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.