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Thursday, April 25, 2002

Session 2: Stem Cells 2: Medical Promise of Adult Stem Cell Research (Present and Projected)

Dr. Catherine Verfaillie

CHAIRMAN KASS: Would the members please rejoin the meeting. While we are waiting in the hope that our straggling colleagues will arrive, a couple of matters of business.

If anyone has not turned in a request for a box lunch, please do so now, and that should be in front of you. We will have lunch in the room just down the hall where we gathered before.

The photographer who has been around here is doing individual photographs for the commission and he will want to take individual photos of members, and we can do that in connection with lunch.

And you will also have in front of you in addition to the materials that Dr. Gearhart provided us, which by the way is — and the lights were out and so you couldn't see, but one could recapitulate his talk with the help of the figures here, as well as checking his article in Nature.

But you also have in front of you a revised version of Bill Hurlbut's memorandum. This has been updated and corrected, and he would like us to substitute it for the one that was sent around earlier this week. Is that correct, Bill?


CHAIRMAN KASS: All right. Well, again, it is a great pleasure to welcome Dr. Catherine Verfaillie, from the University of Minnesota. You have her curriculum vitae in the briefing book, which you can consult.

I won't waste any more of her time by reading from it, and just simply allow her to help educate us on the prospects of present and projected of adult stem cells for regenerative medicine.

DR. VERFAILLIE: Good morning. I would also like to start out and thank Dr. Kass and the council to allow me to present this information on new findings in adult stem cell biology which have been received with great excitement, and correctly so. If they are, and they are actually set upside down, the classical paradigms of biology, and so to be able to do that you have to have full proof to actually be able to be in a position like that.

If they are, and they are actually set upside down, the classical paradigms of biology, and so to be able to do that you have to have full proof to actually be able to be in a position like that.

As Dr. Gearhart already gave in his previous eloquent description of what stem cells are and what they can do, and we will get back to that to some extent at the end, although we are far away from actually being able to use adult stem cells for clinical applications.

But what I would like to do is give you an overview of the greater potential of adult stem cells, which has always been termed adult stem cell plasticity, and what we do know and what we don't know.

And where this may actually lead us. Dr. Gearhart also indicated that embryonic stem cells in humans are fairly or very much in their infancy, the same as we are for adult stem cell biology, too, and so I don't think we are anywhere close to be able to come up with new therapies at this point in time.

I would also like to reiterate that even though my laboratory and our group works on adult stem cells, we have actually actively pursued investigators in embryonic stem cell research, human embryonic stem cells, just so that within the same institution we would have laboratories that have one cell, and other laboratories that have the other cell, so we would be in a position to compare and contrast the potential of the different cell populations, and I think that is very important.

With that, I will actually start my presentation, and I will point out that the work was mainly funded through the NIH, since it is all adult stems that we are working on, and not embryonic stems. And also a number of foundations and one pharmaceutical company.

Dr. Gearhart already gave you an overview of where embryonic stem cells come from, and where primordial germ cells or stems come from. And I am going to reiterate that for you.

I just put up this cartoon that Dr. Weissman published two years ago in Science to point out a couple of things. During development, cells in the inner cell mass make sequential decisions, and each of these decisions is actually accompanied with gain of function, but also loss of function.

The gain of function is that the cells learn how to become a more specified cell type; and on the other hand, actually lose the potential to become other cell types.

And so the decision to be made is somatic or germ cell, and within the somatic lineage doing something that is called gastrulation, cells decide to become the different parts of our body, whether it is endoderm, which is the internal organs, mesoderm, which are limbs and soft tissue, and ectoderm, which really comprise the skin, the central and peripheral nervous system.

And within each of these groups cells again make decisions and learn how to become stem cells for specific organs. And the stem cells for specific organs that has been most well studied is actually the hematopoietic stem cell, which is currently extensively being used in clinical applications for bone marrow transplantations or peripheral blood stem cell transplantations, or cord blood transplantations.

And so that actually has set the paradigm on how we decide what stem cells are. Aside from hematopoietic stem cells or blood stem cells, we have a number of investigators who have identified tissue-specific stem cells in a number of different organs, including for instance the brain, which we until about 10 or 20 years ago thought was a final product when we were born.

But it is now clear that there are stem cells in the brain that can recreate neurons and other components. There is also stem cells in the liver, and stem cells in the gut, and there is stem cells in the skin, and so forth.

The reason why I put this slide up is actually to point out that these arrows have always gone down, and so we have always thought that each time a cell decided to learn something new that it lost the capability of doing something else.

And so if we envisioned beforehand that the arrows would be reversed, we thought that was possible, but we associated that with classical transformation, or actually cancer-forming cells.

So what do we know about hematopoietic stem cells and that is really the paradigm to which I am going to try to talk through the whole field of adult stem cells.

In hematopoietic stem cells, we can actually take a single mouse bone marrow cell that we characterize by proteins on the cell surface, and take that single cell, and for instance you can take it from a mouse that is engineered to fluoresce green under a specific light, and put that in a regular mouse, and ask whether they can reconstitute the blood elements of that animal.

And a number of investigators have actually been able to do that. You can take a single cell, and give it to a mouse that was lethally irradiated so it has no blood, and this cell can recreate the red cells, the white cells, platelets, lymphocytes, for the lifetime of that animal.

And that is really the proof that you have a stem cell that can self-renew, and a single cell can make multiple different things, and it can repopulate functionally the organ that it needs to repopulate.

And so that is really the criteria that we have to hold ourselves to, to actually talk about stem cells, and if you talk about plasticity, you will have to hold us on the same criteria and showing that a single cell can now make two tissues, and that this cell can make two tissues from a single cell, and that these new cells can repopulate a tissue functionally in vitro.

Now, over the last 5 or 6 years, there has been an enormous number — well, not an enormous number, but probably 40 or 50 papers now that have come out in the scientific publications that have used the word adult stem cell plasticity.

And what is meant by that is that you take a cell that was supposed to be a one cell type. For instance, you take a bone marrow cell, or you take cells that are enriched for hematopoietic stem cells.

And it appears that some of these cells may acquire characteristics of cells outside of the organ where they came from. And so it has been shown for bone marrow cells, or cells enriched for hematopoietic cells, that if you transplant these into an animal that was irradiated, and you look in tissues outside of the blood, that you can actually find, for instance, skeletal-muscle cells, heart muscle cells, or endothelial cells, that are now derived from this donor hematopoietic cell.

There is also papers that have shown that if you take muscle from an animal and mix it up in the laboratory, and culture it for a few days, and then use the muscle tissue to give back to an animal, that you could reconstitute the blood system in that animal.

Now, if you think in anatomical terms, this is still within one of the three categories that I gave you at the beginning; mesoderm, endoderm, and ectoderm, and all of this is still within the mesoderm. So this is maybe not so hard to understand.

However, there is also papers that two different cells from bone marrow, hematopoietic cells, and zymogenic cells, which are cells that make bone and cartilage, can give rise to cells that appear to have neuronal characteristics, both neurons and glial cells, that support the structure of the brain.

And there is a number of studies that have shown that bone marrow cells can contribute to liver, skin, lung, gut, and so forth, and so you can pretty much put arrows in whichever way you want.

You know, people have published data that suggests that indeed this may be possible. So obviously this goes against our paradigms and this would say that either something strange is going on, and just something in the last few years is something that we have actually identified.

Now, if we want to talk about blastocyst, I started out with the paradigm of stem cells, and so there is multiple different possibilities here.

Either the bone marrow, which seems to be the organ that harbors the most of these cells, harbors many, many different stem cells, and it harbors the hematopoietic stem cells, but it also harbors the neuro stem cell, and the liver stem cell, and so forth.

And which that would not be bad, but that truly would not be a single stem cell that could be expanded and used to actually transplant patients with all kinds of different organ diseases.

A second possibility is that somehow the cell can be "de-differentiated" and redifferentiated, depending on the environment that it is put in, and that the hematopoietic stem cell can learn how to become a liver if you put it in the liver, or it can learn how to become a brain if you put it in the brain.

Or it could be that it is a remnant of embryonic stem cells or the primordial germ cells that you heard about from Dr. Gearhart that are left around in the body, and that under specific circumstances can be reactivated and contribute to tissues.

And the issue of fusion has been brought up because of the two papers recently in Nature, and the possibility is in theory that what we see is actually that.

For instance, a hematopoietic stem cell fuses with a liver cell, and now you actually have something that is a hybrid, but it has actually liver characteristics.

The other questions that I am going to try to address, and I don't have all the answers for this, is this actually clinically relevant? You know, if you transplant bone marrow into a patient and you find two liver cells that are derived from the patient, from the donor, it doesn't necessarily mean that that is going to help anybody down the line.

So the graft has to be robust and persistent, and there has to really be proved that we don't just see cells that look like a tissue that they end up in, but they also have to function like a tissue that they end up in.

And then the question that I will bring back up at the end, the first question, what is plasticity, and will that matter from a clinical standpoint?

And so we started out in this field — I am a hematologist, and I do bone marrow transplantation as my clinical profession, and I have been interested in hematopoietic stems in the bone marrow.

And about six years ago somebody in our group asked me whether we could grow mesenchymal stem cells, which are cells that may grow on cartilage, to treat children with a specific genetic disease called Hurler's disease.

And when we did this, mesenchymal stems we happened to find, and we went about trying to create these to be in compliance with GMP qualifications, meaning we were trying to remove all sera out of the system, and yet we were trying to use very well defined culture systems.

And so while we were doing this, we came up with a cell that you have heard Dr. Kass refer to as a multi-potent adult progenitor cell, because we don't have a much better word for it.

And it will be appreciated as MAPC, and which appears to have a much greater possibilities than the mesenchymal stem cell possibilities. So we take these cells from bone marrow from humans, and we can also take them from mice and from rats.

And you place these in a culture system that is very well defined, and ingredients, and growth factors, and no serum, and low density, and we expand the cells as much as we can by splitting the cultures on a regular basis.

And if we do this, we have actually found that these cells appear to have an enormous growth potential. And so here on the left-hand side would be bone marrow from an individual, and we start with about 10cc's or a spoon of bone marrow, deplete all the blood elements from the bone marrow, and put it in a culture dish, and then grow the cells for long periods of time.

Classical adult cells would actually not expand much more than 50 times or 60 cell population doublings, just because we have a clock inside the cell that actually causes the cells to become senescent or old once they go beyond a certain number of cell divisions.

And so in the human system, as well as in the mouse and the rat system, we have been able to show that we can create or grow cells that do not seem to conform to this internal aging clock.

And the cells can go beyond that and the human cells are now close to a hundred population doublings, and in mouse and rat, over 150 population doublings.

If you look at the aging clock itself, which are the telomeres, the telomeres are long and they do not seem to shorten in culture, which goes again with the idea that the cells do not senesce in culture.

So in this respect, they have characteristics that are similar to what you would find in embryonic stem cells, but also this internal clock is actually not working.

The phenotype of the cell is strange, and it doesn't really fit anything in particular, but there is definitely no characteristics in these cells.

These cells are blood hematopoietic stem cells, and I am not going to go through all the details here, but if you do an extensive phenotype characterization of the cells, they don't look like blood.

They have some characteristics of embryonic stem cells, but there are a lot of other ones that they do not have. So they have some genes that are turned on that are present also in embryonic stem cells, which are the top two here, and then they have on the cell surface antigens that you really only find on embryonic stem cells, or primordial germ cells.

So in some respects again these cells have some features of embryonic stem cells, even though we got these from the bone marrow of humans, mice, and rats.

We then started trying to test initially all in culture dishes what these cells could do, and we asked whether they could differentiate in multiple different cell types.

And because our initial charge was actually to try to grow mesenchymal cells and make bone and cartilage, that is what we did first. And so what we showed in the culture dish is that if we switch the culture conditions around, and actually use ingredients that are no longer supported for maintaining the stem cells in an undifferentiated state, by actually switch them such that we hope that we can turn on the genetic programs to make bone or cartilage, and so forth, we could indeed do this.

And this is no different than the classical mesenchymal stems that have been described. So we can induce the cells to become bone, and if we say that they differentiated into bone tissue, it is actually a calcified tissue at the bottom of a dish.

We can induce the cells to become cartilage that looks like articular cartilage, even though it isn't very well organized. And you can induce the cells to become lipid-laden lipocytes, and we can induce them to become skeletal muscle cells.

And these cells can actually fuse and make long muscle tubes almost, and we can induce the cells to express a number of muscle markers for the heart, even though we haven't really seen beating cells.

And so we don't really know whether these cells are heart muscle cells. So this is still not that strange, because there is this cell in the bone marrow that has been identified that can do this.

Now, we found three other lineages that are completely outside of the mesenchymal lineage, and some of this has been published, and most of it is actually in press currently.

One of the things that we found is that these cells can differentiate into cells that line blood vessels, which we call endothelial cells. And we have been able to show that these cells differentiate into cells that look like endothelial cells, but also function as endothelial cells.

And as shown in this picture here is actually a blood vessel from an animal that had a tumor underneath the skin, and we actually infused human endothelial cells derived from human MAPCs in this animal, and showed that these endothelial cells seek out the tumor and actually help create new blood vessels in the tumor, which the tumor needs otherwise it can't grow.

And so this proves that these cells that are in the bone marrow can differentiate into cells that can make endothelium. More surprisingly is that the cells can differentiate into cells that look like neutrons, look like astrocytes, and support themselves in the brain, and to some extent function like these cells in the brain.

And so we show here that they differentiated into cells that look like neurons and have electrophysiological characteristics like neurons.

And so this is the second major layer of the embryo, and then we also have been able to show that we can make these cells differentiate into cells that look like liver cells, and actually function like liver cells in a culture dish.

And so this would mean that this cell population, these MAPC cells, can actually differentiate into all of the major components of a human being, even though we only show a few cell lineages here.

I am not going to go through this in too much detail because it is highly technical, but essentially we have not been able to use genetic marking to prove that this could all be derived from a single cell, and we don't depend on population of cells.

So this fulfills two of the criteria of a stem cell. A single cell can differentiate and grow for long periods of time, and can differentiate into multiple different tissue cells.

Two more sets of experiments were done to try to gauge the potential of these cells. The first one was done in an chimeric animal model, in which we took the adult cells, and injected even a single adult cell into the blastocyst of a mouse and asked what would happen in this mouse, and whether we would see contribution to some tissues, no tissues, or all tissues.

So we injected a single cell or we injected 10 to 12 cells, and shown here are two animals. The top one is obviously and the donor cells here have a gene that if you stain it correctly the cells turn blue.

So what we did is we let the animals get born, and we looked at the animals by genetic tools to try to figure out if there were donor cells in multiple different organs.

And we also then took the mouse and actually cut a thin slice through the middle of the animal and asked which organs would have blue cells contributing to the mouse.

The top mouse is an animal that if you looked in the tail by genetic tools that we couldn't find any donor cells, and the bottom mouse here, this is its head, and over here would be his tail, and you can see the spine, and the brain, and all the internal organs.

And you can see that the majority of all the tissues of this animal actually appear to be derived from a single blue adult cell that we have put into the blastocyst.

The efficiency isn't a hundred percent, and this is shown on the bottom here, and so if you look over here, and if you put in one cell per blastocyst, 60 percent of the animals will not be chimeric, but 30 percent or 40 percent of the animals will be chimeric to varying degrees.

If you increase the cell number the chimericism goes up. So this is probably not quite as good as embryonic stem cells, but it is a fairly significant degree of chimericism, and actually the frequency appears to be one in three cells.

So this would suggest that the cells can probably make under the right circumstances more cell types than we have be able to prove in a culture dish.

We can also ask if we now take these stem cells and give them to a mouse that is born, and we give here again cells from the donors' mouse, which again are blue, and we gave these to an animal that was either not irradiated or irradiated with a small amount of radiation therapy in the hope that maybe that would help the cells engraft.

We used an immune-deficient recipient mouse, just because we were worried that the new genes that are in the blue mouse might actually be a basis for rejection. So we don't know what would happen in a non-immumodeficient mouse.

If we do this, what we found is that we do find engraftment in some tissues, but not all. So, for instance, in the top panel, we see that there is engraftment between 3 and 9 percent in the hematopoietic system of this mouse, and we can find the cells, and the blood we can find in the bone marrow, and we can bind them in the spleen.

And if we look in these animals, we can also find over here, and what we did is we actually — the blue color, we used an antibody that is now green, and co-labeled it with a red stain that stains the specific tissue.

And you can see in the liver that there is areas in the liver where donor cells appear to be present. And there is areas in the guts, in the villae of the gut, where donor cells appear to be present.

And there is areas in the lung where donor cells appear to be present. The presence of these cells can be seen anywhere from four weeks after transplantation, all the way to 24 weeks, which is about six months, and the unfortunate thing with the mouse model that we use is that these mice usually die from lymphomas at an early age because of the deficiency that they have.

So we really have not been able to extend the cultures or have the mouse experiments beyond 6 months, and so we are actually trying to go further.

We transplant the cells in an animal that is 6 to 8 weeks old, and so it is not a very young mouse, and it is also not an old mouse. What we showed is that if you damage certain tissues like the hematopoietic system, and the gut system, that you have increased engraftment, which is consistent with the fact that these cells go to places where the repair might be needed.

However, we did not see in this mouse model engraftment in a number of other tissues, and mind you that we gave these cells IV to an intact mouse, which actually was not damaged in any way, shape, or form.

And we don't see engraftment in the heart, skeletal muscle, or brain, and these tissues do not proliferate. We also don't see engraftment in the skin and the kidney, and so these organs we didn't really see very much engraftment.

However, if you infused the cells directly in the muscle, which causes damage, and actually done the cells in response to the local cues within the muscle, appear to be able to differentiate into muscle cells.

So it appears that these cells have the ability and blastocyst experiment to give rise to many, many different tissue types, if given post-natally, and we gave them as stem cells, not as differentiated cells.

They appear to be able to respond at least in some respects to cues that are present in certain organs to differentiate into the cell type that is specific for that organ.

We have looked carefully at the cells in culture and we do not see a significant number of gross genetic abnormalities. We have not looked with a very fine-toothed comb through whether there might be some minor genetic abnormalities over time and culture, and these studies are ongoing.

If we infused the MAPCs in animals, we really do not see any tumors, and so far we have not seen that there are tumors that Dr. Gearhart talked about, and we also have not seen any other tumors.

Obviously if these cells come from bone marrow there is lots of precedent on bone marrow transplantations, where actually if you do this, actually you do not cause tumors in patients.

So MAPC that we have identified in our laboratory seems to be a cell that is not senescing and that can be found in adult tissues of humans, as well as mouse and rats, and they seem to be capable of giving rise to cells from the three germ layers, and it can engraft in vitro in a limited number of tissues.

Now, what I cannot tell is whether these cells actually exist as such in a person, in a mouse, or in a rat, or whether our culture condition is actually such that it, quote, reprograms or dedifferentiates the cells that we take out of the animal, and that then acquire this much more greater potential, and I will come back to that in just one second.

So we now go back to my initial definition of what is plasticity, which is really at the bottom of all of the adult stem cell excitement. I mentioned initially that we would have to show that this is a single cell of a rat, and I think the majority of papers so far published have actually really not been able to prove that a single cell could, for instance, give rise to blood and muscle.

In vitro, we have evidence for that, and in the blastocyst injection, we took a single cell and actually found multiple different tissues. You could ask, well, does it matter?

Does it matter if there are multiple different cell types in the bone marrow, and I think ultimately from an FDA or regulatory standpoint, it will matter, and we will have to be able to say exactly what cells that we are using to be able to acquire a certain function in vitro, and so I think that will be important.

The second question is, is the differentiation or is the remnant ES, and again you could say, well, it probably doesn't matter. But I think at this point in time, I don't think anybody in this field knows whether these are left-over early stem cells like ES cells, or whether these cells are cells that can be reprogrammed, and redifferentiated, and dedifferentiated under certain circumstances.

Now, does it matter? Well, you heard from Dr. Gearhart that embryonic stem cells as such, and not necessarily the differentiated progeny, but the ES cells themselves can cause teratomas, and even though nobody in the adult stem cell plasticity era has actually shown teratomas, it doesn't mean that it might not happen.

If it is dedifferentiation, it means that you reprogram or you change the genetic material in a cell. But if you do that, currently we have no proof that we actually change something and actually cause an oncogene or something like that to be activated, but that is definitely within the possibilities, and that definitely needs to be looked at carefully.

Is it fusion? All the in vitro work that has been published, including the data that I have shown to you today, I couldn't prove beyond any doubt that that is not based on fusion.

Our in vitro data, we have never co-cultured things with anything. So we have single cells that are deployed that can do multiple different things, and so we can't really ascribe that to fusion.

However, in vitro, I couldn't prove it to you today, and we are doing studies to try to address this. I think that fusion might be the reason why some studies in which a lot of pressure has been put on to the system, which is essentially what those two papers had to do in vitro.

So we have a lot of pressure exerted to have that one cell survive after it fuses, and that is a possibility. Also, single cells that are found, rather than whole colonies, may also be the result of fusion, more so than experiments where you see huge colonies arise in an in vivo model.

And so I think we currently cannot exclude the possibility that some of the data is as a result of fusion. Some would say does it matter, and I think it matters a whole lot, even though some investigators say, well, if you fuse the cells and it functions properly, it probably doesn't matter.

But I think ultimately that we do need to make sure that we understand the whole mechanism underlying everything. And is all this plasticity clinically relevant?

And so the majority of studies published to date have actually shown the very low numbers of tissue differentiated cells can be found in multiple different tissues.

A number of papers have been published, two in particular. The paper by Lagasse, et al., where they show that they could rescue an animal with liver failure by bone marrow transplantation, but they have significant degrees of engraftment.

So that definitely was up to 80 or 90 percent of the liver could be replaced by bone marrow cells. And a paper by Don Orlic showing that if they injected stem cells into the heart that was infarcted that a significant amount of donor cells would be found in the heart.

And in the data that I have shown you, that we have up to 5 to 9 percent of the differentiated tissue that seems to be derived from the graft.

However, the majority of studies again haven't really addressed the other question in plasticity, meaning is it in vitro functional differentiation?

And there is really only a single study that has been able to show that, and it is again the same study by Lagasse, et al., who showed that if you did bone marrow transplantation in an animal that had a failing liver, you could rescue the animal and take it off the drugs that kept it alive.

Some studies have shown that there is functional improvement, although the mechanism for the functional improvement isn't completely known, and that is to some extent similar to what you heard from Dr. Gearhart.

And so there is a number of studies who have injected cells in adults in organs and have shown, for instance, that there was improvement in the neuronal function, and that there was improvement in heart function, although there is no proof that the cells, per se, were actually responsible for doing this.

And the question will be is this acceptable from a clinical standpoint, and if you show only functional improvement without knowing the mechanism for knowing why we see functional improvement, and in the long term, again, that is not a tenable situation, and we really have to dig into this much further.

So what can adult stem cells be used for? Well, I think like embryonic stem cells, or primordial germ cells as you heard from Dr. Gearhart, the cells are good tools to study five basic principles in biology.

And we can study self-renewal, and we can study differentiation and redifferentiation if that is indeed the case, and learn what the implications for that are.

And actually try to understand how organs are being created, and what the genetic programs are that you need to turn on. The cells, like other stem cell populations, could be used for drug discovery, for drug toxicity screening.

Adult stem cells could be used as systemic therapies, and currently systemic therapies are done with adult stem cells. Bone marrow transplantation is done every day in many, many institutions around the world, and so we can infuse these cells if we do not think that they make tumors.

So since adult stem cells don't seem to have that as their side effect, theoretically, we could genetically correct cells for patients who have deficiencies of certain enzymes. And the disease, and Hurler's disease would be one example, and a second possibility would be, for instance, in hemophilia, where you need to have a cell that produces clotting factors.

Or other congenital diseases, like Alpha-1-Antitrypsin deficiency, or it could be used for systemic cell therapy, which you would have to treat in many, many different places in the human being. For instance, muscular dystrophy.

So if you had a stem cell that was able to engraft in most muscles, and you could genetically correct it, you could correct that disease in patients with that disease.

Systemic cell therapy may be more complicated with cells that have the inherent capability of making teratomas just because you would always run the risk that teratomas might show up.

And then again if this field progresses further, the same diseases that has been quoted for embryonic stem cell therapies would also be on the list here, and if indeed the cells can differentiate into functional neuron cells, they could be used to treat Parkinson's disease and many other ones.

And since the cells can appear to be able to differentiate into functional liver cells, they could be used either in vivo to replace the liver, but also would be very useful to make bioartificial livers, for instance.

We have shown, and others have shown, that cells from bone marrow can contribute to new blood vessels, and so this could be harnessed to create new blood vessels in vivo, or actually the opposite; lower these cells with anti-cancer agents, and actually use them in a anti-angiogenesis approach for treatment of cancer, and then many other diseases.

Again, we are not anywhere close to being able to do this in any way, shape, or form, and a lot of basic research still needs to go on.

So the first point that was on my previous slide, we really need to spend a lot of time in trying to understand what these cells are and aren't.

And at the same time, start thinking about how we might be able to scale these up under GMP conditions that conform with regulatory agencies, and we will have to ask the question, as with any other stem cell population, whether we will use the cells as stem cells, or as more mature cells that have been educated to some extent to become the final product are totally mature cells.

And then again perform large scale culture systems or develop large scale culture systems. And then the last question is whether we should use these cells in an autologous setting or in an allogeneic setting.

Obviously adult stem cells for a number of diseases could be used in an autologous setting. However, if they were to be capable of repairing hearts, and you have a heart infarct today, we would not have adult stem cells sitting around instead of your own to treat you at that moment in time.

So I think there are some issues, and Dr. Gearhart also brought up the idea that with diabetes, for instance, in Type-1, is an immune problem, and again autologous transplantation may not be the way to go.

I think that for adult stem cells, the initial trials may well be autologous, but that in the long term, to make it more cost effective and more available to many patients with certain frequent diseases, that it might have to be an allogeneic therapy, and then we are actually faced with the same questions that investigators that work with ES cells, and primordial germ cells are faced with. I think I will stop there. Thank you.


CHAIRMAN KASS: Thank you very much, Dr. Verfaillie, for a clear, lucid, orderly presentation, and it is very helpful to us. The floor is open for questions, comments, discussion. Elizabeth Blackburn, please.

DR. BLACKBURN: Thank you. Could I just ask a couple of quick clarifications. Dr. Gearhart mentioned in response to Bill Hurlbut's question the difference between fetally derived human cells and mouse embryonic stem cells with respect to their teratoma producing properties.

And I could not quite gather whether it is human embryonic stem cells that are also known to have any teratoma producing properties. Could you clarify that for me, because you also had mentioned this, and I wasn't sure if you were referring to the mouse embryonic stem cell work or the human.

DR. VERFAILLIE: If you use either mouse or human embryonic stem cells without predifferentiating them into a committed progenitor cell, and you use the stem cells as such, they will form teratomas, because it is one of the tools that investigators use that an embryonic stem cell has that capability. So they will form teratomas.

DR. BLACKBURN: And then post-differentiation?

DR. VERFAILLIE: I think there is very little data on the human embryonic stem cells, post-differentiation in vivo, and whether there is still the tendency for these cells to make teratomas.

DR. BLACKBURN: And the second question, since I promised that I would ask you about, is the fusion issue, and which of course you have raised in your talk as well, but again a question of clarification for me, and maybe expanding on your point that you said, well, fusions are going to be problematic.

I mean, the thing that immediately occurred to me was that these fusions, as reported from the in vitro culture, and I believe from engraftment into mice, that they showed aneuploidy, which of course anybody being a hallmark of tumor cells.

So I wondered if those issues and perhaps others were things you could tell us a bit more about when you mentioned that you had concerns about the fusions.

DR. VERFAILLIE: Well, I think it is something that because of the papers that were published that elegantly showed that if you took a somatic cell, an adult hematopoietic stem cell or brain stem cell, and co-cultured it with embryonic stem cells, and then put quite a bit of selectable pressure on the system in the culture dish, they proved that an embryonic stem cell quality could be transferred to the blood brain stem cell.

And initially they interpreted this as being reprogramming of the cell. But then it turned out that there were four sets of chromosomes, and that the cells fused.

And they took these fused cells and gave them to — injected them into a blastocyst as hyperdiploid as cells with four sets of chromosomes. One group was not able to create chimeric animals, and the second group, under the direction of Dr. Austin Smith, were able to create chimeras in the mice that were what he calls unbalanced, meaning that he saw a contribution to tissues, and that four sets of chromosomes are actually tolerated.

For instance, the liver, where at least 50 percent of the cells, actually half, have two nuclei. So I think that currently no investigator who has worked with adult stem cells has set up the right experiment to actually be able to disprove that it isn't fusion.

I would argue that the data that I showed today in vitro, where single cells make three layers of the embryo, and these were euploid cells, meaning that they had a normal set of chromosomes, and which done in human, mouse, and rat, at the single cell level, we can make the three major layers of the embryo.

So that would go against the argument that at least in vitro, that all of it is caused by fusion. In vivo, in our blastocyst experiments, 1 in 3 cells could do it, which is much higher than the one in a million cells that were quoted in the two papers that were in Nature, but which indicated that one bone marrow cell out of a million could actually make a fused cell population.

And I think one in 50,000 neural stem cells could actually cause fusion. So that was a very rare event; whereas, our events are higher. We are in the process of actually going back to these animals — that we have cryopreserved, to try to identify that since some of the transplants were done female into male, we should be able to prove that we do not find the y chromosome in the engrafted areas and in the chimeric areas, which would get at the question whether it is caused by fusion.

And so I think we really need to set up experiments where we have generic markers on both sides, meaning the donor and the recipient, so that we can prove beyond any doubt that the in vivo results would be the results from a fusion.

DR. BLACKBURN: Yes, I totally agreement with that. I think the in vitro, and I am very impressed by the in vitro results, and as you said, there are questions in vivo.

I think in-part my question was addressing this issue, and I was asking about the tumor forming ability or otherwise, because it was not exactly 4N. It was the median number of chromosomes was different from simply 4N, suggesting that there was aneuploidy, and for example, one might not find Y chromosomes, for example, because those had been selectively lost.

So one would probably have to do much more extensive genome-wide analysis of both of those to be sure that there wasn't some genetic contribution from the recipient cells.

But I certainly am very impressed as you say with the in vitro results, and they seem quite unequivocal, and I guess which is the question that you are addressing, and we will find out as the in vitro —

DR. VERFAILLIE: Yes, and I think we need to set up the experiments where we have on multiple chromosomes genetic markers. You know, sequences that we can distinguish the donor and recipient between. So these experiments need to be repeated.

CHAIRMAN KASS: Questions? Janet Rowley, please.

DR. ROWLEY: Well, I would like to ask a question that will include both Elizabeth, as well as Catherine, because I was struck in the data that you presented on your human cell lines that you had passed for more than a hundred generations, that telomerase was still active.

And I just am curious about that, because many of us do believe that that is, if you will, the internal clock that limits the number of doublings that those particular cells can undergo.

And you derive these from adults, presumably young adults in human, but at least adults, and I am curious as to what you thought about the mechanism of preserving the telomerase activity, and maybe if Liz would have any further comments on that, because again one of the critical features and potential limitations of adult stem cells is the fact that they would have potentially fewer doublings than would those derived from embryos.

CHAIRMAN KASS: Could I ask as a favor to the non-scientists in the group if someone would just give an ABCs on the telomerase matter, and just very, very briefly, so that everybody can understand what the discussion is about. Elizabeth, or Dr. Verfaillie, if you could just give the barest —

DR. BLACKBURN: I am the worst person, because I will fall into expert jargonese and so I will try not to. So, telomerase keeps the DNA at the ends of chromosomes replenished, and such replenishment is necessary, because each time one of our cells divides, the DNA at the end of the chromosome is a little bit whittled away.

So, telomerase keeps putting back a little extra DNA on to the ends of the chromosomes each time on average a cell divides. So the issue that Catherine pointed out in her talk was that if you don't have telomerase after a number of cell multiplications, that whittling away process would have gone too far, and that sends a signal to cells to cease dividing.

And so many, many normal cells in culture are characterized by the inability to keep on multiplying. Did that clarify the question? So many cells do not keep multiplying because they turn the cells' telomerase off as part of their natural differentiated state.

Cancer cells, on the other hand, have telomerase, almost in a great majority of the cases, and very up-regulated, and cells of the hematopoietic system — and I will defer to Catherine on this — have an interesting intermediate situation, where they have regulated telomerase activity that is turned on in a natural and regulated way as the cells multiply in response to signals in the body. Is that fair to say?


DR. BLACKBURN: So I think it is a very interesting question of why telomerases is turned on in those cells that are multiplying so well in culture, and has there been a selective event that has allowed those cells, that for some reason have turned their telomerases on in the culture conditions.

But those are the cells that are outgrowing perhaps others in the population, and perhaps that question might be answered by what is the clonal efficiency with which you get these lines growing out. You may already know this.

DR. ROWLEY: But can I intervene, because you assured that it was often turned on, and maybe these cells are identified because they never turned telomerases off.

DR. BLACKBURN: Yes, and I don't know if that is the typical situation when one puts cells into culture, and I thought that they more often would turn off and an earlier subset would keep multiplying, and again I want you to correct me on that cell growth phenomenon.


DR. VERFAILLIE: So currently we do not know whether it is often turned back on in culture. If we look at the cultures, for the first 40 population doublings, the cells appear to grow slightly faster.

And then a second wave of cells grows out and it grows slightly slower. So initially we thought that maybe the more classical senescing cells were disappearing, and that those were the cells that were growing faster, and the you then select for the cell that has inherent — you know, has the system turned on to not be subject to the clock of aging.

The frequency with which we can grow out the cells from human bone marrow is we believe one in a million bone marrow cells. So it is a very rare event, and so it will be quite difficult to actually specifically ask whether it is turned on and then back off, or turned off and then back on, unless we can actually do some genetic trapping experiments to try to ask the question.

DR. BLACKBURN: I'm thinking of David Beaches' experiments in which he was able to show that cells would spontaneously, if you keep them in culture, turn their telomerases back on, because that gives them some selective advantage.


DR. BLACKBURN: And so I was wondering if such selected advantages occur in your situation?

DR. VERFAILLIE: It could well be, and so the culture conditions are very particular, and so I didn't go into too much detail.

But if you do anything wrong to the culture conditions, we cannot create the cell lines, and so it might well be that it is what we call in my lab a cultural artifact what we see, which would mean that these cells may not exist really as such, but actually are induced to become this long-term proliferating cell by the culture conditions that we put them under.

DR. BLACKBURN: Thank you.

CHAIRMAN KASS: Janet, again, please.

DR. ROWLEY: I have two more questions. One is a follow-up of a question that I asked you about a year-and-a-half ago, on whether out of your MAPC cells you can get hematopoietic tissue.

DR. VERFAILLIE: Well, I think I showed you in vivo that if you infuse the cells into mice that were either not irradiated or sub-irradiated, that the cells appear to be able to differentiate into hematopoietic elements that have red cell, and granulocytic markers.

In vitro, we have had more difficulty to try to do that, even though it appears now that we can at least get for people who don't understand this, but what would be yolk sac hematopoiesis, even though we haven't really seen hematopoiesis that would occur in the embryo proper.

But we can find cells that look like the cells that have been created at the earlier stages of development, where the initial one is made, which is in the yolk sac.

DR. ROWLEY: And the other question is more a more practical question. I don't know precisely how many cells would be required to treat an adult patient with a particular disease, and are the number of cells required, or what kind of limitations, using your system, would be faced if you have not one patient, but hundreds or thousands of patients that could benefit from a particular therapy?

Is this really going to be an applicable strategy?

DR. VERFAILLIE: I think it is a bit too early currently to really be able to answer that question. We have been able to take cell populations and have them undergo 80 to a hundred population doublings, which is really if you were able to do that and not throw cells away along the way, it is 10 to the 50th cells or something like that.

So it is an enormous number of cells that you can in theory create. What I didn't go into too much detail on is that the way that we have to grow these cells is under very low density conditions, meaning that the cells have to be far away from one another, or otherwise they do not maintain their undifferentiated state.

Which is quite different from embryonic stem cells, which tend to grow in tight clusters. From a bioengineering standpoint, meaning scaling it up to making hundreds of millions of cells, will be a major bioengineering question of how we can actually adjust the system to be able to do that.

But on theoretical grounds, you know, if you could overcome all the bioengineering problems, you should be able to create enough cells to treat multiple individuals, rather than a single individual at a time.

CHAIRMAN KASS: Question. Robert and then Mike.

PROF. GEORGE: Just a very quick question of clarification in response to Janet's first point. On this question of whether they were — whether the teleomerases were turned off and then turned back on in the culture.

If it is not that, and if that's not what is happening, the other possibility is that they were never off to begin with?


CHAIRMAN KASS: Mike Gazzaniga.

DR. GAZZANIGA: Again, thank you for a very excellent talk and a cautious talk I thought. I thought it would be helpful for us to understand the new pressures of a biologist like yourself, which are the following.

Here you have this fantastically interesting finding, and up until 5 or 10 years ago, the normal way that such things would be treated is you publish the work in peer review, and then you make the stuff, whether it is reagents, or whether it is cell lines available to others for reproducibility.

And that is a normal sequence of events that we are all familiar with. And now we have the bio-med inserting itself into these laboratories, where all of a sudden it becomes proprietary goods from this work.

When the original media picked up on your story, and I guess it was The New Scientist, there was this cryptic little paragraph in there about how they had seen the patent on some of this work, which is a very complete description, and how does that — what is going on here?

How can — and this is where I would like to go obviously, and obviously it is good for everybody here to get these cell lines that you have out to other labs, and reproducibility, and then the process goes forward.

Are you constrained in some way, and has life been made complicated because you didn't have full public funding and you had to use this other money, or was that your own? What is going on?

DR. VERFAILLIE: So the work was really done at the university with NIH funds and university funds, and so there was really no private funds, except for the small amount from the company that was listed in the beginning, has gone into the work that we have done.

And because of the possible importance of the observation, the university, as well as myself, thought we should get some kind of protection, even though I am not sure that you can truly patent stem cells, because all of us have them.

But just such that we would be in a position to work with biotech companies to be able to produce large-scale numbers of cells and things like that, which is hard to be funded to known private funds.

So there is patents pending on the cell population. Currently, that really has not precluded us of collaborating with other institutions, or investigators within the same institution.

So they have collaborations with 10 or 15 different groups within the U.S., or outside of the U.S., depending upon the expertise that we need, to try to recreate the cells in other laboratories, and actually use their expertise, since I am a hematologist, and not a liver physician or a neuro scientist.

And to actually be able to use expertise in other people's laboratories to move the research forward. So there are some minimal ties attached to working with the cells, but I think it isn't overcomeable, and it really has not been an issue with other academic investigators to collaborate with them.

And teaching people from those labs to come and to grow the cells, and at least start working with the cells. But it is a very complicated and it is a — and I have had myself a lot of problems in trying to find the right patent between potential biotech interests and academic interests.

DR. GAZZANIGA: Right, and you are not alone in that dilemma. So are there other MPAC lines at other institutions now that behave like yours, or is yours still the Golden Grail here?

DR. VERFAILLIE: We have given out the mouse, and to some extent, human MAPC lines to other investigators who are now setting the lines back up. We are also explaining and teaching people how to create them from beginning bone marrow.

And I know that there is one group in Japan who I think pretty much as the system set back up from human bone marrow. You know, they still need to do some additional studies to prove that it is really MAPCs, but we trained a person from there for 3 or 4 months in my lab, and they went back to Japan, and were able to it appears to recreate them.

CHAIRMAN KASS: Could I ask a couple of sort of semi-scientific and semi-practical questions? How hard is it — I mean, you have just indicated that not many people have already been able to do this, but how hard is it to find these cells?

And by which I mean two pieces, and in how many individuals in which you look for them can you find them? And how hard is it to find — how rare are they, and how hard is it to find in any particular individual?

Both of these questions bear upon at least a preliminary assessment of how useful this might be clinically speaking down the road, although things could change where you might be able to enhance the yield.

But could you give us a preliminary sense of this?

DR. VERFAILLIE: I think we have studied now between 70 and 80 normal humans to try to identify the cells. The age range, the youngest donor was two, and the oldest donor was 55. The majority are young adults who want some money to donate bone marrow at the universities.

CHAIRMAN KASS: The two-year old?

DR. VERFAILLIE: No, the 20 year olds. The 2 year old actually did a bone marrow donation for a sibling who needed a bone marrow transplantation. So we have been able to create the cells I would say in about 70 percent of the individuals that we have looked at.

Whether that means that the other 30 percent didn't have it, or there was some technical issue that came about, and we were not able to create them, we start out with 10 milliliters of bone marrow, and we would usually find a few clones that can actually grow out.

And so really the frequency is quite low, and it is one in a million, and that is at least the estimate that we have right now. But there is lots of bone marrow and so one in a million isn't an impossible task to do it.

CHAIRMAN KASS: And could I also follow up on the question of these cells and their promise, assuming the best, and the embryonic stem cells, assuming their best.

This is not a question of whether one should prefer one line of research or another, or whether we should now go ahead with them. But is there anything specific that you could imagine could not be done therapeutically with these MAPC cells that you would then need cells derived from embryonic tissue to do?

Or is this in the rosiest division, is this really a substitute, and one that might even have the rejection problem solved if I am dreaming?

And this is not a question about whether the other research should go forward, but really what is the best promise of this research so that at least we can think about it?

DR. VERFAILLIE: Well, I think that the data that we have in vitro suggests that we can create cells of the three germ layers of the embryo, and so theoretically, you could envision that you might be able to make more than we have done so far.

We have made liver-like cells, and brain-like cells, and epithelial cells, and we have not tried all the other ones. In vivo, the blastocyst experiment, unless that is a fusion event, and if it isn't a fusion event, would indicate that the cells hold the inherent promise of making all the different cell types that make up the tissues, the somatic tissues of an animal.

So again that would suggest that is under — that if we changed culture conditions further that we might be able to, for instance, create insulin-producing beta cells, which we haven't done, or create two heart muscle cells that function like heart muscle cells, and don't just look like it.

So if all these promises hold true, and if we continue the cultures and they can be expanded even further into 80 or 90 population doublings, and so there are lots of ifs here, they may be able to be used to treat a large number of diseases.

The problem at this point in time is that there is so many ifs that it is a very difficult question to specifically answer.

CHAIRMAN KASS: Of course, and I appreciate that, and on the question of the longlivedness, or the half-life of these things, you have gone through — in vitro is what? It is what?

DR. VERFAILLIE: From 80 to 100 population doublings.

CHAIRMAN KASS: And it is obviously too early to say how much longer, and whether those conditions are matched in vivo. But when the people say that the promise in terms of longevity for cells derived from the adults is really much less, is there anything to be said on that question of the basis of knowledge now had?

DR. VERFAILLIE: Human embryonic stem cells have been kept in culture now for 350 or 400 population doublings. So that is 3 or 4 times as long as the adult cells. So we are striving to go there, but we just need time to do that.

Are these cells going to be able to do that? As far as we can tell, after 80 population doublings, there is no shortening of telomeres, and so that means that there is at least another 50 or 60 left.

If for some reason telomerase is shut off along the way for reasons that we currently don't know why that might be, then the longevity would be less than what has been shown for embryonic stem cells.

Now, for classical adult stem cells, if you take hematopoietic stem cells that make blood, but not something else, they would not go for 80 population doublings.

So there is something special about these cells, that they can overcome this senescent block at 60 or 70 population doublings, which is actually long for any other adult stem cell.

CHAIRMAN KASS: Thank you very much. Questions or comments? Janet again, please.

DR. ROWLEY: Coming back again to partly the real world in this iffy situation, and it is a question of the practicality for treatment for particular individuals.

It seems to me that the notion that you might be able to derive these MAPC cells from an individual who had some medical problem might have some limitations because it probably takes 3 to 6 months, or so to get enough cells to then be able to use them therapeutically in that individual, and that is always assuming that the individual has some kind of a somatic disease, and not the basic underlying genetic problem.

So then the way to get around that if it really is 3 to 6 months, and you don't have that window of time, would be to do somatic cell nuclear transplant. Now, have you ever tried that in your MAPC cells?

DR. VERFAILLIE: In collaboration with Dr. Jaenisch, and two weeks from now we will try the mouse MAPC cells in mouse eggs, and ask whether the efficiency of nuclear transfer would be closer to what you would see with embryonic stem cells, and where the efficiency is much, much, much higher than if you use a classical adult cell.

And that might improve efficiency of making cloned embryonic stem cells.

DR. ROWLEY: But I am thinking of the other experiment. You have a patient who is desperately ill, and so you would have cells from that patient, and you would want to use the nucleus of the patient's cells into your MAPC cells, and so that is a different thing.

You have got these cell lines, and how can you make them more compatible with the patient, and agreeing that you can't get rid of the mitochondrial problem unless you do additional manipulations and strategies.

But have you ever tried to replace the nucleus in your MAPC cell with a nucleus from an adult somatic cell?

DR. VERFAILLIE: No, we have not yet.

DR. ROWLEY: Do you plan to?


CHAIRMAN KASS: We could always get everyone at the age of 15 to put away a little bit of marrow for the time that we might need it.

DR. ROWLEY: Another reason to save cord blood.

CHAIRMAN KASS: This is your chance, council members. This is a wonderful opportunity. Questions?

DR. ROWLEY: Well, I would just be interested from Catherine's point of view on her answers to some of the questions, to the two questions that I posed at the end to John Gearhart, and again give her the option to do this as a written response rather than a direct response, but I think it may be easier to — and the second question, which may be very simple to answer in terms of the kinds of restrictions that you find now in funding.

And I would assume since you are dealing with adult cells that there aren't any, but I would be interested in your perspective on the funding, in both government and other agencies.

DR. VERFAILLIE: Well, currently for the work that is ongoing in my group, which works with adult stem cells, actually the amount of funding that has become available through the NIH has increased dramatically over the last few years to support this kind of research.

So that has not been a problem. I have wanted to compare these cells carefully with embryonic stem cells, and so we are in a position currently to do this in a mouse, but mice aren't humans.

And so we have really not been able to do that until earlier this year when human embryonic stem cell research was allowed in academic institutions under NIH funding.

And as I mentioned, we had actually gone out and tried to recruit an investigator with that kind of expertise to be in a position to try to address some of the questions that have come up here, and are these cells going to be equipotent.

And I think to date, even though they are exciting and they seem to be quite potent, I can't really say whether that is the case. And so ultimately we won't be able to answer this question until we can truly compare them and not across country borders, but actually within the same institution, where people can look at the two cell populations at the same time.

And so in that respect, I think that the lack of funding for embryonic stem cell research in humans has made it impossible up until just recently to be able to do that.

CHAIRMAN KASS: Michael Sandel.

PROF. SANDEL: I wonder if I could put to you the same question I put to the previous speaker. Given that some people regard embryonic stem cell research as morally problematic, what would you think of the idea of imposing a moratorium on embryonic stem cell research until we could assess what might be achieved by adult stem cell research?

DR. VERFAILLIE: I think that my answer is very much in line with what you heard from Dr. Gearhart. I think that the main reason why we — to investigate in the field of embryonic stem cell — human embryonic stem cell research is to be able to compare and contrast the two cell populations at the same time.

I also think that what we did in our culture dishes to try to differentiate these MAPCs into liver-like cells or neuronal-like cells is really based on what has been learned from mainly the mouse embryonic stem cell field, where investigators have been able to take these cells and drive them in vitro to become certain cell types, even though that is not a hundred percent fool-proof, and it is not completely figured out how you should do that.

So I think if you have a number of different cell populations at the same time, we try to test all these different questions. What we learned in adult cells might be applicable to embryonic stem cells if they are the cells that ultimately will be the suitable source for our clinical applications or the other way around.

And so I think stopping research in one field actually will slow down research in the other field, and it would be either way. In other words, if you stop our research in adult cells, or embryonic stems, as I think what can be learned in the two systems should be translatable in the other system.

And so I think if you were to ban all embryonic stem cell research, it would really slow down the insight that could be gained in adult stem cell research.

CHAIRMAN KASS: Rebecca Dresser.

PROF. DRESSER: This is unfair, but I am wondering if you had any ideas about the cost of such a procedure? I mean, just based on what you have done in mice, and you mentioned at the very end that to be cost effective that you would probably would have to just have a number of cell lines and not rely on the patient cell.

Is this going to be a very, very expensive technology, and where we have to worry about — well, if all these ifs work out, will we have to worry about who has access, or will it be comparable to the stem cell transplants that we do now with bone marrow now? Or what do you think?

DR. VERFAILLIE: Well, I think it will be relatively expensive if you do it on a single person basis, and you will have to create the cell lines from the beginning, rather than go going to a frozen stock of cells, where you have a very well-qualified product to start with and where you expand cells.

And so you might even have already committed cells frozen as well, and so the cost to get to that point would have to be incurred once rather than doing this over, and over, and over again.

The costs I think — well, it is hard to say, because I am not sure how much of the regulatory issues we have actually complied by at this point in time to actually truly gauge how much it would cost.

But I think that by the time that you do all the quality control tests for infectious agents and things like that, that amounts to quite a bit of money for each cell line that you try to establish.

And in the long term I think it would probably be more cost effective if you would have a therapy for heart infarcts that you could go to a limited number of cell lines. And to put numbers of them, I don't really know.

It would probably be in the range of a bone marrow transplantation currently, which is quite expensive. So it is anywhere between $50,000 and $200,000 per patient.

If you had qualified cells that were frozen, and then you could expand them for a short period of time and do a limited number of tests at the end, the amount of cost incurred would really be all up-front, and then there would be a relatively small amount per patient.

PROF. DRESSER: I guess the other thing is that bone marrow transplants work fairly well with some illnesses and not with others, and would you expect to see those kinds of results with these kinds of therapies?

DR. VERFAILLIE: I think that would highly depend on the type of disease that you tried to treat. You know, you are all well aware of the treatments that have been used for Parkinson's disease, which the trials that were done in Sweden have made little complications.

But when this was extended in multiple hospitals in the West, there were a lot more complications if it was done on a larger scale. So I think that depending on the disease that you go after that it may work better or worse, and it is really way to early to be able to comment on that.

CHAIRMAN KASS: Bill, do you have a question?

DR. HURLBUT: Well, if we have time, I would like to ask a couple of scientific questions if that is all right.


DR. HURLBUT: Do I understand this correctly that you are saying that your MAPC is put into the blastocyst to perform more cell lines than do other adult stem cells?

I thought that adult stem cells generally formed lines in a blastocyst?

DR. VERFAILLIE: There are 3 or 4 papers published on adult stem cells into blastocyst experiments. There is one paper published by a German group, where they took purified hematopoietic stem cells, and injected them into the blastocyst, and what they were able to show was that the cells gave rise to some hematopoietic elements, and that they actually recapitulated the developmental behavior of hemoglobins, which switched at different stages of development during embryos, fetuses, and then adults.

They did not see any contribution outside of the hematopoietic system. The second paper is a paper from a Swedish group, where they had taken neural stem cells that have been cultured, and introduced them in the blastocyst, and as far as I know, they have never had animals been born alive.

And they saw a contribution to a few tissues, but not all tissues of the mouse fetus. And in the last papers, we did a paper by Austin Smith, the one that reported on fusion, where they had taken defused cells and given them to a blastocyst again, and it showed a contribution in one animal that was born, and that was really only a single animal, to the liver and a few other tissues.

But it was not quite the amount of contribution that I showed in the picture here, where every single tissue of the mouse appeared to be having a fraction of the single MAPC cell.

DR. HURLBUT: That is very, very exciting. Another question that I think might be of good general interest to our council, but he question of whether transdifferentiation is occurring, or even the process of embryonic stem cells just differentiating, it is always clouded by the question of how do you know when there is actual differentiation taking place?

In other words, just because you follow one or two gene expressions, you don't know, and one of the problems with embryonic stem cell therapies will be to get the target tissues up to speed, like beta cells producing enough insulin.

I know that there are advances being made on this, but can you just give us a general description of how you identify when you are satisfied that a tissue has in fact been produced?

And maybe tell us a little bit about the — maybe we need a little education on messenger RNA assays.

DR. VERFAILLIE: Okay. The criteria to say that you produced tissue I think needs to include that you turned on the genetic program that is compatible with the tissue that you want to produce.

You find therefore proteins from the genetic program in the cells, and the cells have morphological changes consistent with the cells that you are looking for, and the cells have functional characteristics of the tissue that you are looking for.

So what happens in a cell is that in an undifferentiated state a number of gene programs are shut down, meaning there is no transcription to the messenger RNA, and you will find no protein, and therefore no function.

During a differentiation process, you come in with a growth factor or a cytokine, or a stimulus from the outside, and you trigger a certain set of signals that then open up a new genetic program and the first thing that happens is that you transcribe messenger RNA, that then gets translated into proteins and/or sugars, that then supposedly give a new function to the cell.

So what we have been looking for in vitro, and that is where most of our work has been done initially, is actually taking an undifferentiated cell and showing that a certain genetic program isn't turned on, meaning that you don't find mRNA, and you don't find protein, and you don't find function.

We then switch the culture conditions and add triggers by trial and error, to a large extent to try to activate certain genetic programs. And if we do that, we look for protein and mRNA first.

So we look to prove that the genes are turned on, and then we look to prove that these gene products actually give rise to proteins. We have gone to the next step also and actually tried to then take the cells that we believe that are like brain or like liver, and started asking questions.

If it is a liver cell, it should secrete certain things. It should have the machinery to detoxify blood and things like that. So we have been able to show that in the liver lineage, for instance, that we do turn on the programs to make albumin, which is one of the major proteins that is being secreted in the liver and is present in the blood.

These cells have, for instance, cytochrome p450, which is a massive machinery in the liver that helps detoxify the blood components. And we can show that it is there, and it responds in the correct ways as liver cells would do.

So that is what you do in vitro, and in vivo, it is a bit more complicated, and you really need to use animal models where there is a disease. So you would have to show that the cells ingraft and you can find the donor cells.

You would have to show that they turn on RNA and protein, and therefore have this genetic program turned on. And then function, which means that if you take an animal that has a failing liver, and you give the liver cells to this animal, the animal will now live without having drugs that keeps it alive.

And so that would prove that the cells that you put in have actually acquired the ability to function like a liver cell. And so for adult stem cell research, very little proof of the latter is actually present.

For embryonic stem cell research in mice, there is a lot of evidence, and in the human embryonic stem cells, that evidence is just starting to become available, just like it is with adult stem cells.

DR. HURLBUT: Could I ask one last little question? How many genes are we talking about here; like hundreds, or thousands, and how many do you monitor in fact?

DR. VERFAILLIE: Well, we usually monitor between — well, there is probably hundreds of thousands that get turned on, and so using the new technologies, the array technology, and the proteomics technology, that is one of the things that we are looking at, because it will give us a much better insight in the whole programs that are being turned on.

We just pick and choose the ones that we think are known to be important at certain stages of the differentiation. So, for instance, if you go from a stem cell to a liver cell, we know that you have to turn on X number of genes that happen to be known to be turned on.

So we look at 2 or 3 that are early, and 2 or 3 that are in the middle, and then 5 or 6 at the end. We have not exhaustively looked at all of them yet.

But I think with the human genome being sequenced, we now have the tools in hand to now take cells created from stem cells and look at the whole program of genes that is present, and what we created in a culture dish, compared to what is actually present in real life in vivo, and get a feel of how closely we actually are getting to the real cell.

DR. HURLBUT: Thank you.

CHAIRMAN KASS: Could I — Robby, did you have a question? Why don't you go first, because I have a couple of things as well. Please.

PROF. GEORGE: Actually, I just wanted to follow up the question that Dr. Kass asked earlier just for clarification, and I recognize that there is a great deal of uncertainty as to what the future holds in your area for research, as well as in embryonic stem cell research.

And estimating or evaluating what the prospects are therapeutically is a speculative business, but having all of that in mind — and I was not clear in responding to Dr. Kass whether you identified some areas in which knowing what we do know now about the differences between embryonic stem cells and the MAPC cells, it is possible to identify some areas where we just know that whatever the prospects are for MAPC cells that they won't be able to do, or our therapies won't be able to be developed based on them to do certain things.

And that there is at least a prospect of embryonic stem cells being used to do.

DR. VERFAILLIE: It is so very hard for me to answer that question, just because embryonic stem cells have been worked with for so much longer, and so investigators have been able to, for instance, make cells that secrete insulin to some extent on demand, which has not been accomplished with adult stem cells.

There is a little bit of evidence from pancreatic tissue itself that there might be precursors that can do that, but from MAPCs, for instance, we have not been able to do this yet.

It doesn't mean that we can't. I don't know that answer. So there is a lot more experience with embryonic stem cells and there is a lot more — at least in the mouse system, there is a lot more known on how to try to trigger certain differentiation programs and whether the MAPCs will respond to the same extent and to the same degree.

And I think that currently I can't really answer that question.

PROF. GEORGE: But asking if you look at it and not asking what do we know MAPC cells will be able to enable us to do, and have a prospect of doing, that embryonic stem cells have a prospect of doing.

But if we simply ask the question as do we know just on the basis of the facts of what we know about the differences, and that there are in fact some things that MAPC cells, no matter what, won't be able to do. Or is the answer that we just don't know?

DR. VERFAILLIE: I think we don't know currently, and I can't really answer that question, because we just don't know at this point in time.

DR. FOSTER: I just want to interrupt with this one point. Those questions are really hard to answer, but there is another whole area that is going to impact what you are going to use cellular based therapy for.

And that has to do with good vectors, retroviral gene therapy, and that you are going to accomplish with other diseases that you don't have to use cells for at all.

I mean, the most recent thing in severe combined immunodeficiencies in humans, is you put a retrovirus in, and you put the common gamma chain in for about five cytokines, you know, for these kids. It was just in the New England Journal a couple of weeks ago, or three weeks ago, or something like that.

And they are now two years out, and so there are going to be a whole lot of diseases that you are not going to have to use regenerative therapy or cell therapy. You can't predict those things either at this point.

So I think if you try to jump way ahead of what the basic science is doing, then you are prone to error, and I know everybody wants to know whether an adult cell is better than an embryonic stem cell, or something like that.

And I don't think you can answer those questions, and one of the things that we have heard from both the investigators this morning is that they cross-fertilized with each other.

And so — I mean, that you could not have done what you are doing in the adult cells without what had already been done with the embryonic cells.

So I just would argue against trying to push investigators to say whether an adult stem cell can do this or do that at this point, because we have not even taken into consideration many other approaches to human disease.

I don't mean to be fussy, but I do think that that is an important thing.

PROF. GEORGE: But I was actually asking — well, I think the question I asked was that it really is about what we know now. The question is do we know now that there are certain differences, that as a result of which the prospects for the one area are different from the prospects of the other. And I got my answer. Thank you.

CHAIRMAN KASS: Let me take the privilege of the Chair to expand in a way Dan Foster's comment in a direction that he might not have intended.

DR. FOSTER: That does not surprise me.

CHAIRMAN KASS: Well, I mean, you are a genial sort, and I think you won't — I mean, one of the things that one has to remember in this conversation is that wonderful as the stem cell approach is from whatever source to the treatment of these diseases, that is not the whole area here also.

And that the gene therapy is not the whole story as well, and there are preventive measures, and there are all kinds of other things. I mean, the conversation, because we are taking it up, gives it a certain type of dramatic focus and concentration.

But for the people who work in clinical medicine, they know that this is — that there are lots of ways to try to skin this cat. But I wanted to ask a couple of — to make a comment, and then ask a couple of questions.

You have talked understandably and very welcomely to us about your own very exciting work. There is a great deal of skepticism about many of the published works in using adult stem cells.

And unfortunately, for better and for worse, these reports are caught up in the political controversy that now surrounds us, with people on both sides having a stake in either making the results on one line of work seem better than the other, precisely because they are wed to an either/or choice.

Can you, abstracting from all the political considerations, and the various axes that various people are grinding on these poor cells, can you say anything at all generally about the kinds of initial reports of a clinical sort that we have had with alleged adult stem cells?

Because at least according to some accounts, these have been very exciting, and yet there is a great deal of skepticism about whether these are in fact stem cells that are producing the results.

Can you tell the council anything at all about how we should at the moment regard the news that is coming out to us in this area? How should we receive it?

DR. VERFAILLIE: There have been several publications that came out over the last 1 or 2 years now, where investigators or clinicians have looked at individuals who were transplanted with classical bone marrow transplantations, and looked in tissues outside of the hematopoietic system to ask whether bone marrow derived of donor-derived cells could be found in different tissues.

And the reports that have come out have indicated fairly significant levels of contribution to certain tissues, meaning they have found cells in the heart, and they have found cells in skin, gut, liver, and so forth.

And we really have not looked in the same situation to see whether we can confirm these data or not. I know that some clinical groups have put in doubt to some extent the degree of contribution that has been reported, and it is not quite clear whether the 5, 10, or 20 percent that has been quoted in some papers is indeed actually going to hold up over time.

I think there is some contribution, and the question in my mind still is how clinically important is it what investigators have seen or what clinicians have seen currently.

If you go strictly by the term of stem cell plasticity, none of these studies really show that it was a single cell, or it was a blood cell that gave rise to these tissues, and it might still be that some contaminating cells were contributing to that.

And really none of these studies have shown that this has had any clinical impact on what was going on in these patients. And so they didn't really show that you restored function of the organ that the cells were found in.

CHAIRMAN KASS: I was thinking of a recent report on the Parkinson's cases.

DR. VERFAILLIE: Correct. So the Parkinson's cases were — and that is with fetal brain tissues, and are those the reports that you are referring to?


DR. VERFAILLIE: And so there has been a series of patients transplanted in Sweden with Parkinson's disease, where one team of investigators in a non-controlled study, shows that implantation of the fetal tissue brain — fetal brain tissue into the brain of patients with Parkinson's disease could rescue patients, and could actually correct the Parkinsonism.

And actually have now done so for some patients for more than 10 years. Now, these were highly selected patients, and done by a single group of investigators.

The same was done in the west in 3 or 4 institutions, and some patients got better and some patients did not get better. But I think that gets to the proof of concept that if you have the right cells, and if you can create the right cells, and if it is from embryonic stem cells, or adult stem cells, or from tissues itself, that there might be a way of correcting Parkinson's disease.

But there is again — and I think it would behoove us to really look carefully at exactly what single cell or fused cells that we have to put into the brains of patients with Parkinson's disease to try to correct the disease, and not over correct it as it was done in some of the patients in the U.S., where they had more side effects from the therapy than they initially had from their Parkinson's disease.

And so even though there is an enormous amount of pressure on all of us with stem cell research to try to come up with therapies yesterday, I have been very, very cautious in telling people that do the clinical work that you can't just go around and take stem cells and put them in places in the hope that they will work.

Because we will get into situations like the gene therapy field, where a couple of awful problems have popped up, and have actually halted the clinical potential of these cells enormously.

CHAIRMAN KASS: Could I follow that up, because if there had been more time, I would have asked Dr. Gearhart this question as well, and you are a clinician who deals with patients that are also — now thanks to your new results, and I am sure that you are getting lots of calls as well.

There is an ethical dimension to this area that worries not so much about where you get the cells from, but how we deal with the desperately sick patients looking for any sort of hope.

And let me say flat out that in-part to fend off the opponents, the people in the scientific community and medical community, has to some extent not been adverse to shall I say hyping the benefits here and possibly even taking rather cruel advantage of these hopes.

And from what I hear from you, and from what I hear from Dr. Gearhart, these therapies, there are lots of problems to be solved before these things will be made available.

And that is not to say that there isn't this enormous promise, but what can you tell us, or what advice would you give us about we could responsibly speak about this promise without behaving, let me say, unethically in dealing with the very patients who are coming to us for help?

And I think that's something that you have probably faced directly, and whatever help you could give us on that would be welcome.

DR. VERFAILLIE: Well, like Dr. Gearhart, my e-mail and phone have a lot of messages on them from patients locally, around the country, and around the world who want to bring a child or a parent with a certain disease, and want us to treat whatever disease you can come up with.

And we have to speak the truth, and even though we are excited about the work that we have, and for the work that people do in embryonic stem cells, at this point it is a promise, and I don't think there is any data to say that in the next 1 or 2 years we will actually be in clinical trials with any of this.

So we really have to tell patients, families, and whomever, that currently we are trying to cure mice, but a lot of mice have been cured with a lot of different things, and that doesn't necessarily mean that it will translate into humans.

And so we need to do the regular science that needs to be done to come up with a therapy that is both potentially useful and for certain not dangerous.

And so that the last part of that whole thing is really where everything sits. And so we could go ahead and do things now, but then run into major, major complication issues which would make patients way worse off than they started out.

You could argue that bone marrow transplantation, there was not a whole lot known when the first bone marrow transplants were done, and that is before I started in bone marrow transplantation, and probably some patients didn't fare that well either in the beginning.

But people ultimately still have to learn by doing it in humans, but we have to learn as much as we can in culture dishes, mice, rats, and larger animals before we proceed with therapies for things that are not immediately legal.

And so it is not because you are diagnosed with Parkinson's today that four months from now that you will die from your disease, which is different if you have a acute leukemia, where there is really no other solutions.

And so I also think it will have to be graded depending on the type of disease that you start treating.

CHAIRMAN KASS: And I have one last question, and I don't think we will have another opportunity in this discussion, but this comes to Rebecca Dresser's question about the costs, and how to think about this. And also about the applicability.

There was recently a meeting of the major biotech companies in Princeton, and our scientific director, Dick Roblin, was there, and they were discussing among other things the question of the solution of the immune rejection problem from all these various things.

And all of the ones that were present there are putting their research money not into somatic cell nuclear transfer to deal with the rejection problem, but into other means, for a reason that would have never have occurred to me until it came back from this meeting, which is to say that if you have highly individualized treatments, case by case, that at least under present regulatory systems, if you call these things products, each one of them has to be approved independently by the FDA before it can be used.

And so the question is whether or not — and in partly thinking about the cost and the scalability, and the things that might make things universally applicable, doesn't it make sense more to be thinking more in terms of cell lines, whether embryonic or adult, and that could be made universally applicable, rather than trying to continue to think each person, his or her own replacement, given these practical problems of scale and product approval?

I am not sure that the question was clearly put, but it bears upon the efficacy of this in terms of long term clinical use, and the questions of cost.

DR. VERFAILLIE: Ideally, it would be personalized therapy, and so you would create cells that are completely compatible with the person that you need to treat, except again in situations where there is an autoimmunity issue, which makes it complicated.

And if it is an autoimmune problem starting out, then cell therapy is probably not the best way to go about doing this. For instance, Type-1 diabetes would come to mind, where there is really a rejection of your own islet cells.

I think the costs — and I spoke to that just a little bit before — of creating everybody's own cell line will in the long term will be extremely high, and it will not be a therapy that is suitable for acute events.

So if you have an acute stroke, or if you have an acute heart infarct, and you try to correct that, there is no way that you can clone ESLs to correct that, or you could create MAPCs to correct that within the next one or two weeks.

It just takes too much time to try to do this. Then you could argue, well, I will store our own MAPC cell lines or own ESL lines just in case we need it, which definitely I don't think is financially tenable.

So even though the ideal situation would be to be able to make everybody's own cells, and I think in the long term if the cell therapies are proven to be, for instance, very useful in patients who have a severe MI that you can actually correct them almost immediately after the MI has occurred, or within the next few weeks after it has occurred, it almost has to be done on an allogeneic basis.

And in that case, trying to come up with wise ways of making the cells acceptable to the vast majority of patients, whether it is multiple cell lines and a minimum amount of immunosuppression, or establishing partial chimerism by creating both blood cells and heart muscle cells from the same cell lines, for instance, would be one way to get around that.

CHAIRMAN KASS: Thank you very much. Thank you very much for a wonderful presentation, and a very generous and full response to our questions. If we might take the liberty of just contacting you with some other things.

I know that your e-mail is full, and we will try to add very little, but as we go along, we might have some additional things.

DR. VERFAILLIE: That would be great. Thanks.

CHAIRMAN KASS: Thank you very much. Members are asked to go immediately from here to the other room, where they want a group photo, and the four or five of us who have not yet posed for our individual mug shots, are asked to stay. I think lunch will be served there.

We will reconvene here shortly after 1:30, and let's say about 1:35 or 1:40.

(Whereupon, at 12:28 p.m., a luncheon recess was taken.)

  - The President's Council on Bioethics -  
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