THURSDAY, July 24, 2003

John Gearhart, Ph.D.,
Professor, Institute for Cellular Engineering,
Johns Hopkins University

Rudolf Jaenisch, M.D.,
Member, Whitehead Institute;
Professor of Biology,
Massachusetts Institute of Technology

David Prentice, Ph.D.,
Professor, Life Sciences,
Indiana State University;
Adjunct Professor, Medical and Molecular Genetics,
Indiana University School of Medicine

CHAIRMAN KASS:  Could we come to order, please?  The third session of this meeting is on "Stem Cell Research: Recent Scientific and Clinical Developments."  The most challenging aspect of trying to fulfill our charge of monitoring stem cell research is the monitoring of the scientific research itself.  There is so much of it, it's very diverse with many sources and types of stem cells, and many types of research.  Things are changing amazingly rapidly, and we are trying to monitor a moving target.  The material is highly technical and hard, even for scientists outside of the field, let alone a layman, to evaluate carefully. 

"The Isolation of Human Embryonic Stem Cells," and, "Human Embryonic Germinal Cells," by James Thomson and John Gearhart respectively was reported only five years ago.  The obvious promise of these cells both for gaining knowledge of development, both normal and abnormal, and eventually for regenerative therapies with hundreds of thousands of patients with degenerative diseases or injuries has produced great excitement, much fine work, and to be frank, more than a little hype.

At the same time, reports of previously unknown and unexpected multipotent or stem cells in various tissues of children and adults has produced enormous interest also in so-called adult stem cells, with similar promise, and to be frank, also more than a little hype.

By almost all accounts we stand today in the infancy, not to say embryonic, stage of these researches, and it is surely too early to answer the questions that the layman wants answered.  How soon, for which of our diseases, from what sort of cells, at what cost, and at what risk will the cure be available? 

Yet it is not too early to learn where we are in fact in this rapidly growing field, to try to separate fact from fiction, true promise from hype.  And to do this we have gone to the experts, commissioning review essays, essays that would review for us the published literature over the past two years since the August 2001 decision, covering work in five areas according to the origin of the cells.  "Human Embryonic Stem Cells," a paper by Tenneille Ludwig and James Thomson, "Human Embryonic Germinal Cells," a paper from John Gearhart, a paper on cloned embryonic stem cells, a paper by Rudolph Jaenisch, a review of the adult stem cell research, a paper from David Prentice, and an update on the work with her own multipotent adult progenitor cells from Catherine Verfaillie.  The papers have been sent out with the briefing books, and I assume that they've been read.  We are thinking about another paper on mesenchymal stem cells. 

We're very fortunate and grateful to have with us this afternoon three of the authors, Dr. John Gearhart, Dr. Rudolph Jaenisch, and Dr. David Prentice, who will, in brief presentations, highlight their own papers, after which we will have questions and discussion. 

I would remind Council members that the purpose of this session is wholly scientific.  We want to learn as much as we can about the current and projected state of this research.  This isn't the time for ongoing ethical arguments about the moral status of the embryo, the research imperative, or equal access for the current funding policy, important though these issues are.  And I will simply say I'll use the authority of the Chair to see to it that we try to stay on the topic.

Dr. Gearhart has kindly agreed, by the way, to field questions pertinent to the review essay submitted by Drs. Ludwig and Thomson.  Gentlemen, welcome to you all.  Thank you for your papers, your presence, and in advance for your presentation and participation.  And we will proceed in alphabetical order with John Gearhart first.

DR. GEARHART:  Well, I can't say I'm delighted to be back, but I'm happy to be back.  And to not really dwell on the research that's going on specifically in our laboratory, but to give you a bit of a general overview of where the work is in this field, and I think there will be ample opportunity from some of the things that I say that you can ask questions about it.

I think the first question, and judging from some of the morning topics, that I would like to address is the one before you now, which is, why study these cells?  And what we have heard most frequently are the issues, well, this is going to be the most versatile source of cells for promised cell-based interventions for various therapies. 

But I came at this at a different angle.  I am an embryologist by training.  I've been a student of human embryology for 28 years.  And I'm extremely interested in knowing how we are put together.  What are the events, what are the mechanisms that lead to the formation of the human being?

And over time, as you can imagine, we've studied fruit flies, we've studied mice, we've studied rhesus monkeys.  And I think one of the major, and perhaps the most important bit of information that we're going to get out of embryonic stem cells are going to be the issue of how we are formed.  And this is going to involve cell biology, genetics, on and on and on.  But this is what is going to enable us.  And I think with this information, which at the moment is under the heading of Fundamental Science, or Fundamental Discovery in Basic Science, this, I think, is going to be the lasting contribution of studies from embryonic stem cells.

They're going to have applications, obviously, with that information, and the sources of cells.  And we're going to apply those to birth defects.  We're going to apply it to injuries and disease, because many of the processes are similar.  And we're going to be able to draw on that information in trying to correct diseases and injuries.

True, I think out of this we're going to get sources of cells which, in the short term, are going to be used directly for the cell-based interventions.  And I think through the use of these cells, we're going to discover other important things about these cells, and some in our research we've learned, which we can apply in therapies.

But I want to emphasize one point, and this is my belief, that this period of time that we're going through in studying embryonic stem cells is going to be transient.  And I want to emphasize that I believe it's the information we're going to get out of these cells that are going to be used in the longer term to restore function, to regenerate tissues within the bodies of patients, without the use of these cells.  And that to me, as I look downstream, is going to be the most important outcome of this research.

Now I realize that this quote is taken out of context for these essays that were written, and what was being questioned here.  But I want to make some comments which are important when we consider the use of human embryonic stem cells directly.  And that is, we are finding enormous differences between mouse embryonic stem cells and human embryonic stem cells, not only in practical issues within the laboratory from the standpoint of cell cycle times, et cetera, but also, as we begin to differentiate these cells in a dish, we find some very subtle and some very not so subtle differences between the mouse cells and the human cells to get us to the differentiated functional tissue that we need.

So I do believe it's time that we work directly on the human embryonic stem cells if we want to gain this information on embryogenesis, and if we want to move forward with developing cell-based interventions.

So, the sources that we're going to talk about in general deal with this very early stage here derived from the IVF-donated embryos, and from tissues collected within the early stage of fetal development, the germ cells.  These cells have many things in common, and we've published on this.  You've seen this, probably.  And so we look at them as a group. 

There's a third member of this group, which is embryonal carcinoma cell, depicted here on the right, which has very similar properties to these two other cell types.  In fact, it's the only cell type from which any cell has been derived that's being used in a pre-clinical trial.  And this is on stroke patients at the University of Pittsburgh.

I want to spend time now, or my brief time, on three slides to give you an indication of where the human work is on this class of cells.  What are the questions that are being asked, where are we on some of this stuff, et cetera?

So here now are issues around human embryonic stem cells, or embryonic germ cells.  One of the first questions that's being asked about the stem cell itself is what is meant by "stemness"?  And obviously this, at the molecular level, gets us into genomics, proteomics.  And so there is a major effort around the world at trying to determine the genetic basis of a stem cell.  And the human cells are being used extensively for this purpose.  How many genes are involved, what genes are involved, what signaling pathways, on and on and on.  So stemness is an important issue within this research.

Now, it's a comparative thing.  We can do it also with mouse cells.  We can do it with adult cells as well, trying to find commonality.  What is it that makes a cell a stem cell?  That information will be vital, I believe, in the future, in converting virtually any cell to a stem cell, if we want to go that route.

The second major issue currently are characterizations of existing lines.  As you may know, there's no standardization within the field as to what constitutes a human embryonic stem cell.  We talk about developmental potential.  We talk about molecular markers, biochemical/antigenic markers, this is now being taken up by an international committee under the auspices of the MRC in London to try to get an agreement on these issues from stem cells around the world, human embryonic stem cells around the world.

There's also the issue now of whether or not we should be deriving new stem cell lines.  And in many countries, this is indeed happening.  There are a number of new cell lines coming online.  And what is the importance of this?  Now, many of you have heard at least from what the Europeans refer to as the presidential cell lines, those that are eligible for funding in this country.  Should we have cells that are only grown on human feeder layers?  Most of the cell lines that have been derived are on mouse feeder layers, and there is a concern about infectious agents passing into the human cells. 

I think there's a misconception out there that these lines will be ineligible for any kind of therapeutic use.  I think the FDA has made it very clear that they're certainly willing to consider those grown on mouse feeder layers if they meet certain criteria to also be eligible for use in the clinic. 

But there are a number of lines now that have been derived on human feeder layers, and there are lines being derived that are feeder layer independent, which means it makes it much easier to work with in the laboratory.  You don't have to worry about these other cells that are present in your cultures. 

And there's an attempt now being made to derive these lines in defined media, which means there are no other animal products, no serum, that you can control very carefully the differentiation of these cells by adding specific growth factors in a sequential fashion.

The most important element to this, though, is this top line here.  Many of you may not realize it, but when we say that routinely three or four different mouse embryonic stem cell lines have been used for the last 20-some years, what's the problem here, why can't we use just a few of the human.  Those lines have been selected from a hundred and some cell lines that were made as to being the best cell lines to use for those purposes.  And so I think we're still in this phase of trying to determine what are really the good cell lines, human embryonic stem cell lines that are available.  And thus the issue of generating more.

For the use of these lines, emphasis in the lab now is in the following area.  We're trying to work up high-efficiency differentiation protocols that result in homogenous cell populations of functional cell types.  That is, can we in the laboratory come up with protocols, growth conditions, that are going to get us 10 million dopaminergic neurons, and only dopaminergic neurons?  Most of the protocols we have now result in a mixture of cell types within a dish, and we go in and select out the ones we want.

So there's concern about the heterogeneity of the cells, and the fact that we can't at this point have a high efficiency in getting the cell types we need.  We manipulate the growth environments, and we manipulate the cells genetically to accomplish this goal.

Once we differentiate these cells, and we've had some success in certain pathways which we can talk about, we are very concerned about what we refer to as the authenticity of the derived cells.  That is, is the dopaminergic neuron you've formed, the insulin-producing cell you've formed, the cholinergic neuron, the cardiac muscle, are these actually functional cells that are the equivalent of those that you normally find in the adult? 

So a great deal of effort, both in the dish, in vitro, is being made in different parameters to measure whether or not these are authentic cells.  There are many protocols, as you can imagine, that lead to cells that look like something, but they're not functional.  And this is a major concern.  The other side of this is, the golden test is can you transplant these cells into an animal model of some kind and see that they assume the function that you're hoping that they would. 

But again, these are human cells going into rodents, primarily.  Do we have every reason to believe that they're going to function appropriately?  We don't know that yet.  We're also putting cells into non-human primates to get that kind of a measure as well.

We are in the laboratory contrasting and comparing among various sources of stem cells.  In our laboratory, we work on human ES, human EG, umbilical cord blood, et cetera.  And the importance of this is that you are able to compare and contrast in the same paradigms the sources of these cells to see which ones are going to be appropriate for which tasks.  And I think this is extremely important.

We're also very concerned, as you know, if these cells are going to be continuously grown in the laboratory, there's a finite possibility that you're going to get genetic mutations.  We're interested in the frequencies of this, and the types of the mutations that occur in these cells.  It's a safety issue.  But with our genomic and proteomic studies, we can get a good handle on this at this point in time.

When we get into the grafting issues, which is obviously a desirable endpoint, both in the basic science side, to say you have a functional cell, and then transitioning into any use of these cells in cell-based therapy, we have a number of issues here.

The first, and I think you have to realize this.  There is virtually no appropriate animal model for any human condition.  That's the fact.  We approximate it.  We do the best we can.  But under those limitations, we do the best we can.  And you have to be aware of this.  There's a concern about what animal models you're using for what test.

The issue of what stage of cell differentiation in any of these cells do you use for a specific graft?  We are finding that many things within the central nervous system, you want to use something that is fairly undifferentiated so that the cells can interestingly migrate to where they're supposed to go, set up the appropriate connectivity.  And so if you are working with a motor neuron, you'd like to use a motor neuron precursor.  If you put in a fully differentiated motor neuron, nothing works.  I mean, when you think about the nervous system, it's not surprising.

With insulin-producing cells, we find that you want the terminal cell, at least in the experiments that we are doing.  But for each of the cell types you're working with, we've got to determine this, as well as how do you want to deliver it to those animal models.

The last two are extremely important.  What are the fates of these cells that you've grown in a dish, selected for in a dish, once you graft it into an animal paradigm?  We know these cells like to migrate.  Do they differentiate appropriately?  Do they form tumors?  I remember a few years ago when I was all puffed up that our cells were working so wonderfully, and I went to the FDA for this meeting I thought was going to be one-on-one with members of that group.  And it turned out that there were 40 of them and one of me.  All their questions were about safety issues.  We now spend as much time on these safety issues as we do at looking at differentiations of these cells in a dish. 

Why?  Well, what have we found?  Indeed, in the early mouse work with embryonic stem cells the rates of tumors were extremely high.  Formation of teratocarcinomas, mixed germ cell tumors.  We have at this point, though, grafted well over 3,000 animals with our human cells, and we haven't seen a tumor yet.  And maybe we're just lucky, maybe there's a difference between mouse and human cells.  We don't know that. 

But these experiments are extremely expensive because you've got to serial section through the whole central nervous system if you're putting cells in there.  You're putting them in IP, et cetera.  It's a lot of work.  The FDA, ideally, would like to know if you're putting 300,000 cells into an animal, they want to know where all 300,000 cells went.  This has gotten us into extremely expensive experiments in labeling cells, developing labels for cells that you could follow for a year or more later.  You could go back and find those cells.  So we've employed radiologists, and chemists, et cetera, to help us with these kinds of things.  So it is a major issue.  And in all of our experiments, we look at this.

The last issue, on immune response graft rejection, is obviously to some extent the Achilles' heel of this work.  If we can't provide tissue that's going to be accepted in a graft, then all this work has gone for naught.  So there are issues of active experiments now going on in tolerance, in genetic alterations of cells, the consideration of cell banks, how many different stem cell lines would you need to cover certain populations of people, and something you'll hear about probably from Rudy on nuclear and cell reprogramming, so-called therapeutic cloning.  Is this one of the avenues that we can approach?  So all of these are very active research elements going on with respect to the human embryonic germ cells and stem cells.  We can't divorce them.

Now, in this cartoon, what I've depicted here is one of the more difficult scientific aspects of the research.  And that is that you have in a dish - now this is a bit of a repeat, but it's important to mention - you have in a dish these cells that are capable of forming any cell type that's present in the body.  The major problem here is how do you get them only to form hepatocytes, dopaminergic neurons, heart muscle, blood cells, pancreatic islet cells, solely.  How do you do that?  This is where the real issues are within the laboratory. 

And we try to recapitulate, as I showed you this before - oh, and finally, the issue of - I put this little thing down here to remind me.  Some of you have seen this report earlier this year from Hans Schuler's lab at Penn, that he's been able to find oocytes within embryonic stem cells derived from the mouse.  Now, he did not - now keep this in mind, he did not purposefully differentiate these cells into oocytes.  He came up with a neat little trick of identifying them once they were present in the plate.  Okay, so it's not that he's worked up a condition to differentiate them only to oocytes, it's that they're there.  You can use that technology to pull the cells out, and then further try to mature them, see if they are actually functional oocytes, et cetera.

So we rely in the lab on trying to recapitulate what has occurred to some extent in the embryo by providing growth factors that these cells normally see to get them to enhance, or try to direct their differentiation into the product that we want.

We use selection techniques in the laboratory after getting these mixtures of cell types by different avenues in the dish.  We then come back using genetically selected markers, different growth media, cell sorting, which is a fancy way of sending cells through a beam of light, and if they have a certain antigen on the surface, you tag it in a way that will put them into different pots.  And this works fairly effectively for a number of cell types.  But it's highly inefficient, and we've got to improve this technology.

So what have we done?  What have we been able to do?  We've been able to identify many, many different cell types in a dish.  We've been able to expand many different cell types, whether they're insulin-producing cells, dopaminergic neurons, heart muscle cells, smooth muscle cells, hepatocytes, we're able to do that.  Human.  And we've been able, in many cases, to show that they are functional within the parameters that we like.  And in a few cases we've studied extensively we've been able to graft them into animal models to show that they will work, and can ameliorate a condition in the animal, whether it's, to some degree, whether it's a diabetic animal, or one with motor neuron loss, mouse models of Parkinson's disease, we can do that at this point.

To reiterate, we are concerned about safety issues.  They go hand in hand with all that we do in the laboratory.  It adds, again, expense and time, but we want to make sure we get it right.

Here's another issue of safety.  We find that in many of these cell lines, if you're not careful - now human embryonic stem cells are much more difficult to grow in the laboratory than mouse, and if you're not careful, this can overwhelm your cultures very quickly.  This is an example of a tetrasomy 12p, and also of a trisomy 17, which appear to be fairly common coming out of the human cell lines for whatever reason.  We don't know.   But you have to constantly monitor these cell lines to make sure that they are remaining normal as far as the karyotype is concerned.  Not an unusual circumstance.

Rudy, I'm sure, will deal with this.  But we got highly involved with the issue of somatic cell nuclear transfer, mainly coming in from the desire to have matched tissue for a patient.  But I want to point out two other issues here that I think, in my way of thinking, are perhaps more important.  Rudy will probably disagree.

Number two on this list, to increase the diversity of stem cell lines.  Number three, to facilitate the study of inherited genetic diseases and somatic mutations.  Now, there's been a lot of talk about, with the justice issues, of diversity of cell lines.  We now know how many cell lines we need to cover the population of the United States.  How are we going to get these lines?  We're not going to get them, if we wanted them, by going out and matching up people to get embryos, okay, or even assaying embryos in banks.  The easiest way to do it is you identify a person that has the haplotypes of class I and class II genes, and take a nucleus out of their cell.  And generate stem cells through somatic cell nuclear transfer.  No question about it, easiest fast way of doing it.

The issue of the studying of inherited genetic mutations and somatic mutations.  It means that we can take cells from a patient with a disease, some of them not expressing very much from the standpoint of the pathogenesis of the disease, to others that are full-blown, to try to figure out why some patients have one form of the disease, another has the mild form of the disease.  We can take this directly, and generate stem cells for those studies.  Somatic mutations, breast cancer mutations.  We can generate cell lines - it's the only way we can do it - through somatic cell nuclear transfer.

And then the issue of being able to determine mechanisms of nuclear reprogramming.  We would hope that they would approach normalcy from the standpoint that we may be able to use that information to convert somatic cells into stem cells at some point downstream.  This is why I think somatic cell nuclear transfer into a human are reasons that are quite important.

Well, I think with the issue of the human embryonic stem cells, I think they have uniqueness in certain areas, and I think that what we're going to learn out of these studies is going to be extremely important in the future, not only for deriving cell-based therapies, but in getting information that we're going to be able to begin to instruct our own genes.

And this is something which hasn't been paid a lot of attention, but I think even within this Council, it may be a good idea, that we're on the verge, I believe, of being able to instruct our own cells.  And I think the ramifications of this are enormous.  I mean, talk about enhancement.  One could go on the Internet and get a kit for enhancing any part of your body to a certain degree.  And this is all going to be done, I think, from information that we get out of learning about cell differentiation, how these cells function.  So I think we've got to keep our eye on this for the future as well.

Just in closing, the greatest impetus that I can think of for making advancement in this field is funding through the National Institutes of Health.  This is the only information that I could get my hands on.  It's available.  It's for the fiscal year 2002.  You see how much money is being spent on stem cell research in toto, how much on human adult stem cell research, how much on animal adult.  And up to this point, $11 million.  On the human embryonic stem cell research, I think this year it's going to be more, and we would hope in the future, as the bottleneck for cell lines and more applications come in to the NIH, that these numbers will become robust. 

And I think this will be the key to the dreams that we have, anyway, of learning more about how humans are built, and cell differentiation of human cells on their way to therapy.  So I hope I've given you enough to think about.  Thanks.

CHAIRMAN KASS:  Thank you very much.  Why don't we simply proceed in order.  Dr. Jaenisch.

DR. JAENISCH:  Well, thank you very much for inviting me here.  And I will right away get to the cont roversy. I'm not going to agree with much you said.

So I want to try to develop two arguments.  One is reproductive cloning faces principle biological problems that may not be solvable for the foreseeable future.  And the other one is therapeutic cloning poses no principle biological obstacles, only technical problems.  And this, I think, in my opinion has implications for the status of the fertilized and the cloned embryo.

So you need one piece of data.  When we do nuclear transfer, then it's clear that the donor cell, the donor matters.  So if we use a somatic donor nucleus, cumulus cells, fibroblasts, Sertoli cells, the result to get cloned adults is really low, a few percent.  If you use B or T cells, or neurons, it is very low.  Only with tricks we were able to get animals.  However, when one uses embryonic stem cells as donors, it's an order of magnitude more efficient.  So the conclusion is an embryonic cell is easier to reprogram to support life than a somatic one.  I will come back to this later when I make an argument.

So, let me first come to cloned animals.  If you have an animal born, if you looked at this carefully, you find that approximately four to five percent of all genes, as done in percentile, are not correctly expressed.  Still, the animal develops to birth.  If you look at imprinted genes, it's even worse.  Thirty to fifty percent are not correctly expressed, regardless what the donor cell nucleus is.  So from this we would argue that it's amazing that these defective embryos can develop to birth and beyond.  But you pay for that.  You pay with abnormalities. 

So my argument would be, and I will develop this more, that there may be no normal clones.  Of course, we have the problem to define what we mean with "normal".   But let me then say that faulty reprogramming seems to me is a biological barrier that may preclude the generation of normal cloned individuals, at least for the foreseeable future.

Let me come to therapeutic cloning.  So, as we heard, cloning would involve taking from a patient a somatic nucleus and derive an isogenic embryonic stem cell by nuclear transfer.  This could be used, then, to correct the genetic defect if it was present.  One has to differentiate these cells in vitro, and then transplant it back into the individual and see whether it could improve the condition.

So we have made such an experiment.  I just want to briefly outline the steps we went through.  As a patient we used a mouse, which was totally immune-incompetent.  Due to the Rag2 defect, it did not have any B and T cells.  So that was our patient.

From this, skin cells were removed from this animal.  They were transferred.  A blastocyst was derived.  And from this blastocyst, an embryonic stem cell, which of course is mutant, as the donor.  And then we used just simple homologous recombination to repair one or two alleles to make a functional Rag2 gene, and then differentiate these cells which had problems into hematopoietic stem cells, put them back into the animal, and the result was that these cells could colonize the embryo and repair, in part, the defect. 

So some have argued that this experiment really didn't work.  It really only showed that when you have - I think Dr. Prentice argued that it only showed that one has to use a cloned newborn as a source.  And this, of course, is not therapeutic cloning.  I think that is a misrepresentation of those data.

I would argue that from this experiment we know that somatic nuclear transfer and therapeutic cloning will work because it has worked in the mouse.  So only technical, not principle, barriers exist to adapt it to human use, although these technical ones may be daunting.  Technical, in contrast to principle ones, which we see in reproductive cloning.

So, if I look at the two ways we can think about cell therapy, then we have the nuclear cloning approach as I just outlined, which in the mouse we know works, at least for hematopoietic cells, and I believe will work for other cells.  The alternative is somatic adult stem cells.  We'll hear later from this.

And I believe here there are many, many question marks.  It is a very young field, very interesting, but we really don't understand how to really handle these stem cells, how to propagate those.  And there's really, with the exception of bone marrow stem cells, not really any proof of therapeutic potential at this point.  So I think we need much more to learn here, but this we know will work.

So I want to come to really two key questions.  One is can reproductive cloning be made as safe as in vitro fertilization?  And the other one: Does faulty reprogramming after nuclear transfer, does it pose a problem for the therapeutic application of this technology?  I think these are the key questions in my mind.

So, can reproductive cloning be made safe?  I believe in addition to the technical problems, which are solvable, there are serious biological barriers.  And the main one is this one.  The two parental genomes in all of our cells are differentially modified depending on where the genome came through the egg or through the sperm.  They're epigenetically distinct in the adult.  If you want to recreate that situation, you would have to physically separate the two genomes and treat them independently in an oocyte- or sperm-specific way.

So let me point that out in this very complicated-appearing diagram, but it's very simple.  So let me just go through briefly what's happening during normal fertilization.  In normal fertilization, the egg genome and the oocyte genome are combined.  And they are differentially modified during gametogenesis.  And this is what these lollipops here show.  So they're different in the zygote.  But now something very curious happens.  Within hours of fertilization, the sperm genome is stripped of all methylation.  The oocyte is resistant to this not-well-understood demethylation activity because the oocyte genome has been, of course, together with this activity throughout oogenesis.

So then cleavage proceeds.  And the point I'm making now is that the cells of the genomes in the adult are different, different because of their history.  It's not only for imprinted genes, but also for non-imprinted genes.  Now what happens in cloning?  In cloning, one removes, of course, the egg nucleus and replaces it now with one of the somatic nuclei. 

Now, both of these genomes are now exposed to the egg cytoplasm.  So both become really - they're in identical chromatin state.  So they'll be both modified, and the difference will be equalized.  So the difference you see in a normal adult cell will be equalized, tends to be equalized in cloning.  I think that is a problem among those which we call epigenetic problems.

So in order to solve that, I think one would have to separate the two genomes physically and treat one in an egg-specific appropriate manner, and the other one in a sperm-specific manner.  I think if we could do that, then I think we would have a way to solve that problem.

So the phenotypes of clones, I would argue, is a continuum without defined stages.  So we know the most important stages are implantation and birth, and indeed, most clones are lost at implantation, and then at birth again many are lost.  You end up with very few which develop to late age.  And we know from most experiments, even animals which are one year of age, 80 percent of those die very early with major problems.  These experiments could only be done so far in the mouse because the mouse is the only organism we have old cloned animals.

So I think the problem here is, yes, you might get - because our criteria are not very good here - you might get quasi-normal or maybe a normal individual with a certain frequency.  It's very difficult in animals to test, really, because our tests are limited.  But we cannot predict ever at any of these stages who will be among those outliers, and who will not be.  There's no way to select good healthy from non-healthy clones.  I think that's very important to emphasize.  There's no way we can think of to do that.  So there's no predictability whatsoever.  So from my point of view, this doesn't matter, really, whether one or two percent of potentially normal animals which survive for a long time.  You can't predict who this will be.

So, therapeutic cloning.  So, as I said, cloned animals are abnormal due to faulty reprogramming.  Why is normalcy of differentiated cells derived from ES cells not affected by this problem?  There are two reasons.  One is that we don't involve a generation of a fetus.  And the other one is that the embryonic stem cells lose what I'm going to call epigenetic memory of the nucleus they came from.  So let me develop these two ideas.

So first, it's simple.  In contrast to reproductive cloning, there's no embryogenesis required to derive functional cells in vitro.  So it's totally irrelevant, for example, whether imprinted genes are reprogrammed or not.  Imprinted genes have a function only during fetal development, not in the adult.  So it doesn't matter.  There is one or two exceptions, and those would have to look at.

Very importantly is the cells themselves select themselves in vitro for the cell type you are selecting for.  There's no selection possible in vivo after implantation in a cloned animal.  And then we know that cloned ES cells form normal chimeras, as do any other ES cells.  So biologically there is no difference.

Let me come to the memory question on this slide here.  So we know we can make blastocysts from a zygote from a fertilized egg, or by nuclear transfer from these various donor cells.  Now I would argue the blastocyst remembers exactly where it came from.  And we know that because we implant this blastocyst, derived from a zygote, into the uterus, it will with a high efficiency develop to birth, and will make a normal animal.

If you derive - take a blastocyst derived from an ES cell, and implant it, it will develop with a high efficiency to the newborn stage, but it will make an abnormal animal.  If it came from a cumulus cell, it would be very low efficiency - I showed you this on the second slide - low efficiency to the newborn stage.  It will be abnormal.  If the nucleus comes from B-, T-cells, or neurons, it will be very low.  And it will be, of course, abnormal, if you get it.

So these blastocysts know exactly where they came from.  And we know this, actually, when we look at gene expression patterns in clones derived from ES cells, or from human cells, they're different.  So they remember at birth where they came from.  That's why cloning doesn't work.  That's why these cloned ones develop abnormally, too, in the great majority.

So what about ES cells?  In ES cells, it's a very different thing.  I would argue you erase the epigenetic memory of your donor nucleus.  And the argument is the following.  We know that if you take such a blastocyst, the ES cells are derived from the inner cell mast cells.  These inner cell mast cells express certain set of genes, one of those called the Oct-4 gene, one of the key genes, but another 70.  You put this blastocyst in culture.  All these genes, all these cells array - silence, Oct-4.  This has been published.  And they don't divide.  They sit there.

But over the next days or week or so, some of these cells, one or the other, begins to re-express Oct-4 and another set of 70 genes.  And these are the cells which would proliferate.  And we call them ES cells.  It's a total tissue culture artifact of those cells which can survive under the harsh tissue culture conditions.  So I would argue ES cells have no counterpart in the normal animal model.  They are a real tissue culture artifact, although a very useful one.

Now the same occurs, of course, with these blastocysts.  The efficiency is lower.  But once you go to the selection for the survivors, they're exactly - I think in order to survive, they have to express the set of 70 genes which we call the embryonic genes, which are important for the early developmental stages.  So the point of this slide is really that this selection process, which selects just for the fastest growing cell, erases the memory to where the ES cell came from.  And indeed, when you transplant these cells derived from a B- or T-cell or from a neuron or from a zygote, they have identical properties.  They form normal chimeras, and they in vitro differentiate indistinguishably.

I want to summarize this.  An ES cell derivation selects for survivors.  And I would argue survival depends on an ES default epigenetic state which needs these Oct-4 like genes being on.  Selection process erases the epigenetic memory of the donor nucleus, and the cloned and the fertilized cells form normal chimeras.  So the potential of ES cells derived from an in vitro fertilized embryo and from a cloned embryo is identical by all measures we can do.

So I would conclude, then, it's unlikely, if not impossible, to create a normal individual by nuclear cloning.  The problem of reprogramming may not be solvable for the foreseeable future because of these principle barriers, but ES cells derived from clone embryos have the same potential for tissue repair as those from the fertilized embryo. 

So I think one of the key concerns I can see of this committee is that the derivation of embryonic stem cells by nuclear cloning necessitates the destruction of potential human life.  I think that's a major concern.  And if I just compare now, to my opinion, the difference between a fertilized and a cloned embryo.  The fertilized embryo is created by conception.  It's genetically unique.  There's a high potential it will develop to a normal baby.  The cloned embryo of course has no conception, no new genetic combination.  It is really the product of a laboratory-assisted technique.  Sloppily, we could say it's a laboratory artifact.  But most importantly, it has little or no potential to ever develop to a normal baby.

So I think the embryo, the cloned embryo, lacks essential qualities of the normal embryo, which on this sort of maybe summarizing my thoughts is on this slide where I think there are really three possible fates for a cloned or for fertilized embryo.  We fertilize one of these leftover embryos, hundreds of thousands in the clinics.  They can be disposed of, they can be implanted to form a normal baby with a high probability, or they can generate normal ES cells.  The cloned embryo, three fates.  It can be disposed of.  It can make normal ES cells, as I have argued, but it cannot make with any acceptability efficiency a baby, not even a normal one.

So if this is accepted, instead of disposing these leftover embryos and use them for normal ES cells, which could be used for research, as we heard.  This, of course, to my opinion generates an ethical problem because you destroy potential human life.  I think this - so if this is acceptable to some, I think it should be ethically less problematic, because in this case I think you don't have the potential to form, within acceptable possibility, a normal baby.

So from the biological point of view, I think the derivation of embryonic stem cells by nuclear cloning develops the structure of an embryo that lacks the potential to develop into a normal being with any acceptable efficiency or predictability. 

I was asked - and I want to close with my final slide, which really follows what John said - what are the potential applications of cloned embryonic stem cells derived from a cloned embryo.  I think we talked about therapy.  Clearly, I don't have to dwell on this.  I'm not sure whether this will be really generally available technology.  It maybe too expensive.  I don't know.  And I don't know how fast we could solve the technical problems of adapting this to human medicine. 

However, I think this is the more important point, which I think John emphasized.  I think it allows us to derive genetically identical cells from patients with multigenic diseases, such as Parkinson's, Alzheimer's, ALS, diabetes.  And we can now use this system in vitro to validate the cellular defects of these complex diseases.  So compare ES cells derived from a healthy individual with those from such a patient.  I think there's enormous potential here if we find a difference, to find out why that is so, and is it a screen for potential therapeutics, just in the culture dish.  And as John emphasized, such diseases cannot be studied in animal models, because there are no animal models for those diseases.  But you can really make those in the culture if you just use a cell of the patients.

So I think this is a very important, maybe the most important driving issue, to my opinion, to use this technology.  Thank you.

CHAIRMAN KASS:  Thank you very much.  Dr. Prentice. While we're waiting, Dr. Jaenisch, would you mind just taking one question of just factual information so that we don't lose a lot of time.  This last point about the models for multigenetic diseases.  If someone were to say the same kind of models might be available through, for example, the multipotent progenitor cells that Dr. Verfaillie has, where you could go into the patient and get out progenitor cells.  You might not get all the tissues, but if those cells could reliably be differentiated into islet cells, couldn't you do the studies on the pathogenesis of - in other words, couldn't one, if one had stable and reliable adult stem cell populations, wouldn't you have the same access to the cells from the multigenetic diseases?

DR. JAENISCH:  Yes.  I think if this would work, if this would efficiently work, I think absolutely I agree with you.  I think the problem as I see it now is that, indeed, working with adult stem cells is very difficult.  And the major problem is that we lack the ability in most cases to, for example, propagate them in the undifferentiated state.  For example, with bone marrow stem cells, although they're known for 30 years, this has not been accomplished.  They are very useful for therapeutic applications because they select themselves in the patient, but not easily for this type of research, what you want to do.

So I think there might, at some point, if we learn that in this young field, I agree with you.  Then I think these cells would, if you could differentiate them to dopaminergic neurons in a way that we can do for now from ES cells, as Ron McKay has shown, yes I think that would be useful.

CHAIRMAN KASS:  Thank you.  Dr. Prentice, you're all right?

DR. PRENTICE:  Yes.

CHAIRMAN KASS: Good.

DR. PRENTICE: I think we're ready to roll.  Thank you, Mr. Chairman.  I apologize for the delay up here.  I was fighting off a fever from a respiratory virus, so I probably was in a time warp.

One of the main goals of stem cell research, as you're all aware, is the idea of regenerative medicine with stem cells, the idea that you might be able to take a stem cell from some source, for example here from bone marrow, inject that into an area of damaged tissue in the patient and regenerate or replace the damaged tissue. 

Now, the interesting, confusing thing about adult stem cells has been that it defies what for years we have thought of as the normal developmental paradigm, that as we develop from the blastocyst stage here, that cells follow one of these main developmental trees.  And as a cell would become more and more developed and more and more differentiated, they would end up out here on the tip of a branch and not be able to back away from that particular tip; not be able to back down and take a separate branch, whether it were a nearby branch or one of these main branches.

A lot of evidence now suggests, however, that adult stem cells, at least some of them, can actually move from branch to branch.  Now exactly how they're doing that, the mechanism involved in these types of differentiation or different branches of tissues, is still unknown. 

There's several questions related to adult stem cells, and in fact many of these have to do with the same questions related to embryonic cells.  What is their actual identity?  How could you identify a particular adult stem cell?  What is their actual tissue source?  How do they form these other cell and tissue types?  Do they form actual functional cell and tissue types, as we look at these organs and tissues?  And what's the specific mechanism of differentiation?

They do seem to have some unique characteristics, such as a homing phenomenon, where they tend to home in on damaged tissue.  And most of the results that have been seen in terms of an adult stem cell differentiating into another cell type or tissue type seem to be tied primarily with injury to a tissue or organ.  Usually you do not see these types of changes in the experiments taking place unless there is some sort of damage.  The other thing would be what type of cellular interactions and signalling within the target tissue might trigger some of these differentiative events. 

Now, one way to try and identify a stem cell is with various markers, and I discuss this at some length in the paper.  Typically what people have tried to do is eliminate what are termed "lineage markers" for particular blood cells.  Some people pick a particular marker called CD34, which has been associated with bone marrow.  But others tend to eschew that type of marker.  And I mentioned other markers in the paper that might indicate that an adult cell is a stem cell.  The CD133 marker, or the c-kit marker.

Several people have begun to undertake studies of the gene expression within adult stem cells, and compare them to embryonic cells to see if there is a commonality in terms of all of the various genes that might be expressed that identify a cell as a stem cell.  One of the problems that's been faced, though, is that these markers tend to change over time.  This has been seen in several studies where actually a marker such as CD34, that might have been a chosen marker to identify a stem cell, actually might not be expressed at a later time with that cell.  And then expression of that gene would re-occur, even within the same cell.

So it may be difficult to actually identify one particular stem cell based on some of these markers, simply because of changes in gene expressions as the cells undergo changes in environment, as they may undergo changes in their isolation conditions, and what Theise and Krause simply have called the uncertainty principle, equivalent to the Heisenberg uncertainty principle.  You might at one point be able to isolate a cell with a particular set of markers or milieu of gene expression and say this is a stem cell, but then as you would try to put it into different conditions, or isolate it from different tissues, those particular markers might change, or might be different. 

So it may actually be very difficult with adult stem cells to particularly isolate a particular cell and say, yes, this is an adult stem cell.  It may be more a matter of the particular context of the cell: the tissue they're derived from, the isolation conditions used, or the tissues that they're put back into that may determine their functionality.

In terms of differentiation mechanisms, cell fusion appears to be one particular mechanism by which an adult stem cell may change into another tissue type.  Now there were some in vitro experiments done approximately a year and a half ago that indicated this possibility, but the in vitro experiments were unable to verify whether this was actually a particular mechanism that might be used by an adult stem cell.  More recently, two papers have verified in mouse systems that bone marrow stem cells, at least in these experiments, did fuse with hepatocytes and take on that differentiated morphology and function, even in terms of repairing liver damage.  So this is obviously one possible mechanism with some experimental evidence.

Another possibility might be that a cell would de-differentiate, actually back down one of those branches and go up another branch.  This would involve changes in gene expression to an earlier state, or more primitive type of cell, and then re-expressing specific genes particular to the tissue into which the cell was placed.  Another possibility is what's termed "trans-differentiation" in which the cell apparently is not backing down a branch, but instead simply changing its gene expression so that it now conforms to whatever tissue or cell population that it's within. 

In terms of sources of adult stem cells, not just tissues, but how did they get there?  How did an adult stem cell get into bone marrow, or brain, or liver?  One theory is that some of these cells may be leftover primitive stem cells, perhaps embryonic stem cells, a few that are kept around, maintained in a state so that then they can differentiate into various types of tissues. 

Another proposal is that there may be a universal stem cell, an adult stem cell, not a primitive cell, but one more geared towards maintenance and repair functions in the adult body.  It may arise in one or more tissues, and then may disperse into other tissues. 

Other possibilities are that there are particular tissue stem cells, multipotent, not able to perhaps form all tissues of the adult body, but a limited subset, and that they would be resident within a few of these tissues. 

And then I mentioned here transient stem cells, cells that are just sort of passing through.  And there is the possibility that, especially if we're looking at this idea of a universal stem cell, or even a tissue stem cell that can migrate in some cases, that it may end up passing through a tissue, so you will isolate what appears to be a stem cell from a particular tissue.  But it didn't arise there.  It just happened to be passing through via the circulatory system.

I might mention, too, that some of the other challenges for adult stem cells are very similar to some of the challenges for embryonic cells.  For example, standardization.  There essentially is no standardization in terms of adult stem cell isolation, propagation, differentiation into other tissues at this point.  Long term culture, with a few exceptions, has not been attained.  Catherine Verfaillie's MAPC cells from bone marrow do appear capable of long-term proliferation in culture.  And there are a few other examples, very limited, that I point out in the paper.  But being able to keep these cells growing in culture for a long period of time has been difficult up to this point.

The idea of safety, of course, is always an issue when we're going to be dealing with patients.  Do these cells form tumors?  Do they differentiate abnormally so that you don't get the correct differentiation in the place, in the time, in the tissue that you need?  So all of these need to be faced.

Now this idea of a potential universal or at least tissue stem cell that can migrate primarily has been put forth by Helen Blau of Stanford, the idea being that perhaps the cell is in the bone marrow, but then can go into the circulation and then exit the circulation into another tissue.  Again, most of the studies have seen this type of phenomenon in response to an injury, not in response to the normal physiological systems.

How would we then define a stem cell, especially an adult stem cell, if we have all of this problem with changeability of markers, and lack of standardization?  Moore and Quesenberry have proposed a couple of simple guidelines.  Now obviously, any stem cell has the ability to continue to replicate and maintain a population of cells.  And then in response to some signal, to differentiate into one or more potential tissue types. 

An initial first cut at how to identify a stem cell in the adult would be, can it take on a different morphology in a tissue in which you place it?  Can it take on some of the differentiated cell markers that you would see in that tissue?  Now this is a bare minimum, and it doesn't really mean necessarily that the cell is functional, that it can participate in tissue repair, or perhaps even that it has truly differentiated into that specific cell type.

Supplementing that, if you could demonstrate functional activity, and actual integration into a tissue, this would be a much better marker of a cell as a stem cell.  And certainly, if in an injury situation, in animal model or even in human, you could demonstrate a physiological improvement.  That would be a good indication that you were seeing this sort of stem cell differentiation and repair.

Now, the next set of slides, what I've tried to do is just condense from that rather difficult mass to read of all of these various tissue sources, and cell types that they can turn into, and so on.  I won't spend a great deal of time on these.  I'll just try and point out some particular points on each slide.

With mesenchymal or stromal stem cells from the bone marrow, we need to keep in mind that bone marrow is a mixed population.  We've known about hematopoietic stem cells from bone marrow since the 1960s.  These are well-studied and used clinically.  The hematopoietic stem cell is differentiating primarily, at least, into blood cells. 

Another stem cell that's in the bone marrow is this mesenchymal, or stromal, cell.  As I mentioned in the paper, one way that this type of cell is actually isolated is perhaps first by markers, but then also simply by its ability to form attached adherent layers in the culture dish, once you're putting them into the dish in the lab. 

Now, there have been various studies, and I've just tried to collect a number of them here, showing potential differentiation into different tissue types, either in the lab dish, or in the animal.  Again, the way they're following this in the animal is primarily to use a genetic or a fluorescent marker, follow the cell, see what tissue it ends up in, and look at its morphology, and then also potentially try to analyze functionality and integration into the tissue.

In various disease models, some of these cells have been used.  For example, in models of stroke.  And I want to point out again what Dr. Gearhart said.  Animal models are only an approximation.  This does not tell us the actual situation that we might encounter in human beings.  But at this point, it's the best we can do.  We can see if these cells might participate in cell and tissue repair.  Using rat or human cells, there has been some therapeutic benefit in some of these disease models in stroke with animals.  With demyelinated spinal cord.

Intravenous injection, interestingly enough, of mesenchymal stem cells in the mouse has shown some remyelinization in spinal cord injury.  Adult stem cells are not alone in terms of this ability.  Other cell types have shown this.  And this does not necessarily mean that you've totally corrected the condition.  It simply means that you're able to provide some sort of functional recovering of a bare nerve.

Another interesting point about this particular study was that they did not see a large incorporation of these cells into the spinal cord tissue.  So what benefit they did see did not appear to be due primarily to the adult stem cells actually differentiating into the myelinating cells here.  Instead, what appeared to be happening was the presence of those cells caused a signal for the endogenous cells to start to re-grow and repair the damage. 

In fact, Dr. Gearhart's lab just recently published a paper showing that embryonic germ cells showed in a particular mouse model the same type of repair phenomena.  The mice did show therapeutic benefit.  But what they found was that the cells did not, to a large extent, integrate in and participate in the repair.  Instead, it seemed to be a signalling phenomenon.  And I'll point this out in a couple of other examples along the way.

There has been one clinical trial that I could find in the literature where 11 patients were treated for Hurler syndrome, or metachromatic leukodystrophy using allogeneic, and not from the original patient, but from donor mesenchymal cells.  Now, the study noted that four out of those eleven patients showed a small increase in nerve conductance.  One of the problems with these diseases is a decrease in nerve conductance.  There are other neurological effects, and so on.  So small increase.

And in terms of loss of bone mass, all 11 patients seemed to maintain, at least for a certain time.  But in terms of any of the other symptoms in these patients, there was no effect of the adult stem cells.  Very early study.

Using mixed population of bone marrow stem cells.  So in this case, and again, this lack of identifiability for a particular adult stem cell.  What many people have done is simply take raw bone marrow and either in culture try to get various differentiations, or putting back into the animal in various disease models.  And they've been tried in quite a number of disease models showing some therapeutic benefit.  In this particular case, again in stroke, intravenous injection of this mixed population of cells.  Again, no attempt to purify the cells, or identify the particular cell that's being used for the treatment of the animal model.  But the cells in this case did home in on the damage.  Again, this interesting phenomenon that we've seen.

Various other conditions.  There was an interesting study relating to cardiac damage, where cells from mice were actually put into rats.  And what they noticed was over a fairly extended period of time there did not appear to be immune rejection of these transplanted cells, even though they were even from a different species. 

Now, it's only one study.  Does that mean that these particular cells might be immune privileged and not subject to rejection?  Only more experiments will tell.  Recent study indicated that with diabetes, in a mouse model again, where the pancreas was damaged to induce loss of insulin secretion, bone marrow stem cells injected into the animal could regenerate insulin-secreting cells.  As in the other example I mentioned before, there was very low incorporation of the actual bone marrow stem cells identified as forming insulin-secreting cells.  Instead, what they seemed to be doing in this instance was again secreting particular growth or stimulatory factors to stimulate the endogenous cells to re-form insulin-secreting cells.

And there have been several clinical trials and publications, peer reviewed publications, from those trials, primarily with cardiac damage.  There have been several groups in Germany and a group in Japan that have published their results.  Again, very, very limited numbers of patients, we must keep in mind.  And what they're seeing, though, is some physiological improvement in the ejection fraction from the heart, and when they actually examine some of the damage that seems to be being repaired.  One Japanese group has reported growth of blood vessels in limbs that were threatened to be lost by gangrene. 

Peripheral blood.  Probably we should just skip this category, in a sense, because they seem to be very similar to the bone marrow stem cells.  And it makes a lot of sense that bone marrow stem cells could actually enter the circulation.  The interesting thing is that two groups recently have isolated peripheral blood stem cells from human blood.  One group in particular noticed that the cells that they isolated showed high levels of Oct-4 expression, this gene that's associated with pluripotent status of a stem cell.

In a couple of animal disease models for stroke and cardiac damage, and I put this under peripheral blood because what the workers did was not remove adult stem cells and then re-implant them in the animal.  They injected growth factors to stimulate mobilization of the stem cells, most likely from bone marrow.  And these cells appeared to provide a therapeutic benefit in these conditions.  And we'll come back and talk a little bit more about mobilization later. 

Neural stem cells.  I certainly was taught in graduate school that you start with as many brain cells as you're ever going to have and it's downhill from there.  And on my campus, it's downhill even faster on a good Friday or Saturday night.  But within the last decade or so, the discovery that there are these stem cells within neural tissue that can at least regenerate all three main neuronal types, and perhaps some other tissue types as well. 

Interestingly enough, they have been used in some animal disease models to show some therapeutic benefit.  And there have been some clinical trials, very early.  There is one Parkinson's patient who was treated with his own neuronal stem cells.  The cells were removed, grown in culture for awhile, and placed back just into one side of the brain.  He did see a benefit.  He did see improvement.

In terms of spinal cord injury, there are no peer reviewed publications in terms of actual human treatment.  But there have been reports that at least one group in Portugal are beginning these types of treatments on patients for spinal cord injury. 

Dr. Gearhart mentioned the embryonic carcinoma cells.  And there's one group of this cell termed usually "hNT" that you might say has been tamed in culture.  This particular line that they've come across primarily forms neuronal-type cells in culture, and, as he mentioned, it's being used in a beginning clinical trial on stroke. 

Muscle stem cell can regenerate itself.  It has its own stem cell, primarily dedicated to forming more muscle.  But there's indication that it does have, perhaps, another stem cell present in that population.  Debate as to whether those cells can actually form blood, or if that was one of these so-called transient cells moving through.  The potential though, perhaps, to form bone from this type of cell, and these muscle stem cells have been used in some disease models, primarily directed back towards muscle.  Very interesting one that I came across was to re-form bladder muscle, or at least reinforce bladder muscle, in a rat model of incontinence.  And there's been one published paper regarding a clinical trial using muscle stem cells, one patient, in France, where the patient did seem to receive some therapeutic benefit from the muscle stem cells.

Liver stem cells and pancreatic stem cells.  Pancreas and the liver derive from the same embryonic primordia.  And so it might not be surprising that you tend to see liver able to form pancreas, or pancreas able to form liver.  The interesting thing here is that genetically engineered liver stem cells, simply by adding and activating a gene, have helped an animal model induce diabetes in these animals.  The pancreatic stem cells, likewise, have shown the similar ability.

Corneal limbal stem cells are a very limited subset.  And what we might in one sense call a unipotent stem cell, at least for the most part, other than one or two little reports, so that they would re-form only the tissue in which they're located.  They have been used, however, in corneal transplants in a number of countries.  And there are several published reports on this.  In vitro, one group was able to get them to form neuronal cells in culture, or what looked like neuronal cells.

Cartilage, again, a very limited sort of application, but clinical trials for articular cartilage transplants.  And one group has used them in an attempt to treat children with osteogenesis imperfecta. 

Umbilical cord blood, not a great deal of research has been done, but there are a growing number of publications.  The interesting thing here is, in terms of the disease models, stroke, spinal cord injury, and one particular model of ALS, the cells are delivered intravenously.  So again, this potential to home in on areas of damage.

And then just a list of a number of other tissues here where some type of adult stem cell has been identified, or potentially identified.  And I want to point out the adipose, or fat-derived stem cells.  And I use "derived" because it's very likely this is one of those transient stem cells moving through via the circulatory system.  Interestingly enough, though, it provides a reliable, readily available source, you might say, from liposuction fat.  And one group has been able to see neuronal differentiation in the lab; not in any animal models yet.

Now, in terms of activation and mobilizing these adult stem cells, this may be actually one of the best directions to go in terms of potential clinical treatments.  Simply because it may not be the stem cells themselves that participate in the repair of the damage.  And in that case, maybe what we need to do is simply activate the cells so that they can deliver the signal to the damaged tissue.  And again, there may be various states within the bone marrow as an example of a tissue where you might activate, where the cell could be activated and then mobilized into the circulatory system, and then reach the particular tissue to differentiate.

Again, there were two animal models that had been used, one for cardiac damage, and one for stroke, where there was an indication that this might be a potential avenue of research.  Rather than removing the cells, having to culture them, isolate them, identify them, put them back, simply being able to mobilize the cells within the body. 

Another possibility is genetic engineering.  And in terms of actual animal models of disease, genetically engineered adult stem cells have been used in a few examples in the publications.  In lung, that one particular reference indicated that there was a large contribution of the adult cells to lung tissue.  Again, a damaged tissue system.

Clinical trials, not directly, you might say, repairing a damaged tissue.  But in Severe Combined Immuno-Deficiency Syndrome, bubble boy syndrome, several infants have been treated using genetically modified bone marrow stem cells from the patient.  They seem to have been, quote, "cured," and I use that word advisedly, of the disease.  There have been reports that at least two, and potentially a third infant, has come down with leukemia as a result of that treatment, most likely due to the viral vector that was used to insert the gene into those cells.  Because the gene is not targeted in those instances.  Instead, it may go into an area where it can activate oncogenic genes within the cells. 

In terms of delivering the signal, this might be an even better use of genetic engineering in adult stem cells.  And there have been a few published examples, potentially for use in animal models with tumor therapy, to alleviate some of the symptoms in the model of Niemann-Pick disease for increasing bone healing, and in a Parkinson's model, where the cells were not participating to any great extent in repair of the tissue.  Instead, they're delivering a signal to the endogenous cells there, and stimulating them.

And in terms of that then, perhaps the best avenue eventually to pursue might be trying to isolate what these factors are, so that no stem cells would be needed at all.  Instead, if you can identify those factors to stimulate regeneration within the tissue, they could be delivered directly. 

And there are a few published examples of this, one in which there was some axon regrowth, some, in a rat spinal cord injury model by injecting a couple of different factors.  One mouse Parkinson's model study infused directly a factor into the brain of the mouse model and saw a therapeutic benefit.  Another publication lists a different factor in terms of a model of chronic kidney damage.  And one clinical trial where five patients, five Parkinson's patients were treated with infusion of this glial-derived neurotrophic factor, they did see some improvement.  Not a cure, and it may be that that's not the sole signal that's needed, or the correct signal.  But it did seem to stimulate some physiological improvement in the patients.

Thank you.

CHAIRMAN KASS:  For a young field, there's a lot going on.  And we have also talked our way late into the hour.  Let me make a suggestion, if I might.  Technically, this session was to run till 3:30, with the next to start at a quarter of 4:00.  I'm going to take the liberty of suggesting we spend a half an hour here at least with our visitors to get some of our questions asked.  And if Lori Andrews will forgive us, we'll be pushing back the start of the next session, just so we can get the questions asked.

Michael Sandel, then Jim Wilson.

DR. SANDEL:  I learned a great deal from all of these presentations, as I suspect we all did.  I was especially struck by one feature of Dr. Jaenisch's presentation, and I'd like to put this as an observation which actually will end with a question.

The subtitle of the paper and of the talk, Dr. Jaenisch, and my Council colleagues will appreciate this perhaps more than our visitors.  The subtitle could well have been, "The Vindication of Paul McHugh."  Because when we were discussing the biology of cloning, almost everyone here shared an assumption, regardless of the positions that we took on the ethical issues.  Almost everyone took it for granted that biologically the fertilized embryos and cloned embryos were essentially the same, and the moral argument proceeded from that assumption.  With the exception of one person, Paul McHugh. 

And he introduced a distinction, a distinction, as he put it, between a zygote and a "clonote", by which he meant the cloned embryo, the product of cloning.  And for insisting on this distinction, poor Paul suffered heaps of ridicule and abuse, and, at best, a kind of bemused chuckling, by those of us around the table.  And he argued that there's a difference, a biological difference, with a possible ethical significance between a zygote and a clonote, between a fertilized embryo and an artifact created in the lab.  And he was told that this is an eccentric position.  He was told that this is an off the wall distinction, that it had no basis in biology or science. 

And in a memorable exchange with our colleague Charles, who unfortunately isn't here for the benefit of this discussion, he was asked, "Well, but what if a cloned embryo actually came to term?  Isn't Dolly a sheep?"  And Paul's answer was, "Well, a sick sheep."

And then we read, Dr. Jaenisch, in your paper that a cloned human embryo would have little, if any, potential to develop into a normal human being, that it lacks essential attributes that characterize the beginning of normal human life.  And you explained that it has to do with faulty reprogramming after nuclear transfer.

Now, I don't know whether that's true, whether it's generally accepted, or controversial in the scientific community.  But whatever the case, it seems that what we regarded here as an eccentric, off the wall suggestion, that there is a biologically significant difference between a fertilized embryo and a cloned embryo, now, if I understand you correctly and here's my question, now, you're telling us that far from being eccentric or off the wall, Paul's position may be true.

DR. JAENISCH:  Well, I think that was the whole purpose of going through these arguments for me.  To argue from a biological point of view, it is a continuum.  We have to struggle with that.  And we have, for the early, most abnormal stage, we have good markers.  Death is a terrific marker.  Then we have molecular markers after birth, or we have aging as another marker.

So these are concrete markers, and we know these animals are not normal.  So when it is claimed that cows on the field are normal, which has been published by ACT.  They looked at their one- to four-year-old cows sit on the fields and eat and give milk.  They argued, well, they must be normal.  So they checked that.  And they checked it by checking if it had a heart.  Yes, it had a heart.  A liver, yes they had a liver, and that serum, even.  So they concluded they must be normal and publish this in "Science".

Now, this of course is a very superficial way of looking at it.  And very interesting.  Once this paper came out, six months later, two of these healthy cows, one got multiple tumors, the other one generalized seizures.  Now this is not normal.  You wait, and these things come out.

The point I was trying to make is that these are stochastic problems.  There is not a key master gene which, if this is right then the embryo's right.  No.  The whole genome is stochastically, correctly reprogrammed or not.  And what's amazing to me is that mammalian development is so regulative that it can tolerate so much gene disregulation and still get an animal to birth and beyond.  But you pay for that.  You pay with subtle or less subtle, more or less subtle problems. 

And of course, because it's stochastic, you will arrive at the end at some animals which live a normal life span.  And by all our available tests they might be normal, or you might not see it.  If you look at the primate, or in the human, we might see this because we could look at some much more subtle phenotypes. 

So I think the point I was trying to make is if you want to use this as a technique to generate human beings, there's no predictability.  You cannot do this.  And for me, it doesn't matter with one percent or two maybe something normal, because you can't predict it before.

So I totally agree with what your thought was.

CHAIRMAN KASS:  Jim Wilson.

PROF. WILSON:  Michael asked the same question I was going to ask using virtually the same words.  I guess it's the Harvard affliction.

But I want to just press a bit more.  An oocyte is not potential human life because it's an unfertilized egg.  You can enucleate it and put in a new nucleus, and now it is a clone.  But according to your account, a cloned embryo has little, if any, potential to ever develop into a normal human being.

If you would step back, then, from your biological role and adopt a somewhat more philosophical role, would you say that a clone, therefore, is not potential human life?

DR. JAENISCH:  I would argue it's not potential normal human life.

PROF. WILSON:  Thank you.

DR. JAENISCH:  I think that's the only thing I can say.

CHAIRMAN KASS:  And Dolly still is a sheep?

DR. JAENISCH:  Still was a sick sheep, was a very sick sheep, and which died, of course, predictably very early.

CHAIRMAN KASS:  Janet Rowley.

DR. ROWLEY:  Well, I, too, learned a great deal this afternoon.  I want to make two comments, and then I have a question for Rudy.

The first comment is that I think it is most unfortunate that we're prevented from asking our experts, and particularly John Gearhart, about the status of funding that supports this very critical area of science. 

The second comment is also directed to John.  And I was struck by the specific chromosome abnormalities that you identified in your human embryonic cells, because you probably know that an isochromosome for 12p is the most common abnormality in human testicular carcinoma. 

So it's astonishing that this is the abnormality that you see in these cells, and I'm sure it is extraordinarily important.

The question that I have for Rudy - I was surprised when you said that imprinted genes are only important in embryogenesis, because clearly there are a number of human conditions.

Now, you could say, well, human condition and the malformations occur because of embryogenesis, but there is also this whole question of imprinting and its relationship to carcinogenesis.  So I'd like you to expand on that.

DR. JAENISCH:  This is a very important point you're bringing up, so I was probably a little bit generalized.  So I think most imprinting, as we know, have - so that could be among these things that could be the most interesting, because it's IGF-2, which is a loose cannon. 

IGF-2 is a tumor, an oncogene essentially, so this would be available for cloning, because IGF-2 is very easily dysregulated.  And I would worry if you have both copies active, because that's pretumor status.  So you want to screen for this.

But the other genes - for all of the other genes, for example, an Angelman Syndrome probably really is - there are really problems of fetal development.  So these genes and fetal development - I could go further into this, because it's really growth of the fetus, where these genes appear to play the most important role.

And there's really no evidence, with the exception of IGF-2, maybe KIP-257, which is a tumor suppressor gene, those are the ones you would worry about.  So I think we know only two of those, and those you would test for I think. 

So I agree, which makes this a little bit more restrictive when I say it.

DR. ROWLEY:  Right.  And there probably are others that we don't know about, but -

DR. JAENISCH:  There might be others which we don't know -

DR. ROWLEY:  - you're saying that they would -

DR. JAENISCH:  Yes.

DR. ROWLEY:  - just all be tested for.

DR. JAENISCH:  Those two we know for sure, but others we know they don't play a role.

CHAIRMAN KASS:  I have myself next, if I might.  A question for Dr. Prentice, and then for the group as a whole.  In Dr. Gearhart's presentation, he gave a great deal of emphasis to the importance of the characterization of these cells, the reproducibility, all of these various things.

You had to cover an enormous amount of material in the number of papers, and you also in your presentation alluded to the problem of characterization, though I gather many of these studies seem to be one-shot efforts of throwing some things in and trying to see what their clinical effect might be.

And as I've read the debates, a lot of people wonder, are these really stem cells?  How well characterized are they?  So could you say something about what criteria should be used for these stem cell preparations? 

What criteria would they have to meet before you could satisfy the critics that these are, in fact, fit to be called human stem cells, and fit, therefore, for controlled studies on the kinds of things that you're talking about here?

And kind of a subordinate question, what's the best characterized of, and the most versatile of, these adult stem cell preparations?  Are there any that at the moment meet the criteria that you yourself would advance?

DR. PRENTICE:  In answer to your first question, it's difficult, obviously, because as you point out many of these studies have been sort of one-shot, especially if you look at the mixed bone marrow population studies where they are simply taking a preparation, putting it back into a diseased animal or a diseased model, and trying to come up with some therapeutic benefit.

You know, obviously, what you'd like to be able to do is to isolate the cell or the population, keeping in mind that caveat that the population dynamics may be an important facet of this. 

But let's say there's one particular stem cell that you're after.  Supposedly, if you saw that sort of therapeutic benefit from the mixed population, you should be able to go in and simply take cuts and eventually isolate that one cell, and then be able to grow it in culture.

In terms of the overall markers, that's been very difficult, and there have been a few studies looking at the gene expression common to neural stem cells, bone marrow stem cells, embryonic stem cells.  And there are a subset of those genes that are common to all of those cells.

Now, what you would hope to be able to do, then, is to go look at each of those genes and start using those as guides to come up with a marker for any stem cell that would be targeted for the types of differentiation models that we'd like to see, and eventually clinical applications.

CHAIRMAN KASS:  And with respect to the second question, what is the best characterized and most versatile adult stem cell preparation at the moment?  I mean, can you - is there -

DR. PRENTICE:  Probably Dr. Verfaillie's MAPC cells, and I can't speak for her, but those do show many of these characteristics that ideally you would like - long-term growth in culture, clonogenic growth, and then differentiation.  They've been used in a couple of different animal models of disease to see some sort of benefit.

One of the mesenchymal cell - stem cell lines, if I remember correctly - and there are a lot of things to sort back through there - had been kept in culture, I believe, for about two years.  There was another cell line that - I think it was also a bone marrow derived cell line, but it had been genetically engineered to contain telomerase or additional telomerase gene.  And that had been kept in long-term culture.

I believe also one of the neural stem cell lines has been kept in long-term culture, and actually Dr. Gearhart might be able to comment more on that.

CHAIRMAN KASS:  Okay.  Thank you.

And I have a general question to all of you, if I might.  Since we sent you some general questions that we were hoping to get some help on, to go from where we are now to actually having reliable tissue grafting therapeutic applications in humans, since there's a fair amount of speculation out there - and, Dr. Gearhart, you did emphasize how early we are in the process and the emphasis, really, on learning of the basic biology.

But the public out there is more - much more interested in, you know, where are the cures?  Could you address that particular question of the various sort of stages, and where we are, and what kind of - how one should talk about these things responsibly in relation to the widespread hopes for remedies for which the patient groups are, quite understandably, clamoring?

DR. GEARHART:  The answer to that question is a very complex one for me.  I could run through a number of cell lineages with respect to the human embryonic and stem cell and germ cell lines, where we are in the process, but it's the other end that I want to address.

The most difficult talks that I give anymore are those before patient-based groups where the hopes are high.  They come with expectations that far exceed, I think, where we are.  And this is the result that I think that early on in the field, as you mentioned, the hype was there when circumspection should have been there.  And I don't think circumspection is a retreat from promise.

The reality is when you get into the laboratory, and you are working on cell lines, it seems the more progress you make, the further away the goal is as we learn.  And we can show I think remarkable things in our animal models of putting in cells, and these are almost - they bear some resemblance to the adult stem cell story where you try a lot of things empirically, you get a response of some kind, and you're trying to figure out what it is, why.

But when you superimpose on top of the biology issues of trying to control these cells in a dish, and trying to expand them in a dish to characterize them, to show that they're authentic, that they do work, the issues of safety, which I think we all are in agreement have to be foremost in this, that the time frame is years away before I think any of this will be realized in a clinic.

I think there's misunderstanding, that if all of a sudden you have an insulin-producing cell in a laboratory, which we do, and we're - as other labs have as well - that most people think this is the answer.  You just put insulin-producing cells into a diabetic.  And as you know, this doesn't solve the underlying problem.

We can - so I don't know how to answer the question that you ask.  I mean, I'm certainly optimistic.  I think it's going to be a direct function, and I'll come back to Janet's question that she can't ask or isn't supposed to ask, and that is that obviously more funding into this area - and I know that this isn't a congressional committee of any kind, where we're seeking funding, but obviously the progress is directly proportional to the number of good investigators you have working on projects.

And in this field, we've had a bottleneck, first from the number of lines that could be used, now the NIH funding I'm sure is going to be more robust over time, but this remains not only I think a financial issue but also almost a psychological one - for students getting involved, for post-docs getting involved, for young faculty getting involved in the field, where we don't know what the future will be, to be honest with you, in this country.

We see our colleagues around the world in different laboratories I think making really substantial progress here.  And so I'll get off this part of it, but I think this is an important issue, at least with the embryonic and fetal sources of cells.  And I hope we'd get over that.

CHAIRMAN KASS:  Thank you.

I have Gil Meilaender, Bill Hurlbut, Janet.  Gil.

PROF. MEILAENDER:  Yes.  Dr. Jaenisch, I want to come back to where Michael Sandel started us, because I'm just not certain that I'm clear on something.  And what I want to know is, you know, it's always hard to know when one's speaking as a scientist and when one's speaking as something else.

But as a scientist, there are various kinds of chromosomal or genetic defects that a fetus might have, the result of which would be that it might die immediately after birth or very soon after that.  How would you scientifically describe those fetuses?  Would you say that they have - that they also do not have the potential to be a normal human being? 

I'm trying to get away from the stem cell issue for a moment.  Would the same characterization be the appropriate one there?

DR. JAENISCH:  This a really a question which is not a scientific question, then, right?  An embryo which has a trisomy number, whatever, large chromosomes will die very early, maybe before implantation.  So that has this embryo which is afflicted with this genetic alteration. 

Has that a normal potential development to a human being?  No.

PROF. MEILAENDER:  So it falls into the same - so, in other words, this is a rather large category of embryos and fetuses that - about which we would say this.  We're not only talking about cloned embryos.

DR. JAENISCH:  Well, I think that -

PROF. MEILAENDER:  The same characterization -

DR. JAENISCH:  Yes, I -

PROF. MEILAENDER:  - would fit.

DR. JAENISCH:  I think the difference, from my point of view, if a chromosomal abnormality occurs after normal fertilization, something which is totally unpredictable and is assessed, that is what fate is to genetic game.  With cloning, I think it's predictable, and that's the difference.  It is predictable to a large extent, so I think that would make the difference.

One is spontaneous, unpredictable, it's a risk of normal reproduction.  The other one is predictable.  Otherwise, I really can't - I think this for me is a major difference.  You predictably generate a human being which has no chance to become normal with any acceptable efficiency.

PROF. MEILAENDER:  I think I'll stop there.

CHAIRMAN KASS:  Bill Hurlbut, and then Janet.

DR. HURLBUT:  Did I hear you correctly say you find destruction of IVF embryos morally troubling, but cloned embryos for embryonic stem cell use less troubling?

DR. JAENISCH:  No.  Well, I said that if you accept that instead of throwing away an in vitro fertilized embryo which cannot be implanted because nobody wants it, instead of throwing this embryo away generating to an embryonic stem cell, I mean, people accept this.  I find this acceptable.  But it does pose an ethical problem, because you do destroy potential normal human life.  There's no talking around it.

So if you accept that, then I would argue that a cloned embryo is less of an ethical problem.  That's all that I want to say.  So the potential is not there.

DR. HURLBUT:  And from hearing you and talking with you at lunch, I had the sense that you feel like nuclear transfer actually offers us more versatility and more promise than using IVF embryos, because we can use the genotypes we want and control various things.  Is this fair?

DR. JAENISCH:  Yes, I would think so.

DR. HURLBUT:  So I have to admit I'm troubled by your comment, and not convinced by Michael Sandel's comment.  As much as I like my colleague Paul McHugh, I'm not - I'm troubled by the notion that what you called not potentially normal human life does not have a moral standing.

And the reason I'm troubled by that is - was just alluded to by Gil, that I would not say that a potentially abnormal trajectory of embryogenesis is not a human life necessarily. 

Now, I have a handicapped child myself, and she does not have a problem of development, but she had brain damage at birth.  But she's not what you would call a normal human life in that specifically all-healthy sense.  But she's very human, as human as I am.

I'll let somebody make the joke on that one, if you want.

But the question is this.  I feel for what you're saying.  I feel for what Michael said, and I feel - have been with Paul to some extent on this all along. 

But if we say that natural - the natural meaning of the definition of a human life is not normal, but some meaningful process of integrated - of integration of identity, of continuity, then it seems to me that the clones that have been produced at least with animal studies have to qualify as entities of their species.

So even though, as you said to me even a year ago, a cow standing on a green hill chewing its cud isn't - doesn't make it a normal cow.  I agree.  But I think it's still a cow.

And likewise, the human trajectory of whatever the clone could be is troublingly human-like.  I personally wouldn't feel comfortable saying, for example, as was implied by John Gearhart in his talk, that we could use it to harvest out later tissues for more difficult differentiations.

Well, let me back that up.  All of my colleagues, and my own reading on this, confirms to me that going from embryonic stem cells to useful cells, tissues, and organs, is a long journey, maybe short for Parkinson's Disease, but longer for more complex tissues, and very long for - what we really want is organs and - for transplant, like kidneys, and so forth.

I think there will be a temptation in our civilization to move the trajectory of something that's designated non-human on to further stages of gestation.  Now maybe not gestation in a human womb.  Maybe it'll be in an artificial endometrium or something that can coax it just 30, 60 days longer for good use.  It starts to be troubling, even if you accept your premises.

Let me back it up, then.  I think in principle what you're saying has something to it, and Paul and I have been trying to talk about this, and I've had dialogues with my colleagues on this. 

I put forward in my personal statement on the President's Council Report on Cloning what I called a speculative proposal, where I suggested that - drawing on what Paul had said, that this is an artifact, not a human being, a clonal artifact. 

Why don't we go to the bottom of the problem?  Why don't we say that something that does not have a human potential in any meaningful way, not an abnormal human potential, but no meaningful human potential, might supply us with the essential way to get around the moral problem, at least to get us to stem cells?  And we can deal with the next issues later.

And I want to stay two things before I conclude.  One is in making this proposal, I was very aware that the first issue of what you might call the inviolability of the human life is only the beginning of the questions about the moral use of human process or human tissues or anything.

But what I had in mind was this - that just as we've come to see that a cell does not define the locus of human dignity, nor does a gene, of course - let's start at the smallest.  A gene - a human gene can be put into a bacterium, grown, used to make insulin.  A cell - we do blood cell transplants.  Tissues - we do skin transplants.  Organs - we do transplants and even organ systems.  None of those are the locus of human dignity, human moral standing.

Likewise, in this age of developmental biology, we will learn that partial generative potentials are not the locus of human moral standing.  We will harness these partial generative potentials in such a way that we can do wonderful things with them without violating the integrity, identity, and continuity of human life.

So this is my question to you.  Could we find a scientific way to meet the challenge of our President and our nation and the imperative of our civilization to go forward with the science?  Could we define the danger as we never want to interrupt a normal human life in process, or even one that is remotely normal?

Could we meet that moral objection while at the same time opening the positive future of our science?  And here the suggestion I would have is using something so fundamental, like short, interfering RNA, to preclude the very possibility of anything but the most minimal and genetic mammalian process to the blastocysts from which we could then take embryonic stem cells.

What do you think?

DR. JAENISCH:  Yes.  I think we talked about this before.  I think it's a really interesting possibility.  So what we need to - for a human being to develop two major lineages, which is a trophoblast lineage and the epiblast lineage.  The trophoblast lineage will support the embryo from the placenta.

So certainly there are genes which are only needed, as far as we know from experiments, that are only needed for placental development.  They are not needed for the epiblast lineage or for the embryo.  So one would modify a donor cell - let's say a skin cell that you want to transplant - with inactivating such a gene - I can give you examples of those - and expressing, for example, using the SI RNA technology, which now really becomes - seems to become a routine procedure to inactive genes.

And, indeed, an embryo - a cloned embryo derived from such a nucleus would not be able to form a trophoblast lineage which would be functional.  So, therefore, if this embryo would be implanted, it would definitely fail.  You could predict it would fail much earlier than this continuum, which I outlined for the cloned embryos which are not modified.

So I think, in principle, this is a doable - potentially doable manipulation which would prevent what you are interested in and what you are raising, prevent the development very far in utero.

DR. HURLBUT:  In your opinion, could we reasonably call that not in any sense a human life in process, abnormal or normal, but call it a clonal artifact, a laboratory production for the procurement of cells with a potential for something, but not what we would have to call embryonic cells?

DR. JAENISCH:  Yes, because you would argue this embryo - I mean, the definition of an early embryonic cell, like a two-cell or a four-cell embryo, the blastomere, is totipotent.  These cells can make the placental lineage and the epiblast lineage.

This embryo would not be able to make the trophoblast lineage, so, therefore, it would not be totipotent.  So I think you could argue this would be a real biological difference.

CHAIRMAN KASS:  We do want to go to the next session shortly, but I've got Janet and Elizabeth, and then I'm going to call a halt for this session.

DR. ROWLEY:  I come back to John Gearhart.  And, first, I want to take you to task for comments at the very end of your talk, and then I have a question.  And what I want to take you to task for are your comments, all related to enhancement and how easy it's going to be to just get genetic enhancement of all sorts of features.

And I think - well, you could expand on that or explain what you meant by that.  But this has been such a critical issue for this particular Council - the likelihood of enhancement - that I think that it shouldn't just be left unchallenged.

And the question that I have is, with regard to characterization of current lines, you indicated that that's under the auspices of the MRC in London.  And I'm just curious, are they looking at both so-called presidential lines as well as lines available elsewhere?  Or are they only lines available elsewhere?  And what kinds of characterization are they proceeding with?

DR. GEARHART:  Let me answer the non-controversial question first.  This is a relatively - this panel - there has been, over the last nine months or so, an international consortium that has been meeting on human embryonic stem cells.

The outcome of this was the appointment of a panel headed by Peter Andrews at Sheffield University, who will be - that panel will be setting up the standardizations, if you will, or the criteria for the embryonic stem cell lines.

They will - I think some of this work will be done in the equivalent of what's the FDA in the United Kingdom.  But they will take any cell line that's submitted to them, whether it's a presidential one or one from anywhere else.  They are not a - they are not distributing cells.  They are just testing them for these different parameters.

And, obviously, there will be a number of transcription factors, a number of surface antigens.  This kind of thing will be in that mix.

DR. ROWLEY:  Will they do karyotypes?

DR. GEARHART:  Oh, yes, karyotype is - yes, it's absolutely essential to have a normal karyotype, and one that's stable over a number of cell passages is critical.

The comment I made about enhancements, I just - it may have been out of order here, but I am concerned, I mean, as a biologist I'm concerned.  I'm concerned about a number of issues.  It makes it sound as if most of the time that scientists aren't concerned, that we're just going along in a fashion that, you know, science for science sake.

And I only have to remind you over the last few months to see the use of human embryonic stem cells in mouse chimeras, the use of chimeric human embryos that has been done, that you say there's no reason that these things should have been done.  I mean, there's no scientific basis for this.

And we have to have some degree - and I'm not going to call it regulation, I don't like that term, obviously, but there has to be some consensus as to what should be permitted and shouldn't be permitted in almost a global way.  And I realize that this is very, very difficult.

I've been to two G-6/G-7 meetings in which we have tried to get some kind of consensus even on human cloning among countries.  I mean, let alone the use of embryos for research, and things like this.  And as you can imagine, nothing much happens.  Everyone has their own opinions, and we go away.

When we have the capability - if this work is successful - I'll be frank with you.  If this work is successful, and by success that we are learning mechanisms around differentiation and the development of cell types, we are going to have an awful power that we can instruct ourselves.

Now, we're not necessarily going to be talking about germ line cells or germ line modifications.  Maybe.  Because, obviously, if we can grow oocytes from these cells - there are reports that we can grow spermatogonia and sperm cells out of these - maybe the possibility exists for that at this point.

But I'm just hoping that people are thinking ahead a bit, that, you know, this is something that's going to be on the table in a number of years, and that we should give it some thought - where we're going to go with this.  That's all.

And so I made a comment about, you know, getting it off the internet or something to do it.  Maybe that's an overstatement.  Maybe that's just crying wolf, and I apologize for that.

But I think it's something that we have to be aware is out there.  It's going to be out there.  Not out there now, but will be out there, and we should be thinking ahead.  So I apologize to the Council for making that kind of a comment.

DR. ROWLEY:  Well, I don't think an apology is necessary.  I think what I'm trying to say is - my own personal view is that the Council should be especially concerned about things that are relatively near term.  And by "near term," in such a rapidly-moving field I think a few years is near term.  And if you're saying, well, in 10 years' time we'll be able to do this, we can't see that far ahead.

DR. GEARHART:  Right.  Right.

DR. ROWLEY:  And so that - so what kind of terms are you talking about in terms of time?

DR. GEARHART:  Oh, I'm talking not about 10 years.  I'm talking about a shorter time interval.

DR. ROWLEY:  Okay.

DR. GEARHART:  You know, Janet, I think to be honest with you, the number of federal Blue Ribbon Panels that have been set up, at the NIH level, the National Academy, to discuss issues that seem to be 10 years away, recommendations were made by our leading scientists, our leading scholars, only to be ignored.  And then, we find ourselves in a pot once these things happen.

And it's just, I mean, frustrating as a scientist.  It's frustrating almost as a citizen to do this, where it seems that the scientific community, to a certain degree, is marginalized when it comes to making recommendations as to how we should proceed.

And we are left, then, every time something comes up in this area everything is thrown up in the area as to where we're going, etcetera.  So that's the nature of the comment.  Or, I'm sorry, that's the basis of the comment that I made.

CHAIRMAN KASS:  Last comment.  Elizabeth, if you - did you still want to say something?  You were on the queue.

PROF. BLACKBURN:  I'll return to the science, and I don't know who would like to answer this question.  But it was picking up on the point that I think Leon did raise about asking about the sources of adult stem cells.  And you mentioned that there's really only one good one, or the best one, is that right.  Dr. Verfaillie's.  And -

DR. PRENTICE:  I think Dr. Verfaillie's is the best characterized -

PROF. BLACKBURN:  Yes, best characterized.

DR. PRENTICE:  - in terms of all of these various traits.

PROF. BLACKBURN:  Yes.  So, you know, we're struck - and I think Leon pointed this out, too - by the fact that there's a great deal of research that's been ongoing in the last, you know, couple of years -

DR. PRENTICE:  Yes.

PROF. BLACKBURN:  - on adults.  And yet it seems to me there's a real problem.  And, you know, what's the problem?  Why is there so little known?  To put it bluntly. 

You know, here's 192 publications that you cite, and why is it so difficult?  Are these just inherently really difficult things to work with?  Is this what it comes down to, that people who work with adult stem cells don't really like to say that, or - sorry to cut to the point, but I know time is of the essence.

DR. PRENTICE:  I think one of the reasons comes back to this difficulty in identifying which one is actually the stem cell, how to actually purify it, isolate it.  Some of the cells seem rather easy to isolate.  Certainly, peripheral blood is quite easier, or the adipose-derived cells would be easy to isolate.

But then, how do you really know that one is the stem cell?  And, again, most of the results - the best results seem to come primarily from these mixed populations of the bone marrow stem cells, where they have not intentionally in many cases isolated the particular stem cell.  Instead, they've been geared towards some sort of physiological endpoint.

My assumption would be, then, working back from that, once they could achieve that end point, then they might be able to derive the particular cell.

But I think it also brings up a point I tried to raise before in that it may not be possible, or it may not be preferable, to try to get a particular cell.  It may be the context of these cells with the population and their interactions, exchanging growth factors, altering their own gene expression, and then - in the tissue milieu - that really gives them this ability to somehow cause the repair. 

And I use that term because, again, I think in many of the instances the research is showing that it's not direct integration and taking on the function.  It's somehow stimulating endogenous cells.

PROF. BLACKBURN:  So it may - are you hinting that it might be qualitatively different from what is going on with embryonic stem cells, when those were tried to be used? 

DR. PRENTICE:  Well, I -

PROF. BLACKBURN:  I don't want to get too much into this, but I'm just trying to understand the science here.

DR. PRENTICE:  Yes, I think that might be a good way to put it.  It may be qualitatively different.  You know, it's interesting Dr. Jaenisch mentioned that embryonic stem cells, in a sense, are a tissue culture artifact.  He might just as well, by the same definition, call Dr. Verfaillie's MAPC cells a tissue culture artifact.

And it may be that putting these cells into culture, now changing their context, gives them some of these characteristics that are so sought after.  Our target might be better put in terms of, what is the physiological endpoint that we want?  Not a particular starting cell, but how do we achieve the repair of the tissue damage?

And, again, as I mentioned in the talk and in the paper, it may be that it's not a particular cell that we really need.  Short term, cellular regeneration may be our best goal.  But long term, figuring out the particular molecular and cellular signals - and, again, it may be a context-type signal - cell-cell context or cell-matrix context - that actually can give us that physiological endpoint, and so eventually going to a non-cell-based system, but a signal-based system if you will.

CHAIRMAN KASS:  Paul McHugh insists on a very brief comment, and then we're -

DR. McHUGH:  I have just two -

CHAIRMAN KASS:  - going to break.

DR. McHUGH:  - little tiny scientific questions, but because they are important science that we're discussed here.  The first one really relates to Dr. Jaenisch, and that is, I wanted to know, sir, now whether you could say that the genetic alterations and the problematic genetic status of the fully cloned animal, whether those genetic abnormalities would then pass on to their offspring, and so, therefore, be a contaminant to the whole human genome legacy from then on?  That's the one question.

DR. JAENISCH:  Let me first clarify there are no genetic alterations which are important.  They are all epigenetic.

DR. McHUGH:  Right, yes.

DR. JAENISCH:  So the experiment has been done.  You can take two animals afflicted with large offspring syndrome and mate them.  The offspring will be all (risk?) normal, because you send the genome through the process of gametogenesis, which reprograms everything in a normal way, everything is reset.

Of course, there might be some genetic alterations which are acquired during somatic life.

DR. McHUGH:  Right.

DR. JAENISCH:  Those would not be - they cannot explain the phenotype.  They would be recessive, and so they haven't shown up yet.

DR. McHUGH:  Right.

DR. JAENISCH:  So we don't know if they would show up.

DR. McHUGH:  They might show up later.

DR. JAENISCH:  Right.

DR. McHUGH:  And secondly, to John Gearhart - John, you whine a lot about how troublesome it is to be a scientist nowadays.  I just want to know this.  The President made a very good Solomonic decision and produced the cell lines for you scientists to work, putting the ball in your court.

Are you saying to us now you've used up all of the potential in those cell lines, and now, because of the President's decision, are restricted in your progress?

CHAIRMAN KASS:  You don't have to answer, and he's over time.  But if you'd like to -

DR. GEARHART:  I have to answer Paul.

CHAIRMAN KASS:  - 15 seconds.

DR. GEARHART:  Being a scientist is the greatest thing in the world, and I wouldn't trade it for anything, Paul.  And so I don't -

DR. McHUGH:  I know that, John.  Me, too.

DR. GEARHART:  Okay.  And I'm not here to whine.  The funding issues are improving, and they are improving nicely here.  Okay?  And, clearly, we can learn a lot through the lines that are approved.

CHAIRMAN KASS:  Gentlemen, thank you very much.

Look, we have a guest we've kept waiting for quite a long time.  Would the Council, please, 10 minutes.

Thank you all very much.

(Applause.)

(Whereupon, the proceedings in the foregoing matter went off the record at 4:10 p.m. and went back on the record at 4:23 p.m.)