So here's a glioma cell transfected with a leucine rich protein.  It has all those amide protons on the left.  This is a control plasma.  We turn it on and we see only our tumor lighting up.

            What is the beauty of the system?  It's that we can design a family of reporter genes with all different resonance frequency and we can saturate specific frequencies so we can do multicolor imaging, sort of analog to fluorescence where you have all these different wave lengths.  I think it will be really important in the future to be able, just like immunocytohistochemistry to look at multiple cell populations simultaneously specifically to look at these interactions, also when we co-graft multiple cells.

            So this is a summary of the techniques that are currently available.  It's a very crowded slide.  I am sure they will be posted online so you can maybe look at it later.  I think the three that are most closely to becoming clinically important when we start to use human embryonic stem cell therapies in humans are listed here: positron emission tomography; SPECT or gamma camera imaging for systemic injection; and also MRI as you note that it's primarily for correct delivery and local engraftment, less for systemic.  The resolution are here.  The number of cells, it's possible to see single cells at the right resolution.  I should also say that CT and ultrasound and there are things happening right now with contrast agent that we'll hear more about.

            If you're interested, some of this work is covered in deeper context in this review article that came out last month, if you want to look that up, with a lot of references.

            So, in conclusion, the iron oxide label cells and compatible catheters, and, again, the few centers set up as of yet because the special catheters allows MR-guided realtime targeted delivery and, essentially, accurate cell delivery and it can have various application.  And then after we do that, then reporter genes are needed to serve as a beacon for cell survival, uncontrolled cell proliferation, as well as cell differentiation.

            And I think that at the end it's probably going to be a combination of techniques, so why not put in a PET reporter gene in a cell and at the same time label it with iron oxide so we can do both delivery and follow it.  I think there's an unrecognized potential for interventional radiologists who will do this in their practice or in their academic setting.  That it is something that imaging people always talk about they want to track cells to see where they go, to follow.  Yes, but more important is to deliver them in realtime using specific interventional instruments and catheters and devices.

            So I do think that needs to be an integrated part also with discussions with the stem cell people is how they want to deliver these cells and where because of those issue.

            I should acknowledge a lot of people, also people I borrowed slides, and NIH funding.  We have worked with a company in Baltimore with mesenchymal stem cells.  We have islet cell protocol, got a lot of islets from Justin Diabetes Center.

            And I leave it here.  Thank you.

            (Applause.)

            CHAIR URBA:  So we have time for a couple of quick questions.

            DR. SNYDER:  Jeff, which of the markers that you used to mark cells are already being used in patients and are approved for patients?

            DR. BULTE:  Yes.  So the only one that approved is indium-oxine and they are the ones that have been used in patients just recently the thymidine kinase, the reporter gene from the herpes simplex virus and the Theradex label.

            DR. SNYDER:  And those could be applied to stem cells in your opinion?

            DR. BULTE:  Yes.  There's no difference.  Well, you get a practical issue is that embryonic stem cells may be harder to transfect.  You know, there are other issues there.  They grow in embryo bodies.  Do we get the label.  But, principally, yes, there's no difference, right.

            DR. TAYLOR:   So a number of these MRI studies with Theradex we and others have done and shown that as cells don't survive the label is taken up by macrophages and other cells and actually doesn't accurately reflect the cells you transplanted.  So I'd like your comments on that in terms of clinical relevance.

            And then, two, that's clearly not a quantitative measurement at this point.  So can you talk about what the limitations might be clinically then in terms of how we over or under estimate effects if cell number is really critical to some of the deleterious side effects?

            DR. BULTE:  Yes.  I've heard the question many times before.  There's a limitation by labeling cells with a fluorescent dye, a lipophilic dye, Theradex indium-oxine it leaches when a cell dye is taken up.  In case of the Theradex, the contrast itself disappears quickly since the macrophage is biodegraded.  But the Theradex application is really the correct delivery in realtime, initially.

            After that, we don't know if the cells are dead or alive because the contrast is going to stay there.  Another problem is the label disappears quickly if cells start to proliferate uncontrollably.  So the holy grail is an MRI reporter gene.  It's the holy grail.  And several centers have been working on that, including our artificial approach.  People use ferreting.  We are not there yet, so that's one way.  Yes, so that's a potential artifact.

            Your second question was about -- what was your second question?

            DR. TAYLOR:   How to do with the lack of quantitation?

            DR. BULTE:  Yes, yes.  So the MRI is not quantitative.  Reason being it's very simple, we do not a priori if our cells are clustered in groups or if they homogeneously disperse.  That affects the MRI contrast differently and that's the problem of that.  So the way to do this is indium-oxine or PET bioluminescent imaging, you know semi-quantitative.

            What does that mean?  We do not really know the depth where the cells are, so we have to correct for the attenuation of the signal.  We have to make an estimate so it's semi-quantitative.

            Also, by the way, indium oxine leaches out, binds transferring.  You get a lot of artifacts, liver uptake, spots that the cells actually are not there.  Each technique has it's problem.  I'm just hoping in ten years we have a good MRI reporter gene that's safe.  The reporter genes we are using are artificial, so we're creating things that don't exist.  It's a whole other issue we have looked at and everything looks fine, but I don't know.

            CHAIR URBA:  Dr. Chien?

            DR. CHIEN:  I was going to ask you, this is a two-part question.  One is, what's the minimum resolution that you can get?  I mean how many cells can you pick up?  I know it's obviously not a single cell level detection.  So what is the minimum mass of cells that you can actually detect, 100,000?

            DR. BULTE:  Can you put it on four again, sir, on the visual?  Real quick I can also tell you.  Actually I should point it out because it's a very important question.  It's in this column. 

            The sensitivity, the number of cells, for the reporter genes, bioluminescent imaging, and our artificial , and you go five to ten thousand cells, which you have not talked about them.  PET, actually, I have asked the experts.  I've not gotten an e-mail yet, but I think it's a low number, perhaps 100 or so.  The MRI part with the side, that's a tricky one.  And, again, it's not an easy answer.  It depends on the field strength.  The higher the field strength the better.  These particles use contrast.  It depends on the voxel size, the resolution.

            I would say clinically we can see as much as a 1,000 cells in the lymph nodes in animal systems.  We can use bigger magnetic particles, but we can see single cells in vivo at high field in an animal, but this will be clinically translated.

            DR. CHIEN:  Yes, that's what I thought.  And the other question I had is, don't you think you could, like many other surgical protocols, because a lot of this is kind of surgery interventional, are you going to optimize the entire delivery protocol in animals without having to have all these sophisticated imaging things because then you can use, you know, sort of realtime, single- cell-level-almost analysis with lacZ reporters and optimize that and then just go into humans with an optimized protocol that you don't have to demand realtime feedback of the delivery of the cells while the operator is delivering it on the table?

            DR. BULTE:  Am I allowed to disagree with you?

            DR. CHIEN:  I didn't say that was the case.  I just asked what you thought.

            DR. BULTE:  Yes.  Reporter genes are not realtimed because you get the substrate and it takes a little while, you know, to accumulate in that case.  I think in the case of myocardial infarct, if somebody is poking around at the area of the infarct, at the moment within seconds at least it will be known in realtime and the patient is dying, you know, and is claustrophobic and is in the system, I think it is very important to do that fast, not to see afterwards if the cells were injected at the right place.

            Same with the lymph nodes, you're suggesting it's not important to do in realtime, but --

            DR. CHIEN:  I'm saying is is that once you work out the protocol, okay, so for example, for direct injection in a specific location in the heart for example, that I don't know that you necessarily -- you need readout to know where you're at in the heart, but you may not necessarily have to get realtime disposition of the cells in the heart because you've already optimized the protocol for delivery.

            DR. BULTE:  But if you don't know where you've put the cells, you want to find --

            DR. CHIEN:  Well, no.  You can figure out where you put the cells by electrophysiological feedback, like NOGA and things like that.  You don't need to see it, right?

            DR. BULTE:  Okay.

            DR. CHIEN:  Okay.  Anyway, I don't want to argue with it.

            CHAIR URBA:  Dr. Firpo?

            DR. FIRPO:  You know you mentioned that a suicide gene in, again, acyclovir selection and there's a couple of papers out on that now, more than just the one you talked about.  But are you aware of any studies where people have done selection to kill the tumor and then allowed the mouse to live after that to see if it comes back?

            DR. BULTE:  Following treatments?  Okay.  No, I'm not aware.  I don't know how long the -- you know, if it kills all the cells.  From the signal it looks like they're all dead, but if a few cells remain.  I think it's a matter of dose.  I've heard if you inject small numbers of these cells that they may not form tumors.  It's just like injecting a subcutaneous tumor.  If you inject 100 cells, you don't get a tumor in a nude mouse, but you have to give maybe 100,000 cells.  I don't know. 

            It just depends on how many cells survive.  I guess you could do experiments by dosing 10, 50, 100, 200 and see if they form tumors that the same scenario will apply, that the number of surviving cells could again form tumors.  So it depends on the specific setting.

            DR. GOLDMAN:  Jeff, to just follow up on Dr. Chien's question.  So the MR resolution is going to depend upon the cellular density, of course per voxel.  So just as a base level of maximal resolution, how many cells are required per technique, whether by proton or by polyamide, cells per voxel can be detected let's say in a clinical 3T magnet?

            DR. BULTE:  Yes.  So for clinical 3T magnets I think it's fairly safe to say for MRI within a voxel somewhere between 500 and 1,000 cells, and we're talking here 500 micrometer resolution in each direction, like a cube.  The MI probe is less sensitive.  Currently we can only really do it very well in animal magnets of higher fields.  The sensitivity is about somewhere around 10,000 cells I think at this point.

            The most sensitive tracer are these iron oxides.  They're the most, so that's going to be the limit.

            CHAIR URBA:  We'll take two more questions.  Dr. Woo and then Dr. Weir.

            DR. WOO:  Yes.  We heard from previous presentations that the formation of teratomas is those dependent of the undifferentiated embryonic stem cells, and the threshold may be around 100,000 plus/minus.  And yet in one of your slides, I think it's the first slide of section four, you show a teratoma forming in a mouse and you indicated you only need one cell for causing trouble.  So I'm kind of confused as to what is it that is really needed to form a teratoma.

            DR. BULTE:  No.  There's a misunderstanding and I understand your misunderstanding.  That slide was with one bone marrow stem cell that reconstitutes the entire bone marrow to see the power that when you have one cell that it starts proliferating.  Eventually you can see that.  So that one cell was the bone marrow reconstitution experiment.

            The other one that I followed up was the teratoma slide.  So I understand the confusion, but they are separate studies.

            DR. WEIR:  You've given us a wonderful look into the future.  But I wanted to ask about just conventional radiology techniques as far as monitoring an inject site, for example, for teratoma formation.  Just how sensitive do you think it could be if you were looking at the spinal cord or if you were looking at some other site as far as getting a clue that there was a teratoma being formed?

            DR. BULTE:  Yes.  So currently the way it will be done right now is purely anatomical.  Right?  We get a soft tissue mass or perhaps a skin or keratonin, whatever.  So they'll anatomical at that time.  It's just like, in general, in tumor formations, the whole issue.  By the time a tumor is detected anatomically with MRI, it's already too big.  So you want to have more sensitive methods to detect it in its very early stage.

            So how many cells you need in order for it to detect it?  I think it's the same issue as the sensitivity of these cells perhaps.  So I think the bottom line is, is the sensitivity equal or higher than the number of cells that are needed to form a teratoma.  So if you can detect fewer cells than are needed to form a teratoma, I think that's a good thing.  So at that point you can maybe see it earlier than the cells are able to form a tumor, something like that.

            CHAIR URBA:  Thank you.

            It's time to move onto the public hearing part of the meeting.  I'd like to share this announcement before we start.

            Both the Food and Drug Administration and the public believe in a transparent process for information gathering and decision making.  To ensure such transparency at the open public hearing session of the advisory committee meeting, FDA believes that it is important to understand the context of an individual's presentation.

            For this reason FDA encourages you, the open public hearing speaker, at the beginning of your written or oral statement to advise the committee of any financial relationship that may have with any company or any group that is likely to be impacted by the topic of this meeting.

            For example, the financial information may include the company's or a group's payment of your travel, lodging, or other expenses in connection with your attendance at the meeting.  Likewise, FDA encourages you at the beginning of your statement to advise the committee if you do not have any such financial relationships.  If you choose not to address this issue of financial relationships at the beginning of your statement, it will not preclude you from speaking.

            Our first speaker is Ms. Amy Comstock Rick from Parkinson's Action Network.

            MS. RICK:  Thank you and good afternoon.  My name is Amy Rick and I am actually here in my capacity as president of the Coalition for the Advancement of Medical Research, which is a non-paying position by the way.  But I also serve as CEO of the Parkinson's Action Network.  Aside from that I cannot think of any conflict of interest that I have.  I drove my own car from my home this morning.

            The Coalition for the Advancement of Medical Research is a coalition that was formed in 2001 as a direct response to the President's policy restricting federal funding for embryonic stem cell research, as you all know, for lines that were derived after August 9^th, 2001.  The coalition is comprised of patient groups, individual research institutions, some which are represented here,  as well as associations of researchers.  We have over 100 members.

            Our mission remains fairly consistent, which is to promote regenerative medicine with a prime focus on the lifting of the President's policy on the restrictions for federal funding.

            As I'm sure you all know, human embryonic stem cell research has been quite a focus for patient groups over the years.  With the legislation that has gone through Congress, as well as the scientific breakthroughs that do get a fair amount of media attention, disease groups, Parkinson's, spinal cord injury, diabetes, many cancer, many, many others, the patient advocacy community as been quite focused on the progress.  In fact, to the point that a few years ago when the legislation was a hot topic in Congress, we were fearful that the patient community was at a place where, if the legislation passed, that they would be expecting FDA approval and treatments immediately.

            I think we are not at that place any more.  I find that you have a very educated patient or affliction spinal cord injury population who understand in a very sophisticated way the issues surrounding human embryonic stem cell research, both potential as well as risk. 

            It is quite enlightening to the patient community that, in spite of the President's restrictions laid down in 2001, that the science has moved forward, not as quickly as it would have but for the restrictions, but both with private funding as well as the emergence of -- merging a fair amount of state funding the science has moved forward and it is inspiring to patients and we commend the FDA for actually having the advisory committee meeting to address some of the issue that we hope you will be facing in the coming months and years as you begin to see applications for approval for clinical testing using human embryonic stem cells.

            As you all know, we actually anticipate even hopefully more eligibility for federal funding in the coming months and years as the three main presidential candidates are all from the U.S. Senate.  So we happen to have on record that all three of them voted twice in favor of the stem cell research enhancement act which would have allowed federal funding for human embryonic stem cell research on lines derived, would have lifted the President's restriction if they were left over embryos from IVF clinics that otherwise would have been discarded and a few other limitations.  So given the fact that we have this record, we do anticipate that in 2009 more research will, in fact, be funded using federal funds.

            It is in that context of hopeful anticipation for this field that I want to raise two cautions to this committee.  One would be our request on behalf of CAMR and the patient communities that in spite of the high visibility and great amount of controversy that there has been around human embryonic stem cell research that you not put an extra layer of risk averseness or safety requirements simply because the nature of the visibility or the controversy on the issue.

            It is the risk benefit analysis, if you will, which I understand is extremely complicated on all these issue, not unique to stem cell research, and is not formulaic in any way, but the fact that there's external controversy I would plead that you do not, as scientists, allow external controversy in any way to interfere with your analysis.

            And the second caution that I would raise is, in fact, not as directly related to stem cell research.  But in an article in Bloomberg News this week, actually about this meeting, Dr. Robert Lanza was quoted as saying, in this field there can be no risk whatsoever.  Now, I know that all of us know you can't take that literally because there's risk in everything in life.  But I ask that you -- for the diseases that we're talking about in this room this morning, Parkinson's, spinal cord injury, juvenile diabetes, cancer, if you could, these decisions cannot be made in a vacuum.  We are talking about risks, but you're talking about risks as you know balanced against the life of living with a chronic, progressive disease like Parkinson's.  You're talking about cancer, juvenile diabetes.  There is the ever present, horrible risk of living and dying a miserable death with one of these diseases or with this injury, and if I ask, as you always do, to keep that in mind as you're assessing the risk of some of the very serious questions that we heard about this morning, scientific questions.

            Thank you.

            CHAIR URBA:  Thank you very much.

            Next we'll hear from Dr. Chris Airriess, California Stem Cell, Incorporated.

            DR. AIRRIESS:  Dr. Urba, committee, thank you for the opportunity to speak today.

            First off, I am speaking on behalf of a private company, California Stem Cell, and I'm an employee of that company.  We are actively developing therapies and are engaged in preclinical development currently in spinal muscular atrophy and ALS, as well as spinal cord injury, so some of the diseases of the previous speaker has just brought up.

            The stem cell research field is currently at a turning point.  Research findings enabling the scalable, current, good manufacturing practice production of human cell populations at extremely high purity move the therapeutic potential of stem cell derived treatments from the real of hope to that of practical application.

            At California Stem Cell we have conducted extensive safety testing along the lines that have been discussed here this morning on our human embryonic stem cell lines, as well as the high purity, differentiated human cell products of these lines.  Studies such as these help to minimize the risks of potential therapies to prospective patients.

            Until the technology for safe and scalable generation of patient specifics outlines has proven, compassion compels us now to use existing technologies to develop therapy as addressing devastating and currently untreatable human disorders.

            With appropriate safety testing and careful administration of safe and effective immunosuppressive regimes, emerging therapeutics based on current human embryonic stem cell technologies are an immediate and viable solution for treatment of the widest variety of such conditions.

            We've been highly impressed thus far with the dedication and insights of the team of Mercedes Serabian and her colleagues here at CBER.  Two items in particular that we feel will be conducive to the efficient development of stem cell-based therapies are continued opportunities for early interaction with the FDA through the pre pre-IND process.  This has been very beneficial to us so far.

            We've got a lot of valuable feedback and we encourage the continuation of this process.  And we would like to see clarity, which I'm sure is coming, on the FDA's requirements for preclinical efficacy in safety for stem cell therapies in the form of a formal guidance document.

            Again, I thank you all again for the opportunity to speak today, and we also thank our key partners, the ALS Therapy Development Institute, Families With Spinal Muscular Atrophy, Johns Hopkins University, the University of California, Irvine.

            CHAIR URBA:  Thank you.

            Now, notice of this meeting was made available to the public and anyone wishing to speak was asked to register prior to the meeting.  However, we have a few moments of additional time if anyone else in the audience wishes to address the committee at this time.

            If not, we'll adjourn for lunch until 2:05.  Thank you.

            (Whereupon, the foregoing matter

            went off the record at 1:02 p.m.

            and went back on the record at

            2:07 p.m.)

 

 

 

 

 

 

 

 

 

 

 

 

 

       A-F-T-E-R-N-O-O-N  S-E-S-S-I-O-N

                                     2:07 p.m.

            CHAIR URBA:  If everyone could please take their seats, we'll begin the afternoon session.

            Okay.  So we'll get started with the afternoon session, which, if you remember Dr. Bauer's presentation this morning, was to address three rather broad questions.  And the first question up for discussion is on the slide that's before us.  And I guess just to set the stage, I will read it.

            Inappropriate Differentiation and Tumorigenicity, and what we're being asked to consider and discuss are:

            Criteria for selection of clinically relevant animal species or models that support engraftment of the administered human embryonic stem cells, for example, optimal strategies for evaluating potential host rejection of administered stem cell-derived products?

            What may be the optimal site of implantation in the animals in order to obtain meaningful test results?

            What sorts of study durations are required? 

            And what is the most appropriate dosing method, that is, absolute undifferentiated human embryonic stem cell number versus percentage of undifferentiated stem cells present in the product to extrapolate cell doses tested in animals to plan the clinical dose?

            So that's where we'll start.  And Dr. Goldman, if you'd like to kick off the discussion?

            DR. GOLDMAN:  Sure.  So trying to break that down a bit more operationally, both tumorigenicity and inappropriate differentiation can be looked at as functions of model and disease environment, of course of site, site of implantation especially for nervous system targets, function of the survival and the study duration, the cell dose, of course how that cell dose is calculated, whether before or after transplantation or as a function of both.  And then, of course, both tumorigenicity and differentiation state have to be looked at as a function of the immune state as well whether we're dealing with immunocompetent patients, I mean suppressed patients, or as far as disease models are concerned, immunocompetent, suppressed or deficients.

            So essentially we're looking at a combinatorial function of all those variables and we need to establish an algorithm for being able to apply these as uniform criteria across cell types, and at least in some reasonable fashion across disease models.

            So I'll start just discussing at least the issue of tumorigenicity from the standpoint of just presenting a couple of questions for the committee, and then of course looking at differentiation or inappropriate differentiation from the same standpoint.

            Tumorigenicity strikes me with the most important issue, at least in my own mind from what we heard this morning, is what constitutes a tumor, how to define it?  Of course there was already some debate, if you will, in terms of whether a tumor could be benign, whether a histologic benigness connoted physiologic outcome benign nature.  The issue of whether infiltration and to what degree was a measure of tumorigenicity and to what extent that precluded the use of ES or ES derivatives.

            And then really in a more fundamental level, how looking at histologic tissues can we define a tumor?  Should we look at the division rate, the survival rates over time, the expansion rates of the population over time?  Do we need to assess that from the standpoint of the proliferation or turnover rates of need of cells in the organ into which the cells are being transplanted?  Or as we looking rather for an absolute absence, as the case may be, of proliferation or undifferentiated expansion?  What kind of markers can be used to define anaplasia?  What kind of markers can be used to define undifferentiated expansion?

            Now, of course, we heard the ES markers used as indices of the persistence of undifferentiated cells in grafts.  But worrying about more from the standpoint also of the things not discussed.  What happens as ES derivatives are implanted?  And if those derivatives are still at the progenitor state and undergoing expansion themselves, what allows us to define whether the undifferentiated expansion of already committed progenitors, when does that become a tumor?

            There are instances, some mentioned earlier, some others in literature, of undifferentiated neuroepithelial expansion of ES-derived neuroepithelial cells.  This may be a problem in a variety of organ systems.  So it's not just a question of undifferentiated ES persisting in a graft, but also of their mitotically competent derivatives.  At what level do we need to exercise control over their expansion?

            At what level do we permit the implantation or introduction of any persistent, undifferentiated ES cells, or, as the case may be, still mitotically competent derivatives thereof?  And what kind of markers can we use to define the existence of these cells?  The cancer literature, of course, has a number of markers defined phospho-Akt survive in a variety of proteins that can be used for identification purposes, but they tend to correspond to markers of anaplasia or uncontrolled expansion. 

            Cells that are no longer controlled by normal cell cycle checkpoints.  The issue is going to be, I think, for this field as progenitors are transplanted and then undergo persistent expansion that will not necessarily express markers of anaplastic transformation.  At what point can we define that as normal expansion versus uncontrollable?

            So those are all questions, but they really come down to the point of, what can we tolerate in terms of implantation of potentially undifferentiated or partially differentiated cells that are still capable of expansion?  So I would broaden the issue  behind just ES cells and behind just

ES-derived teratomas or teratocarcinomas.

            So that segues into the issue of differentiation and to my mind it's very much a parallel question.  What constitutes inappropriate differentiation? 

            And so it's essentially by definition.  Ectopic differentiation of a functional, mature phenotype in an area in which that phenotype would not normally be present or in numbers in which that phenotype would normally not be represented, would represent inappropriate differentiation.

            But we don't yet have criteria by which to establish whether an inappropriately differentiated pool is dangerous.  Under what circumstances inappropriate differentiation may be relatively harmless?  In what cases it may be beneficial?  There are no general rules where this is concerned and these outcomes may depend very much on the disease state and the disease target.

            In my own field, if we put glial progenitors into say a demyelinating lesion and we're looking oligodendrocytic differentiation and we see astrocytic, well that would be inappropriate.  If we go into let's say a stoke bed and pick up astrocytic differentiation, well, that would be beneficial.  So this is very much a function of the disease target as well as the cell type that's being implanted.

            Then even appropriate differentiation into a set of phenotypes that is correct, if you will, for the organ may still not yield an appropriate functional outcome, and so the necessity becomes to match, essentially, the quantitative representation of phenotypes generated from progenitors that may be capable of giving rise to multiple phenotypes.

            For islets, for islet progenitors derived from ES, it's one thing to look for beta cells as an appropriate cellular target, but one may expect these progenitors to potentially give rise let's say to alpha or delta cells, but potentially antagonizing the effects of the beta cells.  For example, in the examples mentioned earlier of dopanergic production, well those same progenitors as derived from ES can also give rise to serotonergic and gabergic cells which in vivo/in vitro can potentially antagonize within the steroid of some of the effects of the dopanergic neurons.

            So even when we have ES that are giving rise to the cell types of interests and even the precise representation of cells that would normally be derived from those progenitors, unless the proportions are correct that we're going to need, we may see if not dangerous, then at least counterproductive effects that would potentially diminish ultimately efficacy, as well as in some cases potentially presenting safety issues as well.

            And so we need to define and characterize the state of differentiation that we want cells to be implanted at, what types of lineage potential they have at that point, and it becomes a big of a ying yang in that by the time we have cells that are sufficiently mature to yield the cell type of interest with the highest possible fidelity, in other words the purest possible population of the cell type of interest, well by that point we're far enough down the lineage and the cells may be post mitotic, they may not tolerate the engraftment well, and so we may not have a viable, engraftable cell population.

            On the other hand, the still mitotically competent cells that may have much greater efficacy, as well as survivability upon transplantation, may be those that potentially may give rise still to undesired phenotypes that may still be capable of uncontrolled expansion.  And so this is the, essentially, dilemma I think that we all face is establishing what is the appropriate stage of differentiation for transplantation and how essentially enriched or purified do those populations have to be at the time of transplantation?  So I'll leave those as essentially entry points for discussion.

            CHAIR URBA:  Do you have a couple of comments on what you would do preclinically to identify and answer those questions?

            DR. GOLDMAN:  Well, then it's a question of disease target and, specifically, the cell type of interest.  I think these answer are going to be, to the extent that answers can be derived for any, but I think that conceptions are going to be driven by exactly what disease target and exactly what cell types are being used.

            So, for example, my own target of interest, the hypermyelinating disorders.  This is a set of disorders where glial progenitors, as Jane was discussing before for example, can be productively used.  However, here's little ability to control the oligodendrocytic versus astrocytic differentiation of these cells.  We're still learning what the rules are.

            But we know that by the time the cells are oligodendrocytes, oligodendrocytes  at least primate and human, oligodendrocytes  are every bit as post mitotic as neurons, and so they become very difficult to engraft.  And someone in order to have a working strategy has to be able to give the cells are progenitors.

            And so then the issues of persistent expansion and potentially unregulatable differentiation come to the fore.  And the imperative then becomes matching up the disease target to the phenotypic potential and likely in vivo activity and behavior of those cells.  That can be productively done with some disease targets, not so much with others.  So I don't know that there's a one size fits all answer to these.

            CHAIR URBA:  But if you were going to take the oligo cells to trial, you would take the earlier stage of differentiation, which is more susceptible to some of the problems we talked about.  What preclinical things would you want to see have done before you actually went and did that clinical trial?

            DR. GOLDMAN:  So I would want to see a definition of the expansion kinetics of that cell population over time in vivo in animal models, in animals models that severally replicate the disease target of interest.  The net population expansion from the standpoint of how many cells do you have at given points of time after the initial transplantation, taking out for very long periods of time essentially for the experimental models we typically use, mice and rats, the life of the animals.

            I would want to see what the mitotic rate, the fraction of cells in cell cycle was as a function of time.  And what point the mitotic index of the implanted cell population fell to that of the background, essentially the native cell population of the same phenotype in vivo, in other words of the host cycle kinetics, at what point do they actually match up.

            At that point I would want to make sure that there was no heterotrophic migration of the cells into in this case non-white matter areas, in other words that we weren't seeing heterotrophic foci oligodendrocytes in areas of gray matter for example.  I would want to see that there was no overt anaplastic transformation of any of the cells at any point.  And that there was no evidence of heterotrophic differentiation to non-glial phenotypes, much less non-neural.

            I think all of those are critical safety end points, and that in the final analysis that the overall cell number was in no way perturbed by virtue of the graft, and that the final representation of phenotypes at least was analogous to that of the native tissue that one was trying to either repair or replace.  So that, in effect, we're looking for the establishment over long periods of time of histologic and, therefore, physiologic normalcy.

            CHAIR URBA:  Two other questions I think that tie in with all the other questions and then we'll let other people comment.

            Is the mouse and rat model, is that as far as you go, or do you think you've got to establish it --

            DR. GOLDMAN:  Depends on the disease target.  So, for example, the congenital hypomyelinating disorders, the only models that exist, by and large, are mice.  And yet there's no reason in terms of the known biology, of course we only know what we know and we don't know what we don't, but in terms of the known biology, there's no reason why those models wouldn't be reflective of the congential hymyelinations of humans, of children.

            On the other hand, if one is looking at, for example, models of adult either traumatic injury or stroke, the issue then becomes what the kinds of sizes involved, what the kinds of unique features of the primate and human vascular supply to regions that are being challenged, that one, I believe, does need larger animal, and preferably primate models because there are primate and human specific features of the anatomy, and in some cases of the cell biology, that require large animal, or as the case may be, primate modeling.  So, again, it depends on the disease target that one is approaching I believe.

            CHAIR URBA:  Is there an acceptable rate of tumor formation or does it have to be zero?

            DR. GOLDMAN:  I would say zero, and just getting back to the point that we raised earlier, at least in the spinal cord and brain, there is absolutely no such thing as a benign tumor.  It doesn't matter what they look like histologically.

            CHAIR URBA:  Would the acceptable rate of tumor formation be target specific too, or would you generalize that to all the things you heard about today?

            DR. GOLDMAN:  This, of course, is all personal opinion.  But with few exceptions, the disease targets of regenerative medicine are not ones that, to my mind, should permit the genesis of tumors, of cancers from implanted cells.  When one is talking about biologic therapeutics or any therapeutics in the setting of diseases with short life spans, of course the bar becomes smaller.

            So, for example, one may tolerate the potential development of lymphoma or leukemia decades out in the setting of chemotherapy, radiation therapy of say a child with leukemia, because you're looking at extending the life span tremendously and you essentially are faced with an all or none situation.  Most of the disease targets that I think we're all looking at in regenerative medicine are a bit different in terms of these are disease that often are chronic, they often are involving tissue loss over long periods of time, and so looking at that from the standpoint of playing out essentially the cost benefit over time, from my own standpoint I don't see the risk of tumorigenesis as being really justified.

            Again, I'm speaking with very broad strokes, but in general terms I don't think it's justified.  There are examples, such as Huntington's disease where one may think in terms of inducing endogenous progenitor cells, where the patient may have a relatively short life span and will potentially be using a strategy that may increase the risk of brain tumorigenesis over time.  But then just as the example before with childhood leukemias, if we're looking at potentially extending the life span of that individual considerably beyond what it would normally be, then the risk of late stage tumor formation becomes I think justified.

            But it's for disease targets where the life span is not significantly curtailed or is curtailed but not potentially to the point where the appearance of tumorigenesis would necessarily be where it's need to be, that's where I don't think it's justifiable.

            CHAIR URBA:  Thank you.  Dr. Taylor?

            DR. TAYLOR:   Thank you.

            In thinking about optimal preclinical study design I think you made an excellent point.  I think the first thing is that the preclinical studies have to be clinically relevant.  So they have to be done in animal models that reflect the disease state at which we're looking and they should reflect the questions that we're likely to see clinically with regard to migration, whether route of administration matters more than site of delivery, and I think we're really asking the question, what it really comes down to, what can cells do versus what they actually do, and so we need to design preclinical, in vitro studies to examine both the potential of the cells as well as in vivo studies that show not only the potential, but what actually happens given the population that you ultimately are going to use.

            I wonder if, perhaps, given the preclinical data in mice at least suggest site of undifferentiated cell administration really has an impact on tumorigenicity, and given that we heard this morning that vulnerable sites exist, perhaps site localization studies should be recommended to include direct administration of the final cell product to some of those vulnerable sites, most likely vulnerable sites to see if, in fact, tumorigenesis or some other adverse effect is going to be an issue.

            We've also heard that dosing matters, cell number matters, and I think back to some of the angiogenesis assays and tumorigenesis assays that we all did when we were looking at angiogenic cell types, and there's a standard subcutaneous tumorigenesis assay that we all do to determine if cells are angiogenic.  It seems to me that we can develop similar sorts of assays for the uncontrolled cell proliferation potential of these cells.

            And then, finally, I wonder if -- I think we need to be careful not to add an extra burden on embryonic stem cells, but at the same time recognize that given the fact that we don't yet know to what degree minimally differentiated cells have adverse events, we need to develop strict definitions of what potency and release criteria are going to be, and I'll speak to that more when we get to the next question.

            I guess there is one other thing that I would like to say and that is patient criteria have to fit in here somewhere.  It may be that an age of a patient matters, just like disease matters.  Age may matter, sex may matter, co-morbidities may matter, and so I think as we begin to ask some of these questions about the both positive and negative potentials of these cells, we have to consider the context in which the cells are going to be delivered.

      DR. SNYDER:  I just wanted to just briefly reiterate a point that Steve started to make and Doris began to make, too, and that's that in answer to your question as to what should our cell population be that we put in there and how do we know.  It really so much depends on having really faithful animal models that reflect the real disease.  And that in turn goes hand in hand with a better understanding of what the pathophysiology of the diseases are we want to treat, and that, in and of itself, is turning out to be a moving target.

            You know five or six years ago, for example, for ALS, the slam dunk answer would have been, well, we just need motor neurons.  Now as our sophistication about the nature of ALS is growing, we're starting to realize, well, sure, motor neurons are important, but maybe we actually do need astrocytes, which means that perhaps we need to somehow figure out how to put in a mixed population of cells that'll be necessary.

            This kind of thing I think even extends to our view of embryonic stem cells, and this is a point that Willie and I started talking about.  On the fact of it, to say I'm going to use embryonic stem cells in a neurologic disease, it would be a deal breaker to say, oh, and by the way, I'm also getting some mesoderm, I'm getting some vasculoendothelium, and I'm getting some smooth muscle.  Ordinarily, you would say, well, I guess you can't use your cells.

            Well, with a broader view of how we're starting to appreciate the injury niche, we realize the injury niche is neural elements, and also vascular elements, and some extracellular matrix.  It may be exactly what we want to be able to reconstruct a niche that you have a cell that safety gives you some elements and then some support elements, and Willie talked a little bit about the cross talk.

            So I guess the point comes back to, even as cell biologists, we still have to go back to really good, faithful, animal models.  And that's independent of whether it's a larger animal or a small animal.  We just need models that at least model the path of physiology and then be prepared to revise our notion of what pathophysiology is and what you really want to do to fix something that's broken.

            CHAIR URBA:  Yes.  I think your summary was brilliant.  I think you've really summed it up.  The difficulty, of course, there are so many questions simultaneously.  It's trying to solve a complex puzzle.  So let me just suggest parsing this out into the cell types because each cell, when you examine it, interrogate it, there are different questions.  So ES cell, the intermediate, which would be the progenitor committed but can still have renewal capability in the differentiated cell.

            And so in the ES cell, what's interesting about this is is that your starting material, which would be kind of like let's think about this like a monoclonal antibody, and then you want to purify the soup and eventually get a purified humanized antibody, in this case the definition of your optimal study material is actually a cell that will form a tumor and so that's very different.  So you have to have criteria to ensure that the cell is what you want to begin with.

            So, obviously, first thing, there has to be criteria that it is an ES cell that meets criteria of interest.  That can be done by transcriptional profiling.  It can be done by -- and other issues.  I'm not sure.  I was concerned about the karyotypic abnormalities, but I think that can be sorted out.