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Thursday, April 25, 2002

Session 1: Stem Cells 1: Medical Promise of Embryonic Stem Cell Research (Present and Projected)

Dr. John Gearhart

CHAIRMAN KASS: Well, I would like to ask Dean Clancy to officially open the meeting, please.

MR. CLANCY: This meeting is lawful.

CHAIRMAN KASS: Thank you very much. Apologies to our guests and to members of the audience for the late start. Council Members had to take an oath of office, which should have been administered to us before our very first meeting.

That has been done and we are now legal in every respect. Welcome to this, the third meeting of the President's Council on Bioethics. We are expecting colleagues Krauthammer and George today, and Stephen Carter will not be with us, and Bill May will join us tomorrow.

I would like to introduce a new member of our staff, Judy Crawford, who comes to us as the office manager. Judy, would you please rise so that the council members can know you. We are very delighted to have Judy with us.

We reconvene as the debate about the cloning legislation heats up around us, a debate that we did not begin and do not control. We are in the midst of our own careful and thorough investigation of the ethical, social, and policy implications of human cloning seen in its larger scientific, medical, and human contexts.

We have chosen to proceed in a deliberate, collegial, wisdom-seeking, mode in keeping with our charge to inquire fundamentally into the human and moral significance of developments in biomedical science and technology.

The most challenging aspect of our inquiry to date has been the moral significance of cloning for biomedical research, a topic discussed for the first time at our last meeting, and to which we return later today in the hope of making progress and clarifying the contested moral issues at stake, and in articulating the best possible moral arguments for and again the conduct of such research.

On behalf of the council, I would like to thank the staff for its superb work in advancing our inquiry, and on behalf of the staff, I would like to thank council members for their thoughtful comments and responses. We are in your debt.

The agenda for this meeting brings us into some new, but not altogether unrelated, areas of inquiry. Stem cell research, a topic of our first three sessions today.

Second, the question of therapy versus enhancement as a goal for the uses of biomedical technology, and third, possible regulation of biomedical technology. These topics have been selected with a view to initiating one of our obligatory future projects, stem cell research, and exploring two possible future projects for the council for the rest of our two year charter.

As everyone knows, in his speech announcing the creation of this council, President Bush charged us with monitoring stem cell research, embryonic and non-embryonic, human and animal, in order to assess their progress in gaining knowledge and beneficial therapies, and in due course to offer guidelines and regulations for the conduct of such research.

As I indicated at our first meeting, we have begun to collect data that will enable us to describe, assess, and compare the successes achieved with both embryonic and non-embryonic stem cells.

As we are doing this, however, it seemed desirable for council members to learn firsthand, and from some leading researchers in the field, about the scientific and therapeutic promise of stem cell research present and projected; embryonic and non-embryonic.

And it also seemed desirable to explicitly begin a disciplined conversation about the ethical issues of embryonic stem cell research. Our first three sessions today constitute the official thematic beginning of our project on stem cell research.

We have of course already been deliberating about some of these matters in our discussion of human cloning for biomedical research, a topic that first arose for us as a crucial side question of the larger subject of human cloning to produce children, what to think about it, and what to do about it.

This is therefore a useful juncture at which to indicate the distinction, as well as the connection between these two topics. Many members of the public, including many of our elected officials who are in the process of making policy in this area, as well as some members of the media, have conflated the issue of stem cell research and the issue of cloning.

The issue of cloning comes first to attention as an issue of the ethics of producing children by novel technological means, and the issue of cloning, insofar as it has captured the public attention, is primarily about what to think about the asexual production of new human beings who are going to be genetically virtually identical to already existing individuals.

And the issues there are in the first instance the questions of the ethics of, crudely speaking, baby-making. That is quite different from the question of the ethics of embryo research.

Virtually all embryonic stem cell research now under way, both in humans and in animals, involve cell lines developed from embryos, whether inner-cell mass, or from the gonadal ridge of donated fetuses, that originate from the sexual union of egg and sperm, and very often in the human case using excess embryos produced in in vitro clinics and in all cases from material not produced for the sake of the research. The question of Federal funding of this research that President Bush resolved last summer, this was the question that was resolved last summer, and the research in this area proceeds not only with Federal funding under the guidelines that the President established, but also in the private sector.

The two topics, however, intersect and overlap because cloning to produce children necessarily proceeds through the production of cloned blastocysts, which offer special opportunities for embryonic stem cell and other research.

Some proposals to curtail cloning for providing children would do so by curtailing the initial steps, thus interfering with the possibility of using cloned embryos for research.

And this has given rise to arguments for and against cloning for biomedical research proper. This is where the intersection can be made explicit, and that is where we now are.

In order for us in the other project to continue to make progress, and therefore in order to see what value added might derive from working with embryonic stem cells extracted from cloned blastocysts, one needs to know something about what it would be added to. That is to say, to work on ordinary embryonic stem cells. And in order to see more clearly what the ethical issues are that might come from the question of producing cloned embryos for biomedical research, it would be helpful for us to know something of the ethical issues of experimenting on human embryos of sexual and not clonal origin, and of using extra embryonic — using the extra embryos or fetuses not created for experimental purposes so we can see what different questions arise here.

To help us with our scientific and medical education, we are very fortunate to have as our guests and presenters this morning two distinguished researchers, one who is a pioneer in isolating and characterizing human pluripotent stem cells, Dr. John Gearhart, the C. Michael Armstrong Professor of Medicine at Johns Hopkins University, and the Director of the Institute of Cell Engineering.

And second a person who is a pioneer in work with human multipotent adult progenitor cells, Dr. Catherine Verfaillie, a Professor of Medicine and Director of the Stem Cell Institute at the University of Minnesota.

Each of our guests in separate sessions will make formal presentations, roughly 30 to 35 minutes, after which time we will have a chance to ask questions about the scientific, technological, and clinical aspects of these areas of research.

This is our chance to learn about the wonderful prospects of these investigations. However, let me say that because our guests are here not only as scientists, but also as our neighbors, in a morally aspiring human community, we will perhaps try toward the end to elicit from them their own thoughts about the ethical issues in their own work.

But the purpose of these sessions is primarily our own education about the scientific and medical aspects. With that I would like to turn the meeting over to Dr. Gearhart, and to thank him very much for joining us this morning.

DR. GEARHART: I am certainly grateful to have this opportunity to share with the President's Council my knowledge in a very tiny area of biomedical research, and it is currently quite tiny, but if you read and believe the press, it is obviously going to expand enormously.

Much has been written and much has been said about stem cells, and it seems every morning in the paper there is some article relating to it and continuing the debate.

In the scientific literature, we see virtually in every issue of leading journals a paper dealing with stem cells. An age old dream I think of mankind or humankind has been to replace damaged or diseased tissues with functional ones, new ones, and wouldn't it be nice to be able if you had a damaged liver or kidney to take one off the shelf if you know what I mean.

And this dream I think is going to become a reality, and with some of the advances in biomedical research, and one of the ones that we are going to talk about today, I think this will provide the starting material that will lead to this reality.

The concept behind cell-based therapies — and this is what we are talking about here initially — is a very simple one, and I think that that makes it attractive, and it makes it understandable to the public. And that is that if there is a tissue deficit, why not just replace the tissue. Now, it is easy to say, and it will be difficult to do, but the concept is an easy one.

Cell-based therapy has also been called regenerative medicine, and there are many rubrics for this today. The power of this technology is derived from information inherent in our genes and in our cells, and the recent isolation of these embryonic type stem cells I believe is going to provide the enabling material as I mentioned for this to go forward.

Stem cells are going to serve several purposes, the first of which could be as a direct source in transplantation therapies. That means specific cell types will be grown in culture, such as heart muscles, nerves, et cetera, and transplanted to patients for function.

Or they will be genetically engineered to do exactly what we want them to do and transplant it to patients; or they will be used by our tissue engineer colleagues to construct tissues and parts of organs, which would then be transplanted to patients.

Stem cells will also be used as a source of information, basic science, and this is really where we are at currently. That could be applied to a patient's own cells, such that we could remove cells from a patient and alter them in some fashion to produce the cell types that we want, and then transplant them.

Or ultimately I feel that what we are going to be able to do from the information that we are going to learn on stem cells is that we will be able to work in vitro with patient cells to get them to perform in a manner that we want without taking them out and putting them in culture.This, I believe, is the future. The scientific challenges to attain our goal of producing safe and effective therapies are formidable. It will take the efforts of many scientists and clinicians, in a variety of disciplines, to bring this endeavor to fruition.

Now, the stem cells that I am going to talk about today interestingly really do not exist naturally. That is, they don't exist in embryos or fetuses. They are artifacts of culture.

But we take tissues from embryos and fetuses and they undergo a type of transformation in culture to provide these stem cells. And this source obviously brings with it a number of ethical concerns.

I, as an investigator, who has had to cross this bridge 9 or 10 years ago when I began this work, believe that the ethical issues are manageable.

I also believe that it is the responsibility of scientists to candidly and in a timely fashion present the social implications of their research and its technological applications; to provide assessments on reliability, and to participate in the establishment of ethical guidelines and to work within those guidelines.

For the past 9 years at Hopkins, we have been in compliance with all institutional, State, and Federal policies in dealing with the cells that we work with.

It has not been easy because the landscape has changed in 9 years, and every year there have been new concerns raised, and new issues that had to be addressed, and I think we are keeping up with it.I should tell you also up to this point in time that no Federal monies, no public monies, have gone into our research effort. Now that Federal policy has changed, we do have applications pending before the National Institutes of Health.

I also want to point out something that may be surprising to most of you; that in our laboratory at Hopkins that we just are not concentrating on an embryonic or fetal source of stem cells.

We are studying stem cells from adult sources, umbilical sources, et cetera. This is the only way that we feel that you can have a scientific advance, and that is to be able to compare and contrast the different sources of stem cells.

So side by side, in the laboratory, in experimental paradigms, we are using stem cells from a variety of sources, and this is what I think has to happen to assess which of these sources are going to prove the most effective for any specific type of therapy.

Another thing that I want to point out to you is that the work on the human cells, I do have the questions that came from your committee in- hand, and many of them are asking what is the status of certain types of work.

I just want to point out that this work has been ongoing for a period of 2-to-2-1/2 years, and although we feel that we are making progress, we certainly are going to come up with, well, I don't know as answer to some of your questions.

I just want to let you know that we don't have all the answers to this, and we are very, very early in all studies of stem cells, be they from the embryonic or adult sources.

I would tell you though that to date the work in our lab and others on embryonic stem cells and the results of that work is certainly consistent with the idea that this is going to prove to be a productive line of research.

Well, it is interesting that very few people know you and what you are about, and I think it is important to point out something. My interests, or my research interests for decades, have been in the area of developmental genetics and development biology.

I have been labeled as a human embryologist, and my interests certainly are in the area of how an embryo goes from a single cell to a multi-cellular integrated organism.

And this is where our research has been in the past 25 years, and I have carried this a step further. We are very interested in congenital malformations and birth defects.

I have had a program project through the NIH for many, many years dealing with Down's Syndrome, and we are very interested in trying to determine what the mechanism is that underlies many of the unusual anatomical neurobiological consequences of this extra chromosome in human beings.

And this is essentially how I got into this work. We wanted to have in the laboratory a source of cells in addition that we could study at the site or level of the impact of these extra genes.

And this obviously is a goal, along with a number of other genetic-based diseases and malformations in the human being. So this is what led to our getting into this area of research.

Now, I have to say up front that we are now required by our university to reveal where our monies come from, and these are the sponsors of our research, and there is one sitting in the middle there that I also have to show to you that I am conflicted.

And which means that to the sponsorship of this private company, we have received money for research, for which licenses have been negotiated between Hopkins and Geron, and that I am a stockholder, albeit a few hundred shares of something that is trading now at — and I hate to think about it.

It is not in our possession as you know. It is held in escrow. But nonetheless we do have this arrangement with this company. So I would tell you that this is not the motivation, this connection.

Without the sponsorship of this research, this work would not have gone forward over the past seven years. We are not in this business as individuals to make money.

Well, having said all of that, let's talk about stem cells. The first thing I want to give you is a little bit of a primer on stem cells so that we are talking the same language, and you have an understanding of where I am coming from.

Well, what is a stem cell, and basically a stem cell is a cell that has two properties. It has a property in that it has a capacity for self-renewal, which means that the cell can divide and produce more cells like itself.

And it has some type or some degree of differentiative capability, which means that it can go on to specialize into a single cell type, or it can specialize into a number of cell types.

And in a developmental sense, if we over time at what our research has told us about stem cells, they fall into a number of categories. Early on in developmental practices, we have a cell that is totipotent.

It can renew, and it can form virtually every cell type that is present in an embryo. As development proceeds, its developmental capabilities become more restricted until we get into different lineages, specific lineages, and its ability to divide also becomes more diminished over time.

This has been the classical picture of development. Now, what has happened over the past couple of years interestingly is we find that these restrictions in developmental capability are much more plastic than we had thought.

So out here where we thought that these cells are highly restricted, perhaps they aren't so, and when you remove them from the organism and culture them, they have capabilities of forming other cell types, and Catherine will be talking to you about some of these issues.

Well, we are going to be talking about embryonic stem cells, and what is it about them. Well, interestingly, we know that these cells are capable of producing virtually every cell type that is present in an embryo, a fetus, or an adult, except one.

And that one happens to be the trophoblast cell, which I will tell you about in a moment. So we consider these cells to be totipotent.

They don't have the ability in and of themselves to form an embryo or an individual, okay? They have this other property of self-renewal, which basically with respect to embryonic stem cells means that they will expand indefinitely, and grow indefinitely, and this is a very important property.

It means that within the laboratory from a very few cells that you could grow a roomful of these cells very easily. But there is an issue here that we don't know much about, and that is obviously there is a finite probability that at every cell division that a genetic mutation will appear.

And there was a paper published recently that indicated that indeed this is the case, and the types of mutation, although the mutation frequency and the mutation rate is greatly — by several folds lower than in normal somatic cells, mutations do occur in these cells, and they are of the nature of making these cells susceptible to formation of tumors.

The uniparental disomy appears and it is a condition about which we should be concerned. And up until this point, in the mouse where these cells were first isolated, and for that work the person who did this, Martin Evans, was awarded the Lasker Award last year.

We know that these lines forming whole animals, which is what they have been used for up to this point, in genetic mutations is getting genetically defined strains of mice.

That there comes a time when these cells are no longer productive in doing this and that they lose some quality. So we know that there is going to be a half-life to the use of these cell lines for whatever reason.

I just want to point that out, although they do have this replicative ability. Well, where do these totipotent cells come from, and two major sources. The first is this pre-implantation stage which we are going to talk about, and the second are from specific cells within the fetus.

I also have on this slide, and by the way, I have given you two handouts. One is the slides in the presentation, and another in a fairly recent Nature review of this material, that you can refer to.

I want to point out another source of a cell that is very similar to these two that we have isolated, and that comes as a stem cell for a specific type of tumor called in the old days teratocarcinoma, and now called mixed cell carcinomas.

These stem cells, referred to as embryonal carcinoma cells, were first isolated back in the 1970s when I worked on this, and we thought that these would be the answer to finding cells that would produce a variety of cell types that we could work with within the human.

And I should tell you that at this point in time that there is a clinical trial going on at the University of Pittsburgh using embryonal carcinoma cells that have been selected for a neural lineage, and so that in culture you can derive neural cells and that these have been placed in the brains of 12 stroke patients.

It is a cell that is very, very similar to the two that I am going to talk about. Well, the first source that you are aware of comes from these structures here, which are pre-implantation stage human embryos, and I am sure you are familiar with this.

And where that structure consists of two groups of cells; this outer layer called trophectoderm, and an ectopically placed inner group of cells called the inner-cell mass. It is from this group of cells here that the embryo proper is derived, and it is connected ultimately to this outer layer, which develops in the placental tissue by connecting stock in an umbilical cord.

These cells may number only 15 or 20 in an embryo that may consist of perhaps several hundred cells. And in work in the mouse, and subsequently done in humans, first by Jamie Thomson, was that these cells were isolated, placed in a culture condition, which then permits their growth and their conversion into an embryonic stem cell.

This process of conversion can be highly inefficient, meaning that you would need a large number of blastocyst and inner cell mass cells to derive a few cell lines.

In some people's hands, it can be more efficient, but there is an issue with that. A second source of cells with the same features was identified in the early 1990s, first by Peter Donovan at NCI.

And what they were attempting to do were to culture long term cells that are called primordial germ cells. These are diploid cells that are present in an early embryo that eventually give rise to egg and sperm.

And they isolated, and this is superimposed upon a human fetus, they isolated from the gonads, the gonadal ridges, these large cells, which at the time of isolation in humans are about 20,000 of them present in a gonad, and placed them in culture and essentially ended up with the same type of cell.

This is what a human EG culture looks like, this clustering of cells and I want to point out that there are cells in the background here which are the so-called feeder layers.

All of these cell lines are derived on feeder layers, and all the lines that were approved by Mr. Bush, and all the lines that we have, are derived on a mouse feeder layer, and this is a point of contention, meaning that we are concerned now about the fact of any endogenous viruses being transferred from other animal tissues into the human cells.

And the FDA must deal with this at this point in time, but we do not have permission on the use of Federal funds to derive new lines, avoiding this issue of other animal products.

But they are grown on feeder layers. They are established and grown on feeder layers of other species. If we compare different properties of these cell types, and I bring this up — some of these are of no value to you immediately, but these are the criteria that one must use to say whether or not you have a cell line.

It is very important, and of the 80 some lines that are now purported to be available, I can guarantee you in talking to many investigators from around the world that only a handful of these are bona fide cell lines, and/or available to investigators.

Now, this may beg the point and that that may be enough to serve the purposes in the immediate future. But really the majority, the vast majority of so-called lines available do not meet the criteria that are now used to say whether a line is a line.

Now, how do we — we are very interested then in two things here. One is the basic science aspect of this, and of course what is driving all of this is the hope for some type of transplantation therapy.

Let's talk a minute about the basic science. What we have in the laboratory now are cultures of cells in the plate that can form any cell type in a human body.

Now, the argument is have we demonstrated that you can get out of these all 200 and some cell types? No. You only find what you are looking for.

What we have found though are a large number of cell types that are present in the human body within these dishes. The problem at the moment is getting homogenous population of pancreatic islet cells or blood cells, or muscle cells.

This is the real part of the scientific struggle here, and coming up with the paradigms to say can we take a cell that can form any cell type, and get it to form but one cell type.

And to do this we have to rely upon our knowledge coming out of molecular embryology as to the genetics and what not involved in any type of cell specialization.

And this is really the limiting issue at this point in time, getting these purified populations of cells on demand. There are strategies that are used that we do pretty good at, and we will take the initial populations of cells, and we can change feeder layers, and we can change growth factors, and we can put them in different types of cultures and force them then to begin to specialize.

But they are mixed cultures, and within the same dish you are going to find neurons and muscle, et cetera. And we must then go another step and begin to sort out either through procedures called flow sorting based on what is on cell surfaces to get then pure populations of hematopoietic stem cells, muscle cells, or neuro cells.

And this works fairly well. We can get cultures of dopanergic neurons that are 80 percent pure, and we can get cardiac muscle that is 97 percent pure, et cetera.

But we are a long way from isolating in a homogeneous fashion the various types of cells that we would like to get. Some of them ere doing well at and others were not.

And it is going to require an extensive amount of research to achieve this. Now in going to transplantation therapy — we are going to jump a little bit ahead here, and if we start, this could be ES.

If we start with this population, we do not transplant into anybody, or into an animal at this point, one of the stem cells. You don't do it. The reason that you don't do it is this.

These stem cells are capable of forming a variety of tissues, and they will form tumors, and these tumors are these mixed germ cell tumors that contain a variety of cell types.

They are called teratomas in the old literature. Monster. I mean, they are contained in a mixed array, and you can see teeth, sebaceous glands, hair, bone, parts of the gut, et cetera.

So what you have to do to make this work is you want to at least get cells that you have treated somehow in a dish into some of these more defined lineages that are away from this capacity to form tumors.

So that we then begin to select tissues downstream, all right? Part of the problem, and you will read this in the literature, is how good your selection is, is also indicated by whether or not when you take myocardiocytes that you say, oh, these are all 100 percent myocardiocytes, you transplant them into the wall of the heart, and you end up with a teratoma.

This happens, and we are into the central nervous system, and you end up with a teratoma within the brain. So getting rid of those initial stem cells are essential, and we have ways of doing this genetically, but I just want to point out that this is an issue.

To say nothing about the fact that we do not know whether any cell downstream here has the capacity to revert. We know very little about that at this point in time.

So let me give you an example. There are many of these coming out in a number of laboratories, most of them in the mouse in which lines have been derived in different lineages, and they have been transplanted into animals to show proof of concept, and that you can isolate a specific cell type, and you can transplant it, and it will function within the transplant.

I would like to give you now an example from our work at Hopkins. It is an unpublished work, and it is now under review, but I think it is important because it really illustrates several points that are critical here.

We have taken our human cells and grown them under culture conditions that would select for specific types of lineages, and whether it is neural, or whether it is muscle, et cetera. And now we have, I believe, in our laboratory over a hundred a hundred lines like this, of the human lines.

And in the one example that I want to present to you, which was done with members of our department of neurology, and in collaboration with our lab, is a model, using these cells in an animal model of the motor neuron disease.

And in this study, these animals are treated with a virus that destroys lower motor neutrons, so that animals become paralyzed, and they are paralyzed because they lose the big nerve muscles that in your spinal cord hook your muscles up to the central nervous system.

So that in a period of 10 days following the injection of the virus into the brain, the animals become paralyzed, and we have gone to great lengths to show that it is really the ventral roots that are involved.

You wipe out these neurons and these animals never recover. They never recover. So what we have done is to take our human neural cells out of this and infuse it into the spinal cords of these rats, and to look then for the recovery of motor activity.

This is a rat out for a mid-morning stroll, and this animal is infected with the virus, and it is a virus that really leads to an encephalomyelitis, and within a period of 10 days the animal is paralyzed.

We can document exactly what this paralysis is about. The virus is cleared, and shortly thereafter we will put a cannula into the lumbar region of the animal, and infuse 300,000 cells into the cerebrospinal fluid, and these cells will float all the way up to the hind-brain.

And then we monitor the motor activity of these animals, and within a period of a few months, we begin to see animals that can now place their limbs underneath them, and that can draw them up, support some weight, and begin to push off.

And at the high end, within a several month period, we can have animals that are now walking. And the issue is why are they walking. And what we have learned, although it is not as you can see a normal gait, et cetera, and we have really documented this as well, they are walking.

And why are they walking? Well, initially what we felt was this. This is a panel showing cells within the ventral horn of those animals and I want you to look at this cell here.

This cell, based on its marker, and based on its physical characteristics, and molecular characteristics, is a human motor neutron cell that has been specialized out of these neural precursor cells, that has sent an axon out into the periphery at least two centimeters.

And we have been able to cut the sciatic nerve out on the limb of this animal, place a dye at that site, and that dye is picked up by that axon, and brought back to the cell body that extended the axon.

And it comes back, and this is the green stuff here, and it comes back then into the cell body of the human motor neuron. We have gone on to document how many human cells are present, and what they are as far as the phenotype is concerned, to see — you know, yes, they are forming glia, and they are forming a variety of cell types within the ventral horn of that animal.

Interestingly, and one of the safety issues that we find is that 50 percent of the cells don't do anything. And we are a little bit concerned about that.

I mean, is it good to have all these cells in there that aren't doing anything, but this is an issue that we have got to resolve. Well, it turns out that this is only part of the answer. It turns out that the human cells at the same time are producing growth factors that rescue and enhance the regeneration of the animal's own cells within the ventral horn.

And so this has led us then to set up experiments to try to figure or try to determine what growth factors it is that is causing the growth of axons in those mice and in rats in the ventral horn, and it may be that eventually we can use just the combination of those growth factors to elicit this response. We don't know.

So these cells are serving in a dual capacity, which is somewhat exciting. We have taken the human cells and we now have grafted them into monkeys. They were in monkeys for over a year.

This was a safety study to in fact show that we are not getting tumors formed. I think you can appreciate one of the major issues here that we are going to be faced with, with this type of approach, is animal experiments are of a very short duration. Mice and rats are for periods of several months.

Monkeys we can go much longer. How much data is going to be needed to convince the FDA that this is a safe approach, and this is something that is being debated now within the FDA and it is a difficult issue.

But here we show human cells, and that is these blue ones that have been in this monkey, and in this case for 180 days, but we are now out a year, and we can show that these cells are forming specialized structures and they are non-tumorigenic.

The next phase is to look at a graph model here that is functional.

CHAIRMAN KASS: Can I just ask a question?


CHAIRMAN KASS: What has been injected here?

DR. GEARHART: Oh, I'm sorry. These are the same — what has been injected into this monkey are the same cells that were injected into the rat. The same cells. They were human cells —

CHAIRMAN KASS: Neural precursors?

DR. GEARHART: Neural precursor cells. The same cells, the same culture cells. A major issue that we must discuss and that we are concerned about is graft rejection. Obviously, anything that you grow up, unless it matches the patient, is going to be subjected to that, and now we get into an area which Dr. Kass has mentioned earlier.

But what are our options here? What are the options of being able to grow these cells into any of these lineages and then to transplant them and not have rejection?

Well, there is a long list, and it starts with, well, maybe what we ought to do is derive hundreds of ES and EG cell lines, and then you would have a best match for a patient. Not very practical.

Can we use the patients own cells, and you will hear about some of this shortly. Should we use immunosuppressive therapies. We would like to get away from that.

Can we use what the tissue engineers are referring to as sequestering grafts, and what this is, is you can take grafted cells and put around them matrices that will not permit other cells to touch them, but yet they can produce products, or they can function in a graft.

So you are trying to hide them from the host immune cells. How effective that is going to be, we don't know.

Can we perhaps come in and genetically modify, which is easy to do in these cells with the histo-compatibility genes, so we can make them more like a patient that is going to receive these cells.

Or is it possible that we may end up being able to produce cells that may be universal donors. Again, we are trying this, and at the moment it is speculation.

Clearly the one thing that has worked is the issue of nuclear transfer therapy, the so-called therapeutic cloning, in which as you know the argument is to take a cell from a patient, and fuse it to an enucleated egg, derive a blastocyst, recover the inner cell mass, culture it out, and then these embryonic stem cells would match the genome of the patient.

Is this a pipe dream? The answer is no, and I will give you an example of that in a moment. To get around some of the issues with the human cloning, embryonic cloning in humans, you have seen reports in the Wall Street Journal and other places which I can confirm are real, in which there are attempts now to take human cells, human nuclei, place them for example into rabbit eggs, enucleated rabbit eggs, and grow up a blastocyst, and generate stem cells that have human nuclei and rabbit mitochondria.

And the argument has been made here that, well, these cells would be perfectly fine for an autograft, and this isn't accurate. We know that mitochondria produced polypeptides that are integrated into the cell membrane, and are actually considered to be minor histocompatibility antigens, and will be recognized and rejected by the host from which the nucleus came from.

So this really is not getting around the issue of the graft stuff at all using other animals, and we are a little bit concerned about how this is being handled.

So, let me give you an example, and one which you should read these papers if you haven't from Rudy Jaenisch and George Daley at MIT, using the nuclear transfer therapy, or the therapeutic cloning, to do two things.

What they did was to take a mouse that had a genetic mutation in genes that are important as far as the immune response is concerned. And they took cells from this mouse, took the nucleus out of the cell, and placed that nucleus into an enucleated egg to produce a blastocyst from a cloned embryo.

They took the inner-cell mass cells out of that, and generated embryonic stem cells, that then are the same genome type as this animal, and then went in and repaired genetically the mutation within those cells.

And then differentiated these cells into the hematopoietic stem cell component, transferred them back into this animal that had the mutation, and the transplant took, completing the whole hematopoietic system, and in rescuing that animal.

So this is a proof of concept kind of experiment, and I urge you to read it. It is an extremely powerful illustration, not only of the therapeutic cloning end of things, but also the ability then to come along and correct the genetic mutation and the reference was given to you.

Another argument has been made that we should be using perhaps just eggs that have been stimulated to form embryos, and these are parthenotes.

And the argument here has been that we can then use these directly into the female from which the eggs were taken. I just want to point out that in my opinion that this is going to have very low usage.

You are going to have to recover embryos or eggs from patients, post-pubertal, and pre-menopause. The window is going to be fairly short, I think, for many of the therapies that you would want to effect.

And the other issue is that we don't know much about cells that are derived this way, and how viable, and how functional they are going to be. But this has been used or promoted also as a source, and this is an illustration of where you take those cells.

All of this type of technology, I just want to let you know, and I know that you are grappling with this, but even within the field of the scientists are beginning to argue about what is an embryo and what isn't an embryo.

So any arguments that you have within your council on this, I will tell you is also being held among biologists. I think that my own personal feeling is that anything that you construct at this point in time that has the properties of those structures to me is an embryo, and we should not be changing vocabulary at this point in time. It doesn't change some of the ethical issues involved.

What are some of the problems here, and I will summarize this a little bit. Current research. Well, we have to come up with better ways of having high efficiency differentiation protocols resulting in homogeneous cell populations.

We are dealing with growth environments, and genetic manipulations, and we are trying to define stages of cell differentiation within our cultures.

And assessing whether or not the differentiated cells that we are getting out are normal and completely functional. And this is in a dish.

And let me tell you that there are examples of where you can spend all of an effort studying something in a dish, only to find that if you pop it in an animal that it doesn't behave how you think it is going to behave. We have a lot to learn here.

I think you can imagine that what is going on in a dish is not exactly what is going on in a site where you transplant. The whole issue of grafting, and how you put it in, and the safety issues, and that cells migrate away, and they differentiate, and will they form tumors, and then the issue of the immune response.

These are all, you know, formidable obstacles that lie ahead. I mentioned to you that we can use cells individually, and have been used in a variety of paradigms in our collaborators of single cells, and the tissue engineers are now taking these different cell types and seeing if they can reconstruct or construct organal aids or tissues to do in-grafting, and thee has been some success with this at this point.

Finally, to me, the future is going to be that the basic science coming out of this is the most important element, and that from that information we are going to be able, I think, to take patient cells, where appropriate, and I say where appropriate because if you have autoimmune disease, or in cases where you have an injury, spinal cord injury, or stroke, or heart attack, and you don't have time to take that patient's cells, you are going to have to come up with different paradigms.

But I think we are going to be able to eventually coax a patient's own cells to behave in a manner that we want to, but we are going to learn this I think through the study of stem cells.

The last thing I will say is I know that you want to ask, well, what is the future going to bring, and I am concerned about predicting the future. I can't even do this on a three year NIH grant and this is what is expected of us.

You know, what is going to happen here. I certainly think that everything that has happened up to this point is consistent with success in this area, and I could get into more predictions in a moment.

But we are always asked when is this going to happen, and it is going to be I think based on specific cell types, and on, and on, and on. But the predictive thing is very, very difficult.

Well, I thank you for your attention, and I hope that this was enough of a primer to add more meat to your discussions. Thank you.

CHAIRMAN KASS: Thank you very much.


CHAIRMAN KASS: We were only physically in the dark, but we are grateful for your enlightenment, Dr. Gearhart, and the floor is open for questions, and comment, and discussion. Don't forget that you have to turn your microphones on to be heard. Jim, go ahead.

DR. WILSON: Dr. Gearhart, do you foresee that it will ever make a difference whether cells that are transferred for human cell regeneration come from cloned eggs, or from the retrieval from IVF eggs? Does it make a difference what the source is?

DR. GEARHART: Well, I think in the short term that it will. I think the only way we have around the immune rejection story at this point is from cloned embryos.

For a patient in which you can predict ahead of time is going to need stem cell therapy and you have the time and money available to do the cloned approach.

I would like to think that this is going to be a transitionary period, and that we will not have to rely upon this in the long term, and that we will be able to take for any specific disease a stem cell, or a derivative of a stem cell that may come from the adult source, the umbilical source, the fetal source, or embryonic source.

I mean, whichever presents, and that we will have ways of dealing with this graft rejection story other than through the cloning of human embryos.

DR. WILSON: If I could just supplement my question with a related one to which you referred. What is your current assessment of the value of adult stem cells, as opposed to embryonic ones, as a source of organ regeneration currently?

DR. GEARHART: Oh, I think it is a very viable option and I think NIH should fund it. I think that from what we see in the work, and Catherine will present a nice overview of this, that this is going to be a good source of stem cells.

They have some issues that they have to overcome, issues of expandability, and plasticity, that we feel are — that have not been demonstrated as well as embryonic stem cells, but I think that eventually we will be able to overcome this.

But I think part of the knowledge of overcoming it is going to be coming from our studies of cells that have those capabilities, and being able to transfer that information to those other cells.

So I think we are going to come up with — I believe that in the stem cells, cell-based therapies, that we are going to identify certain adult sources that are going to be good for some diseases, some injuries, and embryonic sources for others.

So I think we are going to mutually proceed on this and benefit from it.

CHAIRMAN KASS: Please, Elizabeth.

DR. BLACKBURN: Dr. Gearhart, you can give us I think a unique perspective on the comparison between adult, and embryonic, and fetal stem cells.

And in particular many of us read the recent papers, the scientific peer-reviewed papers that came out with respect to the adult stem cells, and the interpretation of their plasticity being cast in some considerable doubt by the observation that there was cellular fusion of those cells which had led to in these particular cases examined a mistake in interpretation of their plasticity.

And I wondered if you could give us your perspective on that aspect, which extends Jim's question somewhat.

DR. GEARHART: I will do so in the face of Catherine sitting back here, who is —

DR. BLACKBURN: Yes, I am going to ask her, of course, about this, too.

DR. GEARHART: — actually done those experiments. Clearly the most difficult experiments that we have had to address and interpret are those utilizing adult stem cells that have been placed into the blastocyst of mice to create chimeras.

And in those chimeras, we see that the descendants of those adult cells gave rise to many, many lineages within the embryo, and this was really the issue. How did we explain this.

And from the studies of Austin Smith and others that you are referring to, the implication was that when those cells were transplanted into that blastocyst to generate the chimeras, that a subset of these cells fused with the hosts own cells and it was those fusion products then that gave rise to the variety of lineages.

At the moment that is an implication, and that has not been demonstrated in the embryo. It has been demonstrated in the dish that they had that capacity.

So we are now waiting and putting pressure on Catherine, and Freizen, and others to look into those animals to see if they can recover those specialized cells that were derived from or that had the adult phenotype if you know what I mean, the marker, to say are you truly of the adult stem cell lineage, or do you have other markers present, other chromosomes present, that come from host cells.

So until we see that data — you know, I will wait. That is something that can be looked at scientifically, and that is as far as I would go with you, Elizabeth, at this point.

It is an interesting observation, and we will see if it actually is the answer.

DR. BLACKBURN: And just to extend on what you said, I think what it does now do is to demand that the onus be put on the researcher to show that there has been a plasticity or transdifferentiation, and there are other set of criteria, which would be karyotype and multiple micro-satellite, polymorphisms — sorry to get overly technical — and other genetic markers.

There are clearly tools in hand, and so it seems as if every experiment can in fact be subjected to those sets of analyses now.


DR. BLACKBURN: And will need to be before we can get a good view of this.



PROF. DRESSER: I have four questions, and maybe if I say them all it will be possible to answer some of them together. One, I was wondering if the rats are being given immunosuppressants in this study.

And then you said a problem with the rabbit eggs is that the mitochondrial DNA might cause rejection, and so I wondered if that would happen with a cloned human embryo as well if the egg came from another person, and if you are trying to do a therapy that is compatible with a patient.

And let's see. The feeder layers, I was wondering if they have available feeder layers that do not come from animals or what the state of that development is.

And then finally what about the fact that if you are creating a blastocyst from a patient's cell, and if the patient, let's say, has cancer or some condition that could be related to genetics, would the stem cells somehow perhaps be risky?

DR. GEARHART: There is no question in my mind that the possibility exists that if you are doing an egg donor, and nuclear transfer into an egg, that there possibly exists that that cell — that the embryonic stem cells derived from that could be rejected. Absolutely.

Now, how do you test this? I mean, where do you test it. This almost comes under the same criteria that I have for anyone coming to — if I was on an IRB and they wanted to clone a human reproductivity, what data do you present before you permit it to go.

To me, it is one of these things where you need perfection before experimentation, or without experimentation, which is something in science is anathema.

PROF. DRESSER: Well, you could test that in an animal, right? I mean, you could at least see —

DR. GEARHART: Well, you can, and we could set it up in an animal, but the issue is — I mean, where you are very defined and to demonstrate it by doing it into a different strain of mouse. There is no question about it.

But whether or not that would carry over in polymorphisms that exist in human, again you are still faced with human versus rodent.

The feeder layer issue. It is one that is being taken on, and there is no banning of this type of work with private money, and clearly there are a number of investigators, laboratories, working on establishing feeder layers from human tissue that could be used, and I think that this is very important.

So those studies are certainly under way. We have used a variety of different human tissues as well to look at in our studies. Oh, the very first question that you asked. I'm sorry, it was again?

PROF. DRESSER: For the rats —

DR. GEARHART: Oh, sorry. We did animals that were immunosuppressed and animals that were not immunosuppressed. And we did not find a great deal of difference in the short term, although — I mean, as far as any type of destruction of cells and things like that, although clearly in the animals that were not immunosuppressed that you could see reactive cells present.

So clearly in the monkeys immunosuppressed, absolutely, and so we have done them both. And then the blastocyst question?

PROF. DRESSER: If it comes from a patient with a particular disease.

DR. GEARHART: Yes. Clearly where there is a genetic basis of any type of a disease, you would be concerned about reintroducing the same cells that were subjected to whatever the disease process was.

And I think that this carries over also into, for example, the diabetes work, where if you have an attack on insulin itself, you know, is this going to be a viable alternative, and there are some evidence now that you can alter the insulin molecule to make it not recognized by some of the autoimmune antibodies.

I should say that there are a number of laboratories — and this is one area that is being emphasized in the use of human cells, including our own, with Mike Shamblott, where we have lines that are — human lines that are insulin producing that you can pop them into animals, and demonstrate that they can produce human insulin.

And we are very encouraged by some of these early results. But I would still contend that we have a long way to go to carry that into some type of clinical application. We have a lot of questions to answer.


DR. ROWLEY: Well, I, too, have multiple questions and I want to thank you for a very lucid presentation. That helps a great deal. I would like to first — and I think I will do these one at a time.

It is a substantial question as to what value the embryos that are left over from IVF can play in this whole process as compared with embryos that you develop for either a particular purpose, or just straight off.

And my understanding was that maybe some of the embryos were sufficiently mature so that maybe the cells derived from IVF would not be useful in developing, say, cells lines or things. And I would like your comments.

DR. GEARHART: One of my hats at Hopkins when I moved there in the late '70s was to develop the IVF program. So we are very well tuned into the issues of IVF, and clearly in an IVF procedure the best embryos obtained are those that are used first in first transfers.

So that generally those that are left over are of the ones — we don't want to call it a lesser quality, but at least as far as our eye is concerned, and how we judge grades of embryos, based mainly on morphology to be honest, and more currently we are looking at biochemical parameters that we can measure in the media in which these cells are growing that something has been secreted to have some kind of a measure.

And that clearly those that are the spare embryos generally are those of — let's say, what we deem, and knowing what that means, of lesser quality.

So what does that mean? In most cases, they have not developed far enough along, which means that if they are left over that you take them back out of the freezer, and you try in your culture conditions to get them up to this blastocyst point.

If you can't get them to a blastocyst stage, you can't derive the cells. If there is no inner cell mass, you can't do it. And you find that you are compromised there, and that generally these are not very good embryos.

So one could argue that overall that you would expect to have a low efficiency yield with respect to taking in embryo and deriving a line from spare embryos in an IVF program. That is in general.

DR. ROWLEY: Okay. You mentioned modifying the histocompatibility locus, and I would have thought that there is still so much that we don't know about the MAC that that would — I mean, obviously anything can be done in the future with time, but do you look on this as practical?

DR. GEARHART: Well, back in the ancient days, in the early '80s it seems in this field, Oliver Smithies and others did do knockouts of Class I and Class II genes, in an effort to determine whether or not this could prolong grafts into animals without those.

And that depending on the tissue or the organ, there was evidence that this indeed could be the case, and not that it was an indeterminate thing, but just by days, or weeks, or months, that this was the case.

What they didn't know about at that time were NK killer cells, and those kinds of things, and the importance of other determinants which must be on cells. They wiped everything out.

So some labs are now taking a look at this to see if it is possible then to rebuild back some of these markets. But it is a matter of speculation at this point whether or not this could occur.

Now, what we can talk about I think is it possible to take using the act of transgenesis and things like this, where we could move big pieces of DNA; of taking part of a patient's chromosome-6, you know, and cloning that into a stem cell after knocking out some of it, and we may get some degree closer.

But that says nothing about the myriad of other loci that could be involved as minor histocompatibility problems. So, some of it is speculation, but I think it is also testable at this point in time.

DR. ROWLEY: And my last question is coming back to the 80 plus cell lines, and you raised concerns, which many of us have, as to how useful some of those are going to be.


DR. ROWLEY: Could you expand a little bit, in terms of whether you think they are really not going to be long term cell lines, and that is your concern, or whether there are other aspects.

DR. GEARHART: Well, I have many concerns, and I hope that I can get them all in. I mean, look, we were all thrilled when Mr. Bush made the decision to move forward with this and establish cell lines to permit the work to go forward. There is no question about it.

But as we looked into — and by looking into, it was a practical matter. Many investigators around the world, and I have close contacts with colleagues in Germany, and in France, and in England, and Japan, and Australia, and on and on, as we compare notes all the time on our results of research, as well as on practical things like this, and on political issues.

I mean, there is no question that we have to keep abreast, and what happened, particularly from the German investigators, which is significant, as you know, in Germany, they are not permitted to derive cell lines.

And for a while they were not permitted to use those that were even derived, and recently their parliament voted to permit the use of existing cell lines as of January 2002.

But what happened was that when these investigators set about to import cell lines, and contacted the registry list at the NIH, which continues to grow each day, and more lines are added to it as you know, it turned out that many of the lines were not defined.

Someone just reported that they had a clump of cells growing in a dish, and they didn't have any of these parameters or very few of them done.

And this reduced the list substantially, quite substantially, down to — we are talking about, say, a dozen. And then the issue came up as to, well, are these — can they be imported without a stringent material transfer agreement, and with a reach through clause that would say that anything that you would do with those lines belongs to the person giving you the line.

And this reduced the line substantially. And then other lines are not available because if you needed to get them, you needed NIH funding, and only NIH funding. You could not use private funding with them, and on and on.

And so it drastically reduced down the number of lines that are practically available. Now, whether or not this will have a major impact, clearly the NIH is receiving grants, and we have been reviewing grants, and using the existing approved lines, the few that one can get.

And the work will go forward, and whether or not that will be sufficient, and we recognize that there is going to be a half-life to these lines for various reasons, and that there will come a time if it proves effective in the basic science part of this to move forward, that we should be looking at being able to generate new lines.

And the issue of the feeder cells is a major issue as well, and to begin to establish lines on human cells so that we are not faced with that anything that we derive from this now, and it is important to consider, has to be considered as a xenograft.

Although it is a human line, the FDA requires that if it has seen these other products, it has to be considered a xenograft, which sets up a whole new set of criteria for moving this into the clinical applications.

So I think there are reasons why we should eventually be permitted to derive new lines. Well, I'm sorry. We can do it now on private money, but anything that is derived cannot receive Federal money for support.

CHAIRMAN KASS: There are people waiting in line, but can I get a clarification on this question that came up in your answer to Janet about the durability and longevity of the lines, and on the one hand, one says that the embryonic stem cell lines, their great virtue is that they can be self-renewed indefinitely.

On the other hand, they have a half-life, perhaps because of accumulated mutations. Could you say a little more? I mean, some people claim these are eternal lines.


CHAIRMAN KASS: And could you say something about the possible differences between human and mouse with respect to renewability, because I think it is an important factor.

DR. GEARHART: Well, the issue is maybe they are eternal, but can you still use them. They can still divide indefinitely, but they may not —

CHAIRMAN KASS: But they are no longer the same.

DR. GEARHART: Yes, they are no longer the same, and they may not give you the biologic properties that you need. Strangely enough, Leon, there have been very few publications up to this point, and up to this point there is one that I can cite for you, and I have it in answer to some of your questions by Joe Stanbrook at — Peter Stanbrook, at the University of Cincinnati, in which he looked — these were mouse lines.

And he looked at the frequency and rate of mutation within several mouse lines, and contrasted those with several schematic cell lines that were in the lab as well.

And he found that indeed the mutation rates — and what you do is you pick certain genes to look at changes, and to look at chromosome lost or gain.

This paper was published in PNAS in the March 19th issue for those who are interested, and what he found was that the frequency and rates of mutation were orders of magnitude less in the embryonic stem cell line than in the schematic cell line.

And you are looking at a rate of generally 10 to the minus 6 frequency within any mammalian cell as it is divided. But what he did find, and that was a bit troublesome, was that the type of mutation that appeared in the embryonic stem cell one led to what is called uniparental disomy, which is a situation where you end up with homozygosity across a region, or across chromosomes or regions of chromosomes, that gets rid of really the dominant tumor suppressor genes, which then raises the issue that these cells may be more susceptible to tumorigenesis than others.

Now, that is the only report, and I will tell you that in several laboratories what is being done now with the human lines, and that is using express sequence tags, for example, and you can use 10,000 of them, they are looking at mutation rates at 10,000 loci, if you know what I mean, over time in culture passage, after passage, after passage.

So we will get information on this parameter, and how significant it is going to be, I don't know, but one would predict that clearly there is going to be an accumulation of mutations within these cells.

CHAIRMAN KASS: Okay. Thank you. I have Michael — well, also, was that on this point?

DR. BLACKBURN: Just a very brief clarification. Did the absolute frequency of uniparental disomy go up? Was it an absolute frequency increase, or simply did it relatively increase as you looked at the whole spectrum of mutations in the mouse embryonic stem cells?

Do you see the difference that I am trying to get at?


DR. BLACKBURN: That if it were an absolute increase, that is a reason for concern, much more than if it were simply a relative increase in a number that has already gone down by —

DR. GEARHART: These numbers are rates, and so I believe it is an actual number. In other words, it was a real —

DR. BLACKBURN: An absolute increase?

DR. GEARHART: Yes, an absolute increase.

DR. BLACKBURN: So I just wanted to make sure that I understood the numbers here.

CHAIRMAN KASS: Michael Sandel, and then Frank.

PROF. SANDEL: I would like to go back to the adult stem cell versus embryonic stem cell question, and ask it in a slightly different, and maybe more pointed, form.

As you know, there are some people who regard embryonic stem cell research as morally objectionable. I am not asking you or trying to drag you into that debate. But I would like to know your view on the following scientific question.

If adult stem cell research in the best case scenario redeems its promise, what would we lose medically and scientifically if we ban embryonic stem cell research, or imposed a moratorium on it for a period of time, until we could assess what adult stem cell research could achieve?

DR. GEARHART: I personally think it would be a tragedy, and for the following reason, if this was to happen. I think the length of time that it is going to take to assess whether the adult stem cell avenue is going to provide the potential therapies that we are thinking about, is going to be years.

And I think for us to deny at this point any avenue that has the potential of the embryonic stem cell story is a tragedy to those people who need or who will need these cures.

And I think that it is a time element. If this could be done in a year, I would maybe listen to that argument. But it is going to take years to really assess any of these approaches.

And I really think they should move forward together. I think we are going to learn in both directions how to utilize information coming out of these studies that would benefit, for example, or enable us to understand more about the adult sources if this is going to be the emphasis, and to really make them effective in their use.

So I think that it wouldn't be wise to put a ban on the embryonic source at this point, and wait until another avenue is assessed. The length of time is going to be too long.

PROF. SANDEL: Can you be more specific? Are there certain types of research avenues that you would associate more with embryonic stem cell research, as against adult stem cell research?

Is it likely that success is in particular areas, or is it just that you feel that as a general matter it is better to have more avenues rather than fewer?

DR. GEARHART: Well, I think that one of the messages that I hope that I can get across, and maybe Catherine will, too, is that we are in very early stages in all of stem cell research, no matter what the origin of the cells are.

And to make a judgment as to which of these is already more advanced than the other, it would be a tenuous one at this point, because you have got to remember that there are very few investigators actually working on embryonic stem cells at this point.

The list on the adult side obviously is larger. I mean, as far as investigators are concerned. And I don't think that any of us are really showing dramatic — you know, utilization in the sense that we can say we are going to go to any clinical use of this.

It is going to take years for this to occur. We are in the very early stages and so I would be really hesitant to say that anything is demonstrating anything better.

All I would say about embryonic stem cells at this point in a very positive way is that we know that at this point that out of these cells we can virtually generate any cell type we want in dish and in large numbers.

That is the advantage of this approach. Now, whether this will be surmounted by other discoveries in adult stem cells to do the same kinds of things, I don't want to predict. I hope that it happens.

You know, our — and I also want to emphasize that we — and although we are associated with the embryonic form, we are studying other forms as well. We are not foolish.

As a scientist, you know, you are not going to put all your eggs in one basket here. And so we are trying to move forward on a broad front, and I think that this would be the more rational way to proceed in this arena


PROF. FUKUYAMA: Dr. Gearhart, did I understand you correctly that in the experiment that you headed up with the mouse that it lost the motor function in its rear legs, that you were injecting human stem cells?

DR. GEARHART: Yes. Well, if I could correct you a moment. It was a rat, first of all.

PROF. FUKUYAMA: Okay. A rat.

DR. GEARHART: Rat, too, but the issue is please don't say that you are injecting stem cells. These are derivatives of stem cells. I mean, just so that we know, but they are out of the stem cell line, okay?

PROF. FUKUYAMA: Okay. Fine. But what was the resulting tissue? It was a mixture then of rat and human neurons, or do you think it was simply the stimulation of these other factors that was causing the rat neurons?

DR. GEARHART: Right. That is a good question. We still don't know — I mean, to be honest with you — what the mechanism of recovery here is. We know that sitting in the ventral horns of these animals, and where these big neutrons reside, you now have a mosaic population of host cells, of neurons, inner-neutrons.

I mean, we all — I mean, human and rat, or human and mouse, depending on which one we did. We don't know the relative contributions. We can count cells, but really what is the functional basis of what occurred there.

We know that the human cells are also rescuing the other, but to what degree. This is where the hard work comes in. What was the mechanism, and what really went on or is going on in that ventral horn.

I can tell you in work that John McDonald has done at Wash U, in which they generate a contusion injury in the spinal cord of a mouse or a rat, and then infuse in mouse embryonic stem cell derivatives, and that he is faced with the same issue. He can see that these animals recover to a certain degree, but the mechanism of what is it, of what has really occurred there, is not known.

And I think what we are going to find is a demand that we come up with mechanism in some of these animal models so that we can completely understand what that therapy is going to be if you take it to a human.

And this is going to require a lot of work. Now, some of it you could argue is that you could do it all within animal studies. You know, mouse embryonic stem cells, and you don't have to put the human in.

But I think we are finding enough differences between species that it would warrant at least the study also of the human derived cells in the same paradigms to ask those questions.

PROF. FUKUYAMA: But I am just curious. Are you getting actual tissues in which you have cells from different species that are growing simultaneously?

DR. GEARHART: Oh, yes, absolutely. Yes, sitting in the same — well, you can see in the section here that might be 15 or 20 microns across, you see a mixture of the rat cells or mouse cells, and human cells, functioning.

You know — I mean, this isn't uncommon. We do interspecific grafts a lot in experimental things, and the question is when you do it, and we see, you know, human cells growing in animals very nicely. I mean, as long as there is immunosuppression and things like this occurring.

PROF. FUKUYAMA: But could you go the other way, also injecting stem cells from other species into human beings?

DR. GEARHART: Oh, yes. I mean, this is one of the issues with xenografts. You know, is this something — well, there is a report recently about chicken embryonic stem cells, and the fact that people who had derived these were promoting the use in humans.

Pig stem cells, you know, et cetera, and so it can be done, but a couple of issues, and one of them is the issue of the xenograft itself, of bringing in endogenous viruses, and is this a wise thing to do.

And the other thing that I would ask you, and I won't be flippant about it, is to say that if you — and one of the concerns that we have that maybe this council and others would take up, is long term in a neurologic sense.

If you are putting stem cells in, and you are putting them in between different human beings, what are you doing to that individual. And I would say to you that if you have a stroke, and someone comes along and says, well, we have pig, cow, mouse, human, take your pick, what would you select.

I am not being flippant about it, but I am just saying that I think that we know that human would be preferable at this point in time.

CHAIRMAN KASS: Could I ask a question, and just for clarification again also on your own experiment that you showed us. You said that some of the rats were immunosuppressed and some were not. Is that correct?


CHAIRMAN KASS: And were there functional differences in the results between those two groups, and would that bear upon the question of whether or not the major effect was owing to the action of the human cells, or a stipulation of the endogenous cells?

And lastly, if these animals had come to post-mortem was there a difference? Was there rejection in the non-immunosuppressed animals of the human cells?

DR. GEARHART: It is important to keep in mind the time frame that these experiments are done in. They are of very short duration relatively speaking, in a period of several months maximum.

In experiments that have been done in our laboratory, principally by Mike Shamblott, in taking human cells and grafting, and these are insulin-producing cells, and we have done it in a variety of tissues into rodents, you always see reactive cells, which means that you are eliciting an immune response.

Again, they are short term, and whether you are getting destruction, we see cellular debris, and we see this kind of stuff at these sites. I should tell you a little bit that may be enlightening.

When you do grafts like this, if we say we are putting in 300,000 cells or we are microinjecting in a lot of these cells, many of these cells will die at the time of injection, simply because you have taken them out of one environment and you put them into another, and you see a tremendous amount of cell death.

Very few of these populations of cells continue to divide. In other words, it may undergo one more round of division, and they sit there.

You do see when you come in finally to look at where is the human versus where is the rodent, and you use your human markers. You invariably find a group of cells that you can't phenotype, if you know what I mean, and to say what has happened here, and clearly there are cells being destroyed.


DR. GEARHART: Well, we don't know that. And one of the arguments for many years has been that the central nervous system is an immune privileged site. I don't think anymore that this is something that is believed or subscribed to, and if you have the option of immunosuppression, or of getting around that, that that would be preferred.

And particularly when you are talking about a graft going into a human being that may be there for 20 years, as opposed to a matter of a few months. So I think that this is going to remain a major issue, and there is no question about it.

CHAIRMAN KASS: Thank you very much. Bill Hurlbut and then Paul McHugh

DR. HURLBUT: John, I hear you saying that we should pursue all lines of research, but I want to weigh the different options here and pursue the question of if the lines were restricted what would be gained or lost.

Specifically, I have several questions that hinge each on the other. First of all, the cells that were implanted or tested for their tumorigenicity effect that you spoke of in your paper were the so-called EBDs.

Were those derived only from embryonic germ cells; is that what is implied there?

DR. GEARHART: Yes. In our paper, we took the stem cell itself and plated it out in a variety of culture conditions, some of which are designed to enhance or select for certain types of differentiation.

And we referred to these as embryoid body-derived cells. They came out of this little cluster, and in our field it is essential that we take the stem cell off the dish, and let it form into a little ball, and which is just a multi-cellular structure, called an embryoid body.

Now, this was an unfortunate name that was given to it by a French pathologist back in the '30s, but as you can imagine, when someone in a political sense talks about an embryoid body, they conjure up embryos here.

But these are little clusters of cells, and within those or within that cluster, the beginning of differentiation begins. These cell-cell interactions are essential for this. We have not been able to mimic this in a sheep yet.

So what happens is you get within that ball a variety of cell types being formed, and all that you want to do is to disassociate that ball after a period of time, and select out only those that are going in the direction that you want them to go in.

So this is what we did in that experiment, and so we have now these EBD lines, and in these lines, in these human lines, and these lines have been placed in a large number of animals, in the grafts that we have used, we have never seen a tumor up to this point.

And it may be unique to humans, because human primary cultures are easy to establish and mouse aren't. I mean, there is an issue here that we don't know that you can't do the same experiment in the mouse.

So with our experience with the EBDs, we have never seen a tumor. Our experience in the mouse and using what we thought were equivalent lines, we have seen too many tumors with respect to grafts into the central nervous system.

DR. HURLBUT: Just parenthetically haven't I been reading all along that embryoid bodies are also formed from ES cells?

DR. GEARHART: Oh, yes, absolutely.

DR. HURLBUT: But the point is that your particular lines don't produce tumors, and the ones derived from the primordial germ cells don't seem to produce tumors; whereas, the embryonic stem cell lines do?

DR. GEARHART: Well, the only comparison that we have at this point are mouse ES lines, in which we have derived different types of precursors under different conditions, have been compared to human EG lines that have been derived, or which precursors have been derived in a slightly different manner.

You can't derive them both in the same way. We have seen nothing up to this point on human ES derived lines transplanted. We just have not seen any data on that.

So I don't want to make it clear that there is a difference between the derivation either from a germ cell derived, or an inner-cell mass derived line. Does that make sense? That comparison is not there yet.

DR. HURLBUT: Well, obviously what I have been getting at here is if in fact your cell lines are less likely to cause tumors, then does that imply that there might be some advantage to using your cell lines, and if so, would it in fact be the greatest advantage if a patient's own cell line could be derived from primordial germ cells?

DR. GEARHART: Oh, boy, this committee would — well, wow. Now, think what this means. It means that you would be generating an embryo, and having it implanted. Now, what you don't know is that our fetal tissue comes from 5-to-9 weeks post-fertilization. These are therapeutic abortions.

And which means now that you are way beyond — I mean, the point of where a blastocyst is, and obviously way beyond I think anyone subscribing to that approach.

DR. HURLBUT: You told us that in your paper.


DR. HURLBUT: But is it true that maybe there would be some great advantage if we could find a legitimate way to harvest tissues generated from a specific patient at a later date?

DR. GEARHART: Right. Well, I think it would be terribly risky. We have been asked this question a lot though; is it possible to do a biopsy on a developing embryo, and to remove just a few germ cells.

I think at the stage that we are using these embryos are a matter of — or fetuses are a matter of maybe 6 or 7 millimeters in length, and to do the surgery on this I think would just be impossible without causing harm.

The other issue that I would contend is do you think it would be okay to go in and remove the germ cells from an embryo and let that individual go on and say, well, we have taken your germ cells. Now, we have another therapy for you.

And so I don't think it is a very good thing to do.

DR. HURLBUT: And that is my final point, and I wanted to ask you personally in working with these cells, do you see 14 days as some kind of magic marker moment?

Do you see something crucial about implantation? And you spoke of keeping all options open.


DR. HURLBUT: Why in fact do we allow abortion fairly late in term, and yet now we are speaking as 14 days as the sacred moment? I know that I am opening a very difficult issue here.

But in fact wouldn't we gain a lot scientifically from extending that 14 day limit potentially if we could find a culture median that could sustain the embryo, or wouldn't we gain a lot from implanting, even gestating and harvesting?

And why do we feel that we shouldn't do those things? And I would also be interested in your personal response to these ethical issues.

DR. GEARHART: Wow, you have asked a lot. As you know, stem cells have been obtained from many stages of human fetal development, and have been found to be useful in generating various cell types in culture.

And if we look at a variety of studies, you can find it in the published literature. We have had a number of requests for fetal tissue at different stages, and I think legitimate requests of investigators willing to investigate cell lineages, et cetera, within the embryo.

So people have been thinking about it. I mean, there is no question about that. We have found it difficult enough to be fortunate enough to obtain the fetal tissue that we work with.

I mean, there is a consenting process and we have nothing really to do with other than to make sure that it complies with institutional, Federal, and State law.

To obtain viable tissue from abortuses of any kind is a major concern. When we started our studies, we looked into using spontaneously aborted material, which occurs across the board, but mainly in the early stages.

And we thought that this would be a good source. As it turned out, by the time that we were notified — and this occurs in outlying hospitals, and not at major medical centers, where investigators are — you know, a patient presents with a miscarriage, and it is taken care of in the ER.

And it turned out that it was very ineffective, number one. And, number two, and then I will get back to your question, we found that most of the material that did come to us had chromosomal abnormalities that made it less desirable for use.

Now, the issue of the 14 days, and what does it mean. Well, this was something that really came into play in the United Kingdom when they were trying to deal with this issue.

And it was decided at that point that at that stage the embryo still does not have a central nervous system. It can feel no pain, et cetera. And this was why basically that period of time was set to be able to grow them in culture, or to remove tissue.

We, as embryologists, argue the point all the time as to what is going on in these early stages, and we were always asked these questions. When do you believe personhood occurs and when is it established, and things like this.

To me that is not a biologic question. We don't have a means of probing that. So I think that is why the 14 days was selected, and that's why it is sort of adhered to in a sense.

Do I adhere to that? Well, to a certain degree, no. We take material that is later on, and it is cadaveric fetal tissue. I think that we should be able to utilize any tissue that comes out of abortion if the alternative is that it is just going to be disposed of, which is what happens.

The pathologist takes a look at it to make sure that all of the parts are accounted for, and there is an issue about being concerned about what is left in the uterus.

That is my personal opinion on that. But I don't think that we should be going and establishing pregnancies, and to downstream then utilize that tissue.

I mean, to then stop the pregnancy and then to recover it. I mean, that is my personal opinion. I don't think we should be doing that. As you know, years ago, President Reagan was faced with this, I believe, when he heard that families were establishing pregnancies so that regions of the brain could be harvested to treat Parkinson's disease in the family.

And clearly we don't subscribe to that in any fashion.

CHAIRMAN KASS: Thank you. We are coming up to the break and I have Paul McHugh, Mike Gazzaniga, and we are running a little late because we started a little late. We will take a break shortly. Paul and then Mike.

DR. MCHUGH: My point is very brief, John, because you have touched upon it in several places. But first of all, I want to thank you very much for that coherent presentation, and I especially thank you for showing us experimental data.

And that is what of course generates better questions to ask you. And it is really out of that experimental work that I did have a question. And that is what you showed us was fundamentally a xenograftic experiment using human tissue, human cells, in rats.

And the results were very interesting, and not only was there growth of cells, but you told us that there were trophic factors that were probably acting in this way.

And I then wondered, and you can answer this, why was it necessary to use human cells to demonstrate this phenomenon in a rat, and why weren't you using rat cells to do rat experiments.

And if that is true, that you could do rat cells to do rat things and the like, the development of the question is would it not be wise of us to ask you all to go back and work with your rats and your mice, and your cats and your sheep, and keep going at it, and come back and tell us why you need human stuff to do this stuff, okay?

DR. GEARHART: Okay. We did it first with mouse cells. We don't have rat embryonic stem cells. We did it first with the mouse and it worked.

And in our exuberance, saying, well, would the human cells work, and they did. There is no question that I think that the mouse cells worked better, and the mouse cells were from these neural precursors that we had obtained that I had mentioned that we had this concern about tumors.

But they did work, and so the only two cell types that we have found at this point that work have very similar origins if you know what I mean.

Clearly the paradigm has to be extended to other sources of stem cells, adult and umbilical, and this is planned to say in this particular paradigm will it work.

So, Paul, the answer is that we did it first with the rodent cells, and we could pursue that. I mean, as far as looking for the growth factors and what not.

But we have changed almost completely to the human cells for trying to determine what those growth factors were that were secreted, but we could do that again with the mouse, absolutely.


DR. GAZZANIGA: Just briefly, thank you again for a wonderful presentation. This moves to another level, and that is how big is the American biomedical engine.

And I ask that from the sense of having just taken a trip to China and Japan, and England, and you read that Sweden and Singapore, and India, and so forth, are going ahead.

If America dropped out of this for legal reasons that are on the horizon, how big an impact would that have on the overall resolution and development of these therapies?

In other words, if you just look across molecular genetics and microbiology now, and prior to this issue arising, what is the size and importance of the American effort?

DR. GEARHART: Well, I don't think that there is any question that the investigators funded through the National Institutes of Health, and our academic establishments here, are the engine that drives biologic research, biomedical research, in the world.

There is no question about it. I mean, the volume, the sheer volume of this, is enormous. And if you look at this compared to even in our country to what the biomedical industry, or I mean the private industry is putting into this, it is dwarfed by the Federal funding.

And this is really what is enabling and this is why I think the U.S. has been so far ahead. So it is essential I think to have Federal funding into this area really to reach our goals as quickly as possible.

There is one last thing or one thing that I would like to say to the committee, and it is understandable, but when you are in and start in a business like this, you don't know the impact of it.

The thousands of communications that we have received from patients, and patient-based groups, about our work and about moving the work along, not only is it emotional, it is unbelievable. I mean, from the standpoint of just pure numbers, sheer numbers.

It doesn't just extend within the United States, but throughout the world. In 1998 when we published our paper, within a few days we had 10,000 e-mails alone about it.

And every day I still get hundreds of e-mails relating to this. It extends not only to bona fide — you know, many people don't understand what this work is about.

They are contacting you for a brain, or a uterus, or from some countries we have had requests, hundreds of requests for penises, for example. And you are trying to figure out why — you know, what is the issue here.

We need education and we need informing to say that we are dealing really with cells and tissues at this point. That is what we are really about. It is going to be years away before it goes beyond that.

And so what I am trying to say is that there are requests throughout the world. So that is one issue. I mean, the pressure is enormous, and also people offering you large sums of money to provide them with cells outside of the arena that it should be done in. Do you know what I mean?

There is desperation, and you see this, and it is tragic, and as a researcher this is new to you. This is something that you are not accustomed to and never will be accustomed to handling.

So I just wanted to let you know what that pressure is like. It is enormous. I have boxes full of these things. I don't know what I am going to do with them, but you try to respond.

There has been an issue with brain drain. We know that there has been one investigator from the University of California system that went to the U.K. and received one-and-a-half million pounds to pursue this work in the U.K.

Well, this happened here. I will tell you that — and I am talking to students in our own group, you know, go to Europe for your post-doc, and go to England for your post-doc if you want to continue in this thing.

And I think you will see more of this, and whether major investigators will leave, I don't think so. I think we will get through this, and I hope that we will get through this period in this country.

There are many, many investigators, many investigators, and I can't tell you what it is like not to be able to give a cell to the person next door to you because of a policy.

I mean, this is just an incredible situation. I think we will get through it, and I think we will be okay. But I am still concerned about it. Sorry for the editorial, but I think it is important.

CHAIRMAN KASS: Charles, did you want a quick word?

DR. KRAUTHAMMER: If I could just ask a very quick question. You said that you would oppose and you supported the opposition of creating a fetus for, say, harvesting the brain cells, and you talked about the example in the Reagan years.

On the other hand, there is no difficulty, at least in your estimation, of using tissue from a discarded fetus already aborted, and tissue which would otherwise be thrown away.

Would you apply that same distinction to the embryonic stage? In other words, you now use — you develop embryonic stem cells from discarded embryos from IVF clinics, and would you be equally opposed to the creation of embryos specifically for their use as sources of embryos using that same analogy?

DR. GEARHART: No, I would not be opposed to that. I don't give the same moral status to that entity.

CHAIRMAN KASS: Well, we have — let me just make mention of one matter. Janet Rowley has submitted in writing, and I would endorse, these questions if we had enough time.

We would like your comments on what kind of regulation you think might be or should be developed for this area, and what is the status of government support for what kind of research, and what are the limitations that are counterproductive.

If we could invite — if you would be willing, and these are hard questions and they are big questions, but if you would be willing to respond if we put these set of questions to you, and perhaps some others to you in a letter?

DR. GEARHART: Absolutely.

CHAIRMAN KASS: I think the committee would be very grateful for your help in thinking through the regulatory questions, which are at the moment not what we have here.

DR. GEARHART: Absolutely.

CHAIRMAN KASS: I just want to thank you very, very much, for an instructive morning, and also for the wonderful spirit in which you presented your remarks and engaged the questions. I am very grateful to you for coming.

We are running about 15 minutes behind, and we will reconvene at a quarter-of. We have an hour-and-a-half for the second session this morning as originally planned.

(Whereupon, at 10:33 a.m., the council was recessed and resumed at 10:49 a.m.)

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