and you can measure that on a graph, and perhaps the amplitudes can be
used as an outcome measure and we could possibly apply our imaging techniques
to optimize that.
Now, the reason why I showed this
example is that I'm fascinated by the histology shown here that these cells, in
this case they're human fetal glial progenitor cells, they're stained here in
red, for human nuclear antigen.
The question really is, what are the
limits of cell migration and cell proliferation. We have here a mouse brain, if we transplant
these cells in humans or in the spinal cords, are they at some point going to
stop? Where do they stop? So these are questions we cannot answer by
taking biopsies. We have heard
immunohistochemistry or PCR. Of course
that's being very complicated.
The relevance of imaging, and this
is in general for any sort of stem cell specifically for human embryonic stem
cells we're talking about, is the imaging, once we know in animals that they
work and we can measure, let's say, the outcome measures such as the BBB score,
that we correlate that with this
accurate cell delivery, the cell migration, and correlate the therapeutic
effect and then we can optimize the number of cells, the delivery routes, the
timing, and the dose. We know very
little about this currently, and, eventually, we can hopefully apply it
clinically.
Now listed here, and this list might
not all be inclusive, are ten techniques that are currently can be used, and
I'm talking about noninvasive imaging.
Not all of these can be used clinically, and I will show some examples,
and at the end come back to these, what the different advantages and
disadvantages are.
The only FDA-approved cell tracker
as of today is indium-oxine. It's
primarily used for immune cell trafficking.
The half life is short. It's 2.8
days. I'm showing here an example of
mesenchymal stem cells that have been labeled with indium-oxine. They have been administered IV. And what we do then, we image IFE Spect and
the SPECT/CT, so I'm starting right away with two imaging techniques. One only gives the hot spots; the other one,
the CT, gives anatomical information, and we fuse and superimpose them so we
can interpret the hot spots within its anatomical location and what you see is
mesenchymal stem cells are large. They
will clog up the lungs.
So you have to be very careful. You don't want to use emboli. You have to inject single cell
suspension. And after two days, you can
see here a hot spot where we have induced in dogs myocardial infarct persisting
for five days.
We have found some benefit. It's very hard to validate that because
myocardial infarct is very variable. But
what's interesting, that out of 10 or 20 million cells we inject, we can
quantify only about 50,000 arrive in the heart, and the rest is all staying in
the liver and the spleen being trapped.
And apparently a small number may be enough to induce a beneficial
effect.
Much of the work that I've started
with and I think clinically will be the way that cells are going to be
delivered involves stem cells with a magnetic dye, and super paramagnetic iron
oxide is a material that's also found in birds that migrate along the
geomagnetic field, dolphins, salmons, it's sort of like magnetic colloidal
fluid. It's FDA-approved as a liver
agent. How does it work? This is before, after injection. These particles IV are taken up by
macrophages. They have scavenger
receptors. These particles are opsonized
with proteins, and then the problem we face in the MRI and you get black spots.
So here is normal liver that
contains Kupffer cells, which are the scavenger cell, the macrophage that turn
black, and you can see the isointense tissue is a tumor. So macrophages take this up.
Stem cells are non-phagocytic and
there are now a number of techniques that introduce these particles into cells
most commonly using transfection agents.
So I come back to the three
themes. The first one, are the cells
delivered correctly? I think that's the
most important thing to start out with.
For the brain, we have different routes of injection, one is
stereotactic parenchymal injection. Such
is the case of Parkinson's disease.
The other one is intravenous infusion
that I have shown. It's less invasive,
but small numbers may reach the target area as compared to parenchymal
injection, but that may not always be without risk.
Another method is intraventricular
injection. I think this is a good route
of administration for diseases that are located throughout the brain, close to
the ventricles such as multiple sclerosis, the entire area can be
targeted. And, for instance, in cases of
stroke, what about intra-arterial infusion on the side of the stroke so we can
target ourselves?
Shown here is an example of the
latter of cells were injected in the carotid artery on the site of the middle
cerebral artery occlusion, and you can see here a moderate graftment on this
side with some cells also localizing in the hemisphere. And one of the things we have done is we have
monitored the injection process using laser Doppler flow monitoring. You can see here we stop our infusion, the
signal goes up and then it slows down again.
So if you have cells in the brain, they're going to impede the blood
flow.
And in this case here you can see a
massive engraftment here on that side.
You can see the LDF drop significantly and then we stop the
injection. So even if certain
administration routes IV where we inject larger cells, we have to be very
careful that we are not going to clog up capillaries when we target these areas
because actually we can induce mini strokes.
We can with MRI, shown one
hemisphere on the bottom, we can look in 3-D where our cells are localized, in
this case primarily in one hemisphere as you can see.
Now, not many places are able to do
MRI-guided injections. That will be the
future to deliver cells. We have
catheters that are compatible if the MRI that are steerable, they can be
targeted towards the area of interest.
I'll show you an example of work on
here with Dara Kraitchman in a myocardial infarct. We can see the MRI. We can inject the paramagnetic tracer
gadolinium that is bright. When tissue
is -- it goes in but it takes a little bit longer to go out because it resides
there. And we can precisely place a
catheter near the infarct so we can see.
And this is a frame movie. In
realtime it's much better. We see this
in realtime on the screen with special software and then we can inject our
cells right here and we can see that.
Now, mistakes are going to be made
by let's say less experienced radiologists and at this point we do not know
what the best grafting site is. You can
imagine if injected at a myocardial infarct itself it's a very hostile
environment. There's low oxygen, low
nutrients. Not too far away, perhaps at
the border zone it's best.
So if you can see where to locate
it, then we can correlate it to our outcome measures and the good thing it's
done in realtime. So if the therapy does
not work and the patient is being taken out of the magnet, goes home or in
intensive care, if it doesn't work it's not the injection itself. It's the cells that are failing, perhaps not
enough.
If we don't do this and it doesn't
work, we do not know what the problem is.
It may be that the injections were done incorrectly.
So I will show an example where it
really is important. This is the first
clinical study on MR tracking of cells performed by some colleagues with me in
the
So you can see here cells are
labeled with iron. They turn blue. We can stain that, inject it in the draining
lymph node, that of stage three melanoma patients; lymph node is resected; and
the resolution is really very high of MRI.
You can see here the cells through vessels going to lymph node from
another lymph node.
Now what have you learned from the
study? One is yes, in a clinical magnets
the patients with few numbers of cells we can see migration of the injected
nodes to nearby lymph nodes, and this is validated here with the
scintigraphy. So I think that's
important, but it's translational from the animal work.
What the real surprise was is that
in half the patients the cells were misinjected completely and nobody knew that
it happened until the MRI was done. So
they're not set up in the
So here's the lymph nodes where
the cells need to be injected in, and
dendritic cells need to stimulate T-cells in the medulla. You can see it injected in the subcutaneous
fat. So with indium-oxine you can't see
that. You see a cloud of radioactivity,
but you do not know where you are.
So I think this illustrates perhaps
also with spinal cord injections, you want to inject cells in a myocardial
infarct or in the striatum. It's
important to know where they are. So the
second thing is, where do they go?
This is an early example done with
oligodendrocyte progenitors, worked on with Ian Duncan. You can see here the injection site. You can see here the cells and they also
myelinate sort of like a B-line migrating away we actually sorted about 40mm
away from the injection site and it's interesting that the MRI distribution of
these cells also correlates to the new myelination that the cells induced. There's no normal myelin in these animals.
We can also do that in vivo and we
put cells in the ventricles so we don't put it in the parenchyma. We can see cells moving out of the ventricles
into the brain. They myelinate and we
can correlate with histology.
Now we can look at 3-D
reconstruction. Now this is an example,
this is Evan Snyder's cell line, the C17.2 of the cell line that is
immortalized. So it continues to
proliferate. It does not form
tumors. So I think this is benign
proliferation. But you can see here, in
3-dimension, you can see those fingerlike projections, how they grow into a
glioma, like a glioma also into the brain, and I think it's very hard with
histology to really look at these invasive patterns that pop up again. You see here all these fingers coming out
where they migrate probably along white matter tracks.
Now we can also, when we do our
histology, this is an ex vivo image. So
it's noninvasive. We can determine the
exact plane of cutting. If we don't know
a priority of where our cells are, we may be unlucky and cut the wrong
plane. We can do this in any dimension. We can slice it up. We have the computer NCR cells there, that
black spot, so we know we can focus in on that area.
The resolution of MR is such that
when we look at cancer vaccines we see here cells homing into the medulla. There is some vaccine injected into the food
path, so the direct cells, immunosentinel cells, they pick it up and they go in
the lymph node and we can see here very small numbers of cells. And this is a lymph node of a mouse so if you
know how small these are.
Now, what about quantifying the
local number of cells? With MRI it's
very difficult in certain cases. It does
work in this case. It was very homogenously
distributed. So we sampled the number of
black pixels on the MRI. We made single
cell suspensions off these nodes, and, because of the iron oxide labeling, the
cells were magnetic. We could isolate on
a magnetic cell separation column and we, in this case, actually can use MRI to
quantify cells.
I'm not going to show it. We are looking at immunoadjuvants to see to
enhance the trafficking and it's very nice.
We can do this over time non-invasively.
What can we learn from islets
transplantation studies? MR tracking has
also been pursued for a group in
So the problem of islet cell
transplantation is really the immunosuppression, which is very toxic to the
beta cell. We don't know how long the
islet survives.
Another approach is to protect these
cells in semipermeable alginate capsules so the insulin can go out but the
cells are protected from antibodies. And
there is now some clinical work ongoing in
This is, again, an example of how
these things can be infused. This is an
interventional radiologist, Arepally and Howard Hughes Fellow Brad Barnett, so
they can be targeting the portal vein and then we can engraft these capsules in
the liver and we can exactly follow the grafting process. We can do that in realtime. I don't know if the liver is the best place,
but it's an example how we can monitor the grafting.
So now one of I think the most
important question once the cells have delivered correctly, that's the most
important thing. But the most important
question is, what do they become? And we
need a reporter gene for that.
I've heard a lot about cell
survival, how many cells survive. A
technique that's already mentioned is called bioluminescent imaging that uses
the luciferase from the firefly, we get luciferin and photons are being emitted.
And it's interesting some work I'm
doing with Doug Kerr is when we started these experiments, now almost two years
ago, that we had a very hard time to get good survival of cells, and we found
out that immunosuppression, the direction you choose, is a major
determinate. And standard cyclosporine
that people use from other studies is actually not best. There may be other regiments that enhance
survival. So I think it's very important
that you can monitor cell survival and use it.
Now, unfortunately, it cannot be
used clinical. Why? Light has a limited penetration depth. You can also only do this in white mice. If there's pigment, melanin, it blocks the
light, so there are limitations here.
The other thing you can do is you
can put the luciferase on the reporter gene.
For instance, we have done that on the GFAP when they become
astrocytes. So if you don't put it on
the promoter and you have glial-restricted precursor cells and the control is
kidney cells, they all light up because there's no promoter. But then you put it under GFAP, you can see
that only the glial-restricted precursor cells will light because they did then
change to astrocytes and not the control cells.
You can do this for many reports in
the interest of the neurons, of course.
I think the key is you can see when it happens. Because this is noninvasive, you can stick in
the mice two times a day if you want. Some
gas anesthesia, they can handle that, and you can follow this over time.
Bioluminescent imaging has been developed
by Christopher Contag at
Do they form teratomas? And I learned something new today. There was an auto teratocarcinoma and I
exchanged it here. I forgot to do it in
the first one. So the issue is, and I'm showing
the example of one cell, let's say people say adult cells are much safer than
human embryonic stem cells. People do
magnetic cell sorting or perhaps flow cytometry, but I think if there is only
one cell that is undifferentiated, that one cell can still cause trouble.
This is work done also at
So this is the bioluminescent
experiment. So you can see that both
tumors grow. They make teratomas. They increase proliferation to get more
signal.
Now, at this point, again,
ganciclovir is given, right, and only the one with the thymidine kinase, which
is a triple one, will act as a suicide gene.
So you can see the tumor disappearing in here.
So I think this is so cool that you
have one reporter, you can image it, and at the same time you can intervene
with the drug if it gets out of hand. So
this is the thymidine kinase system.
You can also monitor with positron
emission tomography like you see here, the tumor growing and then it goes away
with the ganciclovir.
Now, where is this clinically, there
are now four clinical studies including islet cell transplantation. As far as I am aware, and I may be wrong, I
know of one study with thymidine kinase also performed at Stanford by Sam
Gambhir and others. They have checked
the T-cells in glioma patients for immunotherapy. You can see here T-cells homing. There are
two tumors here and some peripheral artifacts the system.
Now, so these other techniques I
think that are clinically now being implemented at this point, in 2008, 2007.
There are a lot of things brewing
around the corner. One of those I think
is the first time that it's being done.
We have cloned an artificial reporter gene not found in nature, based on
rational design, and this reporter gene is chock full of amide protons which
have a different resonance frequency. We
can saturate those protons. They
exchange with water. We always image
water with MRI. We can give a specific
radio frequency pulse that turns that on.