9UNP0161SD_ Jon Odorico_WordPress

- Welcome, today's DART Talk will be given by Jon Odorico from the School of Medicine. He'll be discussing "Cell Therapies and Transplantation." 

- Good evening. Thank you Adam, for that introduction. Judging by the turnout today, which is pretty good, the weather didn't keep too many people away, but thank you for inviting me to the DART Symposium. I'm happy to address you tonight and the viewers online about our research in the field of stem cell therapies for treatment of diabetes and transplantation. So you are all the stalwarts and the diehards, so I think you'll be rewarded for coming out on a nasty night like tonight. I hope you will be anyway. What I'll try to cover tonight, in the series of about 50 minutes, is where we are in the field, current status basically, of differentiating embryonic stem cells to pancreatic beta cells, or insulin producing cells, for a replacement therapy for patients where they've lost insulin production or need additional insulin production to treat or reverse their diabetes. 

I'll talk about some of our research, some other research in the literature. And we'll also talk about somatic cell nuclear transfer derived human ES cells as a means of generating genetically matched human ES cells, so that one might obviate the process of rejection and not need immunosuppressive medications after transplantation of such a therapy. We'll talk about a few of the innovative ways that scientists have thought about to get around the ethical issues with regard to somatic cell nuclear transfer derived human ES cells, or therapeutic cloning. And then we'll finish with some lessons from pancreas transplantation, and whether the aspects of autoimmune may play a role. So by way of introduction, I think it's fairly well-known by many that there's a huge public health impact of diabetes. It's extremely costly. It's extremely morbid for patients that develop diabetes. There's a high mortality. There are many complications that develop. In fact, it's the leading cause of blindness and kidney failure in the United States. 

It's one of the most prevalent diseases in westernized countries. It costs, and you can see some of the numbers here, enormous amounts to take care of these patients, for our government and for our healthcare system. And there's an epidemic of diabetes. And it's estimated that the incidence or prevalence of diabetes may increase 40% in the next 50 years. So it's a huge problem. And it affects both the young and the old. There are several different types of diabetes. Diabetes can be loosely defined as an absolute or relative insufficiency or deficiency of insulin producing beta cells that are usually present in the pancreas. The main two types are Type 1 and Type 2. Type 1 is usually juvenile onset. 

It's usually associated with an absolute deficiency of insulin and a destruction of beta cells by autoimmunity. And most of those patients require insulin, exogenous insulin or insulin injections from the very beginning of their disease onset. In contrast, Type 2 patients, initially, it's more associated with an insulin resistance. And that is that the end organs, muscles, heart, brain, etc., do not see or perceive the insulin that the pancreas or islet beta cells are producing and therefore, the pancreas increases its output of insulin. But that is insufficient, and results in hyperglycemia or elevated blood sugars, which then over time is thought to cause burnout or apaptosis or death of those beta cells. So ultimately those patients may also require insulin therapy and be relatively insulin deficient. But it takes some time, usually, for that process to develop in any particular patient. In contrast to the autoimmune ideology of diabetes in Type 1, Type 2 is a polygenic and familial component. Also, many patients that may not require insulin are often treated with diet and exercise and hopefully weight loss, as ability to control their diabetes. 

So the two types are very different, but there are other types that might represent a mixed phenotype. There is also gestational diabetes. And there's also something called maturity onset diabetes of the young, which is associated with genetic deficiencies of some of the pancreatic developmental transcription factors. So diabetes is a multipresentation type disease. For the purposes of the therapies that we'll talk about for the rest of the talk, we'll be mostly focusing on clinical application of Type 1 diabetes. And that is because there's an absolute deficiency, and one wants to replace those lost beta cells. So primarily, we're talking about therapies for Type 1. So what are the current therapies for Type 1 diabetes? These two images represent the two accepted clinically accepted therapies right now in 2008. On the left, you see an insulin pump. 

This is a multigenerational insulin pump. It has a censor that senses blood sugars. That's implantable. It has a delivery system that's the size of a pager. It has a reservoir for insulin. It delivers it through that small catheter directly into that subcutaneous tissue. And this is an advanced technique that has shown a lot of promise, but has become accepted in some centers as the state-of-the-art. However, it's still associated with some complications. And many patients still have imperfect blood sugar control. And in some patients, it's even more dangerous than just standard injections. 

So this is a therapy that has some advantages and some down sides. Pancreas transplantation, on the other hand is a therapy that's also effective; 90% of patients, after a pancreas transplant do not require insulin, and basically have a vacation or cure from their diabetes by one year after transplantation. And many of those pancreases continue to function for many years. Patients can remain off insulin from one or two days after the transplant, for 10 or 15 years in some cases. However, this too is associated with some side effects and potential complications. Whereas insulin pump therapy may not require major surgery, pancreas transplantation requires major surgery, and is, therefore somewhat risky, associated with bleeding complications, technical complications, return to the operating room for various corrective procedures, as well as the issues with immunosuppression. Patients, after transplant, require immunosuppression to prevent rejection. And that's associated with short-term side effects, long-term complications, including increased risk of infections and malignancy. So those are two therapies that are clinically available. Neither of them are perfectly effective. 

Although pancreas transplantation can absolutely normalize blood sugars. However, it's more risky and in summary, insulin pump therapy may be less risky, but it does not absolutely prevent metabolic complications, such as retinopathy, neuropathy, ischemic changes in the limbs, nor does it absolutely correct blood sugars. In many patients, despite their best efforts of a physician and the patient, they can still have erratic blood sugar control. So these are imperfect therapies, but it's what we have today. Patients and physicians are looking for something better. And as is often the case in medicine, we're looking for less invasive ways to do things. So a less invasive way to correct blood sugars with automatic minute-to-minute control, with the production and release of insulin, is to, in addition to a pancreas transplant is an islet cell transplant. Islets can be isolated from the pancreas, according to the diagram shown here. And this has now been done in many hundreds of patients and humans. You have a donor here. 

And the pancreas is removed and then through an enzymatic and mechanical digestion, and then a purification process, the islets can be purified and then put in an IV bag and then infused by gravity into one particular site that's used clinically, in general, is the liver. There they are carried by the portal blood flow or the liver blood flow. And they lodge or embolize in the liver and they take up function. Now this is a therapy that is used clinically, although it's still considered experimental. It's also been used in patients that have been transplanted at the University of Wisconsin. In fact, we did our last islet cell transplant just yesterday. The islet cell transplantation is proof of principle that a cell therapy can normalize blood sugars. And here we have some curves of hemoglobin A1C, which is glycosylated hemoglobin and is a measure of long-term blood sugar control. High is bad, in the 8-12 range. Six is normal. 

And you can see that after islet cell transplantation, most patients can return to normal glycemia. This is a glucose curve over approximately a week and a half in one of our transplantation patients. And you can see that after their islet cell transplant, they've had very stable blood sugars. So this has been done in many centers across the world. It is still experimental, however. There are some limitations of the therapy. There's a shortage of donors. And this is the same for pancreas transplantation. There's also islet damage that occurs during the process and the engraftment period, such that if you transplant a million islets for instance, probably 300,000 of them survive. There are scientists and investigators working on ways to improve and prevent that damage and improve engraftment. 

However, the current state-of-the-art is that there is a lot of wastage in the system. Islets, also, just like a pancreas transplant are highly immunogenic, meaning they stimulate the immune system to attack it, i.e., the rejection process. The shortage of donors and the islet damage that occurs during and after transplantation accounts for the fact that the success rates may be reasonably good in the short term; 80% of patients may come off insulin at some time after islet transplantation. However, in the long-term, by three to five years, most patients have returned to insulin therapy, unfortunately. So it's good in the short-term. It may not be durable, but science is studying that, and hoping to advance that and improve its success rate. But the fact that you have islet damage, and these come from donors, means that none of the islets that are produced are of absolute, off-the-shelf quality in all cases. So it's not quite reliable, the product that's produced during the isolation process, although it's still a state-of-the-art process. Or it's still an art, I should say. 

So with this proof of concept, human islet transplantation as a cell therapy, one can ask is an embryonic stem cell, if it can produce beta cells, or as an adult stem cell, if they can produce beta cells, can they be used as a cell source for transplantation. And in many ways Type 1 diabetes is a stereotypical disease amenable to a cell based therapy. As I told you before diabetes is due to loss of a specific functional cell type, the beta cell is damaged by autoimmunity. It's a simple protein factory, in a sense, in a very simplistic sense, so there's low anatomical complexity of the system. We don't need integration of the cells in a host environment, like a neuron would have to integrate with other neurons to take up functions after it's transplanted. Likewise, a heart cell needs to function in beating, if it's transplanted. That needs to be in synchrony with the other heart cells of the host. So there's low anatomical complexity and an absence of a need for integration in the host tissue. In addition, we have the islet transplantation as a cell based treatment proof of principle that's successful in some cases, but not in all cases. There's a shortage of donor cells. 

And as I mentioned, there's a variable quality of islets recovered or isolated from the cadaver donors. So Type 1 diabetes is a stereotypical disease. Other diseases have been thought of, of course, for stem cell therapies are Parkinson's, ALS, Alzheimer's, heart diseases and vascular diseases as well. Many others, you can pick an organ system, and it's possible that stem cell based therapies might be useful to treat diseases in that system. All right, if we are thinking about embryonic stem cells and differentiating them toward beta cells for a diabetes therapy, you have to think about development a little bit. And this is a schematic of development. And you can see here the blastocyst embryo producing, going through gastrolation and developing of the three germ layers, which the differentiated cell types present in our body are derived. And embryonic stem cells are a culture derivative of these inner cell mass cells of the blastocyst. When one wants to think about differentiating them, one wants to think about forcing them through this pathway, through developmental signals and other mechanisms. So we asked the question a number of years ago, simply, initially not to drive differentiation towards beta cells, but can ES cells enter this pathway of development, again, once they're in culture. 

So the answer to that question is yes. ES cells can become endoderm, from which the pancreas is derived, and they can become beta cells, which are one of the endocrine cell types of the pancreas. These are a number of reports now, that have appeared in the literature and data, including our own, that says that yes, human and mouse ES cells can become pancreatic beta cells and produce insulin. To tell you a little bit about our data, I wanted to just give you a brief background of pancreas development, a very brief background and tell you about one molecule called Pancreatic Duodenal Homeobox 1. PDX-1 is a homeobox transcription factor that is essential for pancreas development. In the early stages of development, when the gut tube, or uncommitted endoderm is becoming or thinking about becoming pancreas, or liver, or thyroid, or lung, or the other gut derived tissues, which are derived from the endoderm. It has to express some transcription factors that are specific to the pancreatic lineage. This is one of them. And PDX, as I said, is essential for pancreas development, in that mice who are born without it fail to develop a pancreas, and are born with neonatal diabetes. There is an analogous situation in humans that's been discovered, mutations in the PDX gene. 

So this is an important gene in human development as well. And we also know from mice experiments that PDX-1 expressing progenitor cells can give rise to all of the functional cell types of the pancreas, which I'll show you in a minute. So in green here, you can see the PDX expressing cells in the pancreatic buds. There are two buds in fact, which bud off of the endoderm tube, which is shown here across the middle, and the dorsal bud here which forms first and the ventral bud here. So PDX are these green cells highly expressed in the pancreatic buds, fated to become pancreas. Now, PDX is also important for insulin production in the adult islet. Shown here on the right is a human islet isolated from a cadaver donor. And it shows in red the PDX expressing cells. And so PDX transactivates the insulin locus and therefore is very important in the expression and production of insulin. Now the adult pancreas in fact, has two functions other than blood sugar control for which the endocrine cells are primarily responsible, but it also has functions to secrete enzymes into the GI tract to help us digest food and to produce bicarbonate to buffer stomach acid, the function of which is taken up by the acinar cells and the exocrine cells, enductal cells. 

So the islets only make up one percent of the pancreas. Therefore, it's a fairly rare cell type in our body. The pancreas is not a particularly large organ either. An interesting fact that may come into play later is that endocrine function of the pancreas is not necessary during development to control the embryo or fetus' blood sugars. In fact, the mother does that in utero for the fetus to promote growth. But the blood sugar control in the embryo does not require the presence of insulin cells during development. So we wanted to look at, can embryonic stem cells differentiate into beta cells. And a number of years ago, some scientists in my lab set out to test that hypothesis question and spontaneously differentiated embryonic stem cells in culture, first through an embryoid body phase in suspension culture, where they make these embryo-like structures, and then with subsequent differentiation in the presence of serum, but without any added growth factors. And what we found was that, in fact, progenitor cells do appear in these cultures marked by PDX expression in red here. And within those clusters, emerge a subset of cells that express some of the endocrine hormones, insulin and glucagon. 

At a later stage, some of those PDX expressing cells express insulin in the cytoplasm in granules and they also continue to express PDX. And, in fact, many of them co-express the marker C peptide, which is a marker of endogenous production of insulin, and is denovo production, and is a critical marker, and demonstrates bona fide production of insulin. We found that in mouse ES cells. In human ES cells as well, we could get PDX insulin co-expressing cells. That also expressed C peptide. And these three markers together are highly reminiscent of an adult beta cell. Now, we've done this many times and others have reproduced this work. However, the observation was made that it seems that there were a large number of PDX expressing cells that could emerge in these cultures, but very few endocrine producing cells. This image on the left is a large patch of PDX expressing cells. But however, as you can see, only two cells within that cluster are also expressing insulin. 

We counted cells in many cultures of different cell types, insulin, glucagon, etc. We saw that there were very few endocrine expressing cells within the cultures. In the range of 10-100 cells expressing some of these hormones out of several million cells. So there were quite few endocrine cells. And, in fact, there were too few endocrine cells to really test their function, because the assays used could be inhibited by the other cell types there that might take up insulin or affect the insulin output. Because there are few numbers, it's been difficult to study the function of the cells. However, again, in summary we do know that mouse and human ES cells are capable of differentiating into pancreatic progenitors, and that islet hormone expressing cells can be produced, and that the phenotype of these cells is very reminiscent of adult beta cells. However, we also learned, and I don't have time to show you, that serum inhibits the production of the cells. And if you withdraw serum at a later stage, and reduce the serum component in the cultures, you can enhance the endocrine differentiation. So how do we then, if we have a few endocrine cells and we want to make more endocrine cells, if we're going to think about generating millions of cells for millions of patients. 

How do we direct the differentiation of embryonic stem cells into beta cells and only beta cells? How do we get rid of those other cell types? Well, in these cultures, I didn't show you, but there are probably other cell types within the cultures. We're not interested in getting those. We're interested in enriching for a cell type of the pancreatic lineages, and that could be either a differentiated cell type represented in green here by the terminally differentiated beta cells that are fully functional. Or it could represent a precursor cell at a variety of stages shown there in the other colors, which might then differentiate then along that lineage, but they'd be relatively committed and therefore generate primarily endocrine cells, or at least pancreatic cell types. So how do we get to this type of approach? How can we direct the differentiation in culture? Well I think it's important to think about, and what the field has come around to is that we need to take clues from development. And we need to take clues then, from developmental studies in lower organisms, such as mice, and zebra fish, and other lower organisms. 

So we need to learn from those mouse and zebra fish studies and apply what we learn there to human ES cells and culture. However, we know that human ES cells don't necessarily behave like mouse ES cells and don't respond to the same growth factors. The mechanisms controlling pancreas development in mice might not be the same as pancreas development in humans, although there are certainly some similarities. So the same growth factor that works for mice that's been discovered in a series of studies in lower organisms, might not function the same way in human ES cells and culture. It's certainly worth testing, but we have to be wary of the fact that we might come up empty or be misled. So some degree of empiricism is probably necessary in a series of studies to balance rational approach, take clues from development and empiricism, just a shotgun approach in figuring out what are the growth factors or combinations that might promote differentiation towards a pancreatic cell type. Keeping that in mind, if we want to investigate a large number of developmentally relevant growth factors, or unknown growth factors, looking at multiple different combinations and concentrations, we're talking about a large array of experiments. So we and others are working along those lines, combining both a rational approach and an empirical approach. But we're also doing some other things. So in addition to looking at what are the intercellular signaling molecules that are elaborated by cells and tissues during development to drive and induce differentiation to various cell types, we're also looking at transcription factor directed differentiation. 

That is, can the over expression of a particular key transcription factor, like PDX in embryonic stem cells or subsequent transcription factors are necessary for later stages, can those be used to drive differentiation. We're also looking at ways of purifying cells in the cultures by pulling out the cell type of interest using magnetic bead sorting or flow cytometry. What I'm going to tell you about today is mostly focused on developmental signals. So there's been a series of studies in the literature that address this. There are some very elegant studies and represent the state-of-the-art. So this is one study where a series of investigators in Sweden said, we don't know the signals. Let's take the embryonic pancreas and mix that with ES cells; and maybe there will be something elaborated in the embryonic pancreas, an unknown signal that's produced that will direct differentiation. So this group took human embryonic stem cell cultures that were partly differentiated. In a nonspecific fashion, they collected certain areas of those cultures that were relatively enriched in or pancreatic endoderm and expressed PDX, but were not able to express insulin in culture for those investigators. And they took those derivatives and co-transplanted them into mice with a mouse embryonic pancreas. 

So what did the pancreatic signals do? The pancreatic signals appeared to stimulate the differentiation of the human ES cells into insulin producing cells and other cell types. So we have here a series of immunostains for markers of insulin, a human nuclear factor. And we can see that there are insulin cells that are detected in this tissue underneath the kidney capsule that are also expressing a marker for a human nucleus, so they are human derived rather than mouse derived. Similarly for glucagon and similarly for amylase. You can see that there are other cell types within this tissue that also have a human nuclear marker, and are derived from human ES cells that are not expressing these markers, so it's possible that there are other cell types here as well. But nonetheless, they demonstrated that there's some signal within the mouse embryonic pancreas that seems to drive differentiation towards an endocrine cell type. And as their control, they used a human -- and that did not have the same effect. So this was effect specific for the embryonic pancreatic tissue. So that suggests that there's some differentiation promoting factor, some signal in this early stage pancreas that is promoting differentiation. 

Other scientists have gone a bit further and have looked at specific growth factors and a cocktail of growth factors. These growth factors were tested also based on a large volume of developmental literature and a specific factor that has been shown now and reproduced in several labs to promote differentiation towards endoderm. And then with subsequent additional growth factors toward a pancreatic lineage, they showed that activin was an important growth factor in this. Activin is a factor that stimulates nodal signaling. By promoting nodal signaling, they can promote definitive endoderm differentiation. So their culture protocol is shown here in yellow with activin and some of these other factors shown here, including FGF10 fibroblast growth factors, cyclopamine, which inhibits sonic hedgehog signaling, retonoic acid, and a number of other factors in specific sequence at specific time points appear to optimally promote endoderm, then pancreas, then endocrine differentiation. With such a protocol, they were able to show that insulin producing cells were enhanced and were dependent on a high dose of activin. I should say that although it's clear that activin is important, the absolute relevance and importance of some of these other growth factors that they used in this culture protocol has not yet been determined. So with activin, they showed that high doses of activin and induction of nodal signaling can promote endocrine differentiation, and specifically beta cells. And here is a culture in the center treated with high doses of activin. 

It shows in the green cells that there are more endocrine cells in the cytology panel below that shows approximately 7% of cells are producing insulin. So this shows that activin and nodal signaling are important for endoderm differentiation. You can modestly enhance the prevalence of endocrine or insulin producing cells in the cultures derived from human ES cells. However, this is still not a purified culture. And other functional studies they did showed that the cells do not behave necessarily like mature beta cells in response to glucose. In response to high glucose, they do not release a large amount of insulin, although they do express it. That's really the current state of the field at the moment. And where do we have to go to next to think about a cell therapy? What do patients want? They want a relatively purified population. 

It doesn't have to be absolutely purified, but it has to be at least highly enriched. We need to think about, can we enhance that differentiation with matrices and tissue engineering constructs, additional growth factors, a degree of empiricism and rational approaches. And then we need to really show that these cells function well in vitro and in vivo. For that, there are a number of animal models of diabetes that are available. There is chemically induced diabetes, that is established model systems in mice, and rats, and primates. In addition, there are autoimmune diabetes models in mice and rats. So those would be model systems to test this cell therapy. And not until we develop a cell therapy that has a robust ability to restore normal glycemia or good blood sugar control in animals with diabetes, can it then move forward into the clinic. So as one thinks about delivering this to the clinic, again, we have to think about balancing efficacy and safety. And before the FDA or any regulatory agency would approve an initial phase one clinical trial, there would have to be a series of tests to look at efficacy and safety. 

From the safety point of view, we want to make sure that these cells don't develop tumors, or malignancies, or are transformed, or are unstable genetically after transplantation. That's an important consideration. We also want to make sure that there's an absence of transmission of unusual infectious agents that might be carried along in the cell line, unrecognized in culture. So we'll have to go through a series of tests. Most of the ES cells to date do not have any infectious agents or any unusual viruses. They have been tested extensively, but they'll need to be tested again immediately prior to going into cell therapies. We want to avoid some cell based adverse events or adverse effects. In islet transplantation, when you put islets in the liver, they cause focal fat deposition in the liver, it turns out. So that might not be the best site. And we don't know what that focal fat deposition would have in terms of the long-term effect, whether that would be a problem. 

Unlikely to be a problem, in fact, but there are other cell therapies like delivering cells into the heart that might cause some problems, namely, arrhythmias. Neural cells put into certain areas might create problems with integration and synapses with the wrong cell types, which might be problematic. So all those things will need to be tested and optimized before advancing to later stage clinical trials. So those are some of the hurdles and the challenges moving to cell therapies. There's a lot more to be done in the diabetes field. The earliest trials of human ES cells are phase one trials that are just getting underway now for spinal injury with some investigators in California. And we're anxiously awaiting the early results of those. So, moving on to the next segment of the talk, we've covered current therapies for diabetes and what's going on in my lab, as well as what's going on in the literature and the current status of the field. I'd like to move into the next part of the talk, and talk about generating genetically matched human ES cell lines. Of what is the benefit of that? 

Why are we interested in that? This will deal with a little bit the ethical aspects of somatic cell nuclear transfer. So one's interested in generating genetically matched human ES cell lines, because one might get around the issue of histo-incompatibility and rejection. And therefore a patient who gets this therapy might not reject the cells without immunosuppression and immunosuppressive medications, or anti-rejection medicines might not be required. Is a patient that gets cell therapy from a human ES cell possibly in the future, would it require a genetically matched cell line to advance to clinical therapy? I would say probably not, although there are some advantages and some applications. But I'll mention a little later, there may not be as much of an advantage in the application of diabetes. I'll talk a little bit about that in a little while. But first, what are the strategies that people have come up with as ways to generate histocompatible cells for transplantation? Well, histocompatibility is largely determined by the HLA or human leukocyte antigens that are present in all somatic cells. 

Those are the transplantation antigens, so one could think about generating cell banks, or HLA specific cell banks of ES cells, much like a bone marrow bank. However, that hasn't really solved the problem of histo-incompatibility with bone marrow transplantation, so you'd need millions, and possibly billions of different combinations of different ES cells to really establish a bank that was useful in all patients. One could think about deleting or inserting these HLA genes that are specific for the donor, or are specific for the prospective recipient and deleting the ones from the donor or inserting new ones that are specific for the prospective recipient. That hasn't been accomplished yet, but I'm confident that it might be in the future. And I'm sure some investigators are working on that. Another potential way to do this is to think about generating hematopoietic stem cells from ES cells, transplanting those into a patient, let's say, with diabetes, and using the human ES cell line to make those hematopoietic stem cells. Creating a state of chimerism, such that the recipient no longer is going to react or reject the cells from that particular human ES cell line. And you could make a beta cell perhaps from that same line, and therefore they wouldn't reject it and come back with a therapy for a beta cell. So that's a possibility, but I think there's a lot of work that needs to be done there. Nuclear reprogramming has been from very earliest days within a year or so after Jamie Thomson discovered or isolated human ES cells, nuclear reprogramming was thought as a way to generate genetically identical human ES cells. 

And that's the idea of therapeutic cloning. So therapeutic cloning, though, is not reproductive cloning. It is not likely to have the disastrous consequences that some believe, yet there are ethical challenges and issues with it that many have had. So what are we talking about with therapeutic cloning? Therapeutic cloning can be described by this diagram here. And what potentially would be involved, is you have a patient that's a prospective recipient, or potential recipient of a cell therapy. You take a skin cell or a somatic cell from that individual, and then go through the methodology used to clone Dolly, you would recover the nucleus from that cell and genetic material, get an oocyte from a third party individual, usually a volunteer, a woman volunteer, who has allowed recovery of oocytes. Then one removes the nucleus, inserts the donor nucleus, go through a period of development in vitro, generate an embryo. And then, from that blastocyst, generate or isolate the inner cell mass and generate the human embryonic stem cell line for the purposes of differentiating those cells in vitro, to generate a cell type that would be useful for that patient. And then transplant those cells back into the patient. 

So as I said, there are a number of ethical issues with this. However, first I just want to tell you that somatic cell nuclear transfer derived ES cells do show some evidence of histocompatibility. Although some people feel that, or there is evidence that there may be some histo-incompatible component to somatic cell nuclear transfer derived human ES cells. That would still require anti-rejection medicines if transplanted. And the reason that's suspected is that the clones might inherit mitochondrial DNA from the oocyte, and therefore might present antigens, either directly or indirectly to the host immune system that are foreign to that nuclear donor or prospective recipient. And therefore, they may not be purely incompatible. But studies that have been done so far, although not a series of perfect studies, but the studies that have been done so far in mice, pigs and cows, suggest that nuclear transfer derived tissues are genetically compatible, or histocompatible, with the nuclear donor. So it's likely that they would require very little immunosuppression, or none at all, if such a thing could be applied to humans. Another thing that I wanted to tell you is that when one thinks about histocompatible tissues derived from somatic cell nuclear transfer derived human ES cells, they're useful, potentially for cell therapies. But they're also potentially useful for other types of therapies, such as is outlined here in a very elegant experiment done by a group of scientists in Boston, which worked toward correcting a genetic defect in mice. 

So, such a therapy could be applied, not only to a patient that needs a new cells because of deficiency or organ failure, but they could also be applied to patients that have genetic defects in the following way. So what these scientists did is they had an immunodeficient mouse, up here in the upper part of this diagram. They took some skin cells or fibroblasts from that animal, cultured them. And then used somatic cell nuclear transfer to take the nucleus from those skin cells that are syngeneic to that animal, and then generate embryonic stem cell, in this case, a mouse embryonic stem cell from those skin cells. And those skin cells, of course, were genetically deficient. They had this genetic defect of the RAG2 deficiency, which made them immuno deficient. And because they have this RAG2 deficiency, they are unable to make T and B cells. And the animals don't have an immune system. So they set about repairing this deficiency by putting a wildtype gene into these ES cells and correcting the genetic defect with -- recombination using a wildtype locus. Then they took those genetically corrected ES cells, differentiated them into embryoid bodies, then over expressed another gene, which drove hematopoietic cell development or blood cell development. 

Then they took those blood cells and put them back into the immunodeficient animal. And new syngeneic blood cells were formed in that animal, partially correcting the immune deficiency. So this is an example of how a genetic defect, an isolated genetic defect could be corrected by a combination of gene therapy and stem cell therapy, and somatic cell nuclear transfer. I think a very elegant experiment. Why do people have ethical problems with somatic cell nuclear transfer? Well, it all hinges around these two stages. The ethical challenges are, the main probable is that one is generating a embryo for the sole purpose of research or destruction. And that, in essence, is one of the major problems with somatic cell nuclear therapy. Another major issue, another ethical issue is that in order to get a large number, one needs to get a large number of oocytes to make this happen. The process, as we currently know it, is extremely inefficient. 

There's a lot of wastage such that a large number of oocytes are necessary to do one experiment, to generate one embryonic stem cell line that might be genetically matched by somatic cell nuclear therapy. So where does one get a lot of oocytes? One gets them from women volunteers who have been hyperovulated or hyperstimulated with hormones, and then doing an ovary recovery procedure, which is although not extremely risky, and is done routinely for in vitro fertilization procedures, does require a procedure, invasion of the patient, and has some potential risks. It's certainly not without risk. It's associated with some pain and discomfort, as well. So if one thinks about doing that, one has to put many women simultaneously through that process. And that's what was done in Korea a number of years ago. That was considered ethically very questionable in many western countries. Nonetheless, we need to improve somatic cell nuclear transfer so that perhaps one can do this without an oocytes or the volume of oocytes that are necessary, perhaps the ooplasm might be used. However, one still has issues with generating a human blastocyst or embryo for the sole purposes of destruction. 

Most cases, ethical and political debates about science come after a major discovery. It's fairly unique that the ethical and political debate surrounding somatic cell nuclear transfer have generated a whole new industry of science, and that is to develop innovative methods for creating histocompatible matched human ES cell lines without destroying an embryo, or without needing a large number of oocytes. So since a few years ago, a number of approaches have been put forth. Really, a whole cottage industry has arisen around this. These are some of the methods that have been published for generating histocompatible human ES cell lines, without the process of producing a human embryo and without some of the ethical issues. I won't go through all of these in a lot of detail, but one of them is an altered cell nuclear transfer. The idea here is to generate an embryo that cannot implant. The way to do that, the way the scientists did that is to knock out the CDX2 gene, which is required for trophectoderm or placental development. And, therefore, although you've got a functional intercell mass from which one could derive ES cells, you would not get implantation of that embryo. However, one still produces an embryo earmarked for destruction, albeit an abnormal embryo in this method. 

In addition, it still requires a large number of oocytes. So in my mind, it does not get around the major ethical issues. Another way to think about doing this is to not destroy the embryo. And what one can do, as is commonly done in prenatal genetic diagnosis, associated with in vitro fertilization, when you generate the in vitro fertilized embryo in culture, you can then sample it and take out one cell of that embryo or one blastomere, and then test that for the genetic defect that might run in the family of that family that's having the in vitro fertilization. So you can keep embryos that do not have that genetic defect and then implant those. So some scientists went ahead and did that, and showed that you could generate a human ES cell at least transiently, that has some phenotypes of a human ES cell, from a single cell or a blastomere, and that does not destroy the embryo necessarily. Although in this publication, the embryos were not implanted and did not go on to have normal development. So that's a little bit of an unknown, yet it does show the principle that possibly one could develop human ES cells of a single cell of the embryo without destroying the embryo. Still, a large number of oocytes at the current rate of producing those embryos in vitro would be necessary to do that on a wide or large scale. Another potential method is parthenogenetically derived embryonic stem cells. 

Those were recently described for human ES cells, and previously in mouse and primate ES cells. Parthenogenesis is the process of activating development without a sperm, that is, auto-activation in culture of an oocyte. One of the down sides here is that the oocyte, or parthenogenetically derived human ES cells would be genetically identical only to a woman. So it would be a therapy one could develop cells, then, only for females and not for males. The last method, just recently described here by Jamie Thomson and Dr. Yu at the University of Wisconsin is induced pluripotent stem cells. I'll go into that methodology, or technology, in a little bit more detail. Again, as a potentially useful way for generating genetically matched human ES cell lines. So what was the concept here is that one can take a somatic cell, a skin cell for instance, as they did in their publication, from an adult. In their publication, they used some neonatal skin cells and some foreskin cell lines rather than adult cell type. 

We'll get into the differences there. But taking that somatic cell or skin cell from an adult, and then modifying it by over-expressing four key pluripotency genes in that somatic cell, at least transiently, showing transient expression and then that would promote dedifferentiation or reprogramming towards a pluripotent stem cell like phenotype. The genes they used were Sox-2, Oct-4, Nanog and Lin-28. Others in mice and humans have shown some other genes may play a role. The minimal set of genes has not actually been determined yet, But the IPS or induced pluripotent stem cells that were generated by this group are really strikingly similar to human embryonic stem cells. They have high telomerase activity. They express some of the phenotypic markers of human ES cells. They can differentiate into all the germ layers and teratomas in mice. When compared to human ES cell lines by a study looking at hundreds and thousands of genes, they were virtually identical. So their study is shown here. 

This was published in Science this past year, approximately six months ago. They used these four genes and over expressed them in these foreskin cell line, IMR90 shown there in panel B, in this image taken from their publication. And you can see that in the cells that had all four genes expressed, they had the largest number of IPS colonies that were produced. And they sequentially subtracted one of each individual genes and showed that the two or three key factors were really Oct-4, Sox-2 and Nanog, which are common pluripotency genes that have been described to be expressed in human ES cells and in mouse ES cells. So you see that we can induce a pluripotent state by over-expressing these genes and somatic cells. So the question next is, will these IPS cells be useful clinically in the future? I think the question remains-- the answer to that question still remains as we don't know. But many are excited by this development, though it still does not obviate the possible utility of wildtype human ES cells and improvements in somatic cell nuclear transfer or therapeutic cloning, primarily because these are genetically re-modified cells. So one of the issues with IPS cells is they did this with a neonatal and foreskin fibroblast cells, and a foreskin fibroblast cell line. When one wants to think about therapy, we need somatic cells from an adult patient, an aged patient, perhaps. 

Someone who has cells that are undergoing senescence. So what the right cells are, and whether it would be possible for the skin cells from an adult is not exactly clear at the moment. But it will be an important thing to test. These cells are also highly genetically manipulated, and have lentivirus inserted into their genome. So because of that, there's potential for transmission of virus, and some of the problems with gene therapy, and some of the risks of gene therapy associated potentially with transplanting these cells. So it will be very important to find other ways to insert and express these genes. And it will also be important to test these cells for safety prior to clinical therapy. The other aspect is a more mundane question, but a simple question. Because they have the insertion of these genes in a certain area of the genome, have that insertion or that process disrupted differentiation potential for differentiation into differentiated cell types? That is, will it prevent differentiation or lead to incomplete differentiation? 

And we don't know that. But that is potentially surmountable by generating multiple cell types from a somatic cell for that particular patient and then testing them. But nonetheless, that will be important to test whether they have the same differentiation potential as wild type human ES cells. So in the last couple minutes, I just want to talk about whether genetically matched human ES cells will be useful clinically, for specifically patients with diabetes. And the reason why that might be a question hinges around the autoimmunity issue. Patients with Type 1 diabetes, who might be recipients of this therapy have autoimmunity. They have lifelong autoimmunity, meaning that they still retain memory T cells that are poised to attack self-cells, if new self-cells could or would be transplanted. That is, many years after transplantation, they still have active autoimmunity as part of their immune make-up. The reason why we know that is from mouse studies, and from some human experiments in the early days of pancreas transplantation. In the early days of pancreas transplantation, before immunosuppression, some segmental pancreas transplants, or partial pancreas transplants, were done between genetically identical twins that were discordant for diabetes incidence. 

One had diabetes, one did not. But they were genetically identical twins. The transplants were technically successful, again this was before the era of immunosuppression, so the patients were not immunosuppressed. The patients remained off insulin for several months. But within three months, or four months, the patients returned to insulin therapy and lost insulin secretion. And when those grafts were looked at, they had the pathological findings of recurrent autoimmunity. There was immunological attack on those beta cells. So it's clear that autoimmunity may destroy self-beta cells if allowed to develop from an embryonic stem cell or transplanted from an embryonic stem cell in a particular patient. However, we also know that in a small group, a series of patients done at the University of Chicago, that twin transplants with low amounts of immunosuppression, less than is needed for normal organ transplantation can be successful. That is, only a small amount of immunosuppression may prevent that autoimmunity attack from attacking a new pancreas, but not as much as is necessary for a typical organ transplant. 

So I think I'll stop there. We've covered a lot of things. I hope you got something out of that. I just acknowledged some of the scientists in my lab who have done some of the work I showed you, showed there in the upper right, and some of the people that have helped us with the research, some of the studies I have not showed you. And our funding agencies for which we are very grateful for their continued support. So I'll end there. I'd be happy to take any questions. 

- For the islet transplantation, what does that accomplish that pancreas transplantation doesn't? 

- It accomplishes all the same things, hopefully, a pancreas transplantation does. However, it's a less invasive procedure. 

The patient isn't under the knife for six hours. There's no big scar. There's less risk of bleeding. So it's a much less invasive procedure, and perceived to be a little bit more safe in that regard. However, on the down side, at the current state of the therapy, it's less durable than pancreas transplantation. Whereas islet cell transplant recipients may achieve insulin independence for a number of years, most return to requiring insulin within five years, whereas that's not the case with the pancreas transplantation. Those patients generally have preserved function, the majority of patients have preserved function for more than five years. 

- You talked about Type 1 and Type 2 diabetes. For Type 2, it is obviously much more prevalent. But were you saying that that's basically, Type 2 diabetes is basically that you become resistant and your receptors don't really work to bind insulin anymore? 

- That's generally felt to be the main mechanism. Although it's not really known, and it's clearly multifactorial and takes on different forms in different patients. So scientists are still studying what exactly are the mechanisms of that insulin resistance, or the causes of Type 2 diabetes. However, most patients, or many patients do end up having a relative deficiency of insulin as time goes on. And many patients, although right after their diagnosis of Type 2 diabetes, can be treated with diet or exercise or pills, eventually go on to require insulin therapy. So there are some similarities in terms of the end result. So when people think, however, about stem cell therapy for diabetes, for the most part, they're focusing on Type 1 diabetes. And if a therapy of any sort might be found to be effective for Type 1 diabetes, then I would envision people starting to apply it to a subset of patients with Type 2 diabetes. But one would require a lot more production of insulin, a more robust production of insulin, because that potential for insulin resistance. 

- Focus on maybe, finding a way to re-express the receptors or you know, find out exactly what's wrong with them, because I feel like since it's so much more prevalent, like everything I hear seems to do with Type 1. 

- Well there's a lot of research going on for Type 2 diabetes. Much of the focus of the American Diabetes Association, in fact, is on Type 2 diabetes. Basic science, clinical aspects, a variety of things. Whereas the Juvenile Diabetes Research Foundation primarily focuses on Type 1 diabetes, but not exclusively. So there's a kind of separation there, if you will. But there's a lot of work going on with looking at the nature of the disease of Type 2.