Hello. My name is Elaine Fuchs. I'm a professor at The Rockefeller University in New York City, and I'm an investigator of the Howard Hughes Medical Institute. And today I'd like to tell you about stem cells, about their biology and about their promise for regenerative medicine. So, let's go to the very beginnings of stem cell biology. The word stem cell itself is a relatively recent word. It was only in 1877 that Ernst Haeckel, a German scientist, coined the word stammzelle, or stem cell, a fertilized egg that gives rise to all the cells in the body. That was the initial definition of stem cells. And then the word stem cell became popularized by EB Wilson, a famous cell biologist in the late 1800s, who wrote about it in one of his books. So then, it was about 30 years later when this person, Alexander Maximow, was a... he was a Russian scientist. He fled the Russian Revolution and joined the ranks of the University of Chicago. He was a cytologist. And just looking underneath the microscope at bone marrow cells, he saw some cells that seemed undifferentiated. And he predicted, at that time, that perhaps the bone marrow contained a cell that can give rise to all the different hematopoietic cell lineages. And that was the first notion that stem cells might not simply be restricted to embryonic stem cells, the cells that can give rise to all the cells of our body, but actually that different tissues might actually contain stem cells, adult tissue stem cells that had a more restricted potential, being, in this case, able to give rise to the hematopoietic lineages. But that was just a postulate. And we now have to fast-forward some 60 years later, when these two individuals, Ernest McCullough and James Till, back in 1961, did the remarkable experiment that demonstrated the existence of adult tissue stem cells. They took a laboratory animal, and they irradiated its bone marrow. And then they took a healthy bone marrow, and one by one, put in the cells from the healthy bone marrow until they found a single cell that could reconstitute the entire hematopoietic cell lineages of the bone marrow. And that was the first demonstration that tissue stem cells existed within adult... the adult. And that really opened the door for some really exciting biology to come. So, let's fast-forward now, again, to... another 15 years or so, after the pioneering work of Till and McCullough, and now it wasn't until the mid 1970s when this person, Howard Green, who recently passed away... he was then at MIT, and he took a piece of human skin, and was able to culture and passage cells from the piece of human skin, and he could passage them endlessly under conditions where the cells could still make tissue. This established the existence of the ability to culture adult tissue stem cells, in this case stem cells coming from the skin. So, let's take a look at some of the experiments that were done in those early days. It was possible to effectively culture the embryo, in this case the adult skin stem cells, and passage those cells. And you see in the middle panel the chromosomes, the dark chromosomes within the cells. These cells were rapidly dividing in culture. And if you look at the bottom panel, what you see is actually reconstituted skin, in this case reconstituted epidermis, that was generated entirely from these single stem cells that were cultured. In fact, we didn't call them stem cells at the time. We called them keratinocytes, epidermal keratinocytes. But effectively, these were tissue stem cells. So, Howard Green very soon recognized the power of the technology that he had just developed for the treatment, in this case, of burn patients. He took skin from the unaffected area of a burn patient. And then cultured those cells, grew sheets of epidermis, and then grafted those cells onto the burned area of the patient. Same patient, just taking good skin and expanding the cells in culture. And the reason why that worked is that you could use growth factors and nutrients that allowed the cells to grow fast, generate sheets of skin, in order to be able to be used for burn therapy. In those early days, Dr. Green was able to effectively cure or treat patients that had burns up to 95% of the total surface of their skin. Remarkable. All generated from a few stem cells that were endlessly passaged in skin. And we've learned a very important lesson from this. Effectively, now, we have 35 years of success of the use of stem cells in a clinical setting. And I think what one of the most remarkable aspects of that is that those patients whose skin was almost entirely reconstituted from stem cell therapy, if you will, never showed signs of abnormalities, never showed signs of genetic alterations in the skin, or of cancer, that might give us an idea or a notion that culturing those cells long-term might be deleterious for the cells in some way. Remarkably, the skin of those patients still produced healthy epidermis. This ability to culture stem cells in the... in the laboratory turned out to be the foundations for embryonic stem cell research, which was going to come another five years after Howard Green's pioneering work. And there, the breakthrough was really the realization that stem cells cannot survive on their own. They require other cells for their sustenance. And what Howard Green did in his breakthrough work to culture embryonic... to culture human skin stem cells was basically the realization that epidermal stem cells rely upon dermal cells in order for them to grow and be maintained. So, he used what is called a fibroblast feeder layer, an irradiated layer of fibroblasts, in order to be able to grow the human epidermal stem cells on top of it. And that research then served as, also, the foundation for embryonic stem cell culture, which also succeeded because of the addition of the fibroblast feeder layer. So, what are stem cells, then? Let's get down to the basics and the definition. Basically, stem cells are cells that are able to make an animal, if we're talking about embryonic stem cells. The embryonic stem cells have all the power to reconstitute an entire animal. Stem cells have the ability to make and replenish a tissue, if we're talking about adult stem cells. So, adult stem cells replenish the tissue. Embryonic stem cells have the capacity to make the animal. And effectively, what we know about stem cells -- and particularly stem cells of adult tissues -- is that they have the ability to be able to generate not only self -- so, that means that they can self-renew -- but they also have the ability to produce short-lived progenitors. These are often rapidly proliferating progenitors that then go on to make the differentiated cells of a tissue. So, in this case, it would be the epidermis. And it's often, then, the short-lived progenitor cells, or these transit-amplifying cells, that have the ability to generate the bulk of the tissue. The stem cells only divide relatively sparingly in most situations. So, what are the differences, then, between embryonic and adult stem cells? Embryonic stem cells are pluripotent, and they can generate all of the cells of the animal except for the support cells or the placenta. In the case of adult stem cells, these are usually multipotent, and can generate several different tissues of our body. Sometimes, they're unipotent, and can only generate a single tissue or differentiated cell of our body. So, in the first part of this series of lectures, I'd like to concentrate on embryonic stem cells, on induced pluripotent stem cells, and on their applications to basic science, medicine, and the pharmaceutical industry. So, where do embryonic stem cells come from? If we go to the early developing embryo, the blastocyst, that's just a few days old, it consists of several hundred cells at the most. And at this point, the blastocyst has a group of cells which are called inner cell mass cells. And those are the cells that develop into the fetus, or the embryo. The surrounding cells -- this single layer of cells that surround the inner cell mass -- are called the trophectoderm. The trophectoderm is going to develop into the fetal support tissue for the embryo. So, those cells do not give rise to any part of the embryo, but basically they're necessary in the womb to be able to develop the fetus. So, scientists learned that it's possible to take the inner cell mass cells, then, and put those cells into a petri dish on a fibroblast feeder layer, or with the appropriate nutrients that support the inner cell mass cells. And now they can culture those cells. That's the culture that are called cultures of embryonic stem cells. That's where embryonic stem cells come from. And thereafter, those embryonic stem cells can be passaged effectively endlessly, just like, as I mentioned to you, epidermal stem cells were initially. And these cells, now, though, have much more power than the cells of the skin. So, why is this important? Well, let's think about the future for regenerative medicine. There are many different cell types for which we don't have very much information, and for which there are many different types of human genetic diseases. For nerve cells, we could generate, potentially, nerve cells, possibly for the treatment of Parkinson's disease or Alzheimer's disease. Scientists are excited about the ability to generate nerve cells for the treatment of spinal cord injuries. Scientists are excited about the ability to culture pancreatic islet cells for the possible treatment of diabetes. Muscle cells for muscular dystrophy, and sudden death heart syndrome. Immune cells for immunodeficiency disorders. Or, thinking about the treatment of cancers, it's possible to generate the stem cells that derive or produce the cancers, and that's also possible in culture. And scientists are very interested in utilizing these types of technologies in order to be able to understand more about the biology. So, it's not just the treatment of patients, but it's really understanding the biology of how these cells develop and what gives them their properties, to be able to ultimately come up with new cures and treatments for various different types of diseases. And that involves not only the clinician and the scientists, but also the pharmaceutical industry or the biotechnology industry. So, let's take a look at a couple of examples of what embryonic stem cells can do. In this case, human embryonic stem cells were treated with particular growth factors that allowed these cells to differentiate into muscle cells. And now, looking at the embryoid bodies -- and this is the work of Gordon Keller and his colleagues at the University of Toronto -- you can see that the cells undergo this beating feature. And you can see a few examples of that, of the cells basically in the culture dish, now, undergoing the contractions that heart muscle cells do. And interestingly, they undergo them in a synchronized fashion, in the way that our heart cells do in our body. So, in one of the very earliest experiments, done, now, a decade ago, scientists took embryonic stem cells and differentiated them into neurons, and used them to treat paralysis of a rat, in this case. And here was the rat before the injection of embryonic stem cell... stem cells differentiated into spinal cord neurons. And here is the rat after the treatment. Now, these were early studies. Scientists have gotten much better at these sorts of treatments. But again, these kinds of treatments are what ultimately will be necessary in order to move the field forward, and in order to think about therapies for the future. So, let's now go to 2017. What are scientists doing now? Well, now it's possible to differentiate the embryonic stem cells not only into a neuron but into specific types of neurons. There are hundreds of different types of neurons in the body. In this case, they took human embryonic stem cell-derived cortical neurons and grafted them into a mouse cortex. So, the importance of this work I'll describe in a moment. But these are the studies of Lorenz Studer, a scientist and one of my colleagues across the street at Sloan Kettering Cancer Institute. So in this case, the cells that you see in white, now, are human cortical neurons. And the slice of tissue that you're looking at is a mouse brain. Three months after the graft. And even later after the graft. Six months after the graft, there are still human cortical neurons that are existing in the mouse brain. And scientists are currently trying to understand, are they working in the right way? Are they behaving as cortical neurons? And this is important for the treatment of a whole number of different disorders. And it allows scientists to learn more about the biology of Alzheimer's, of autism, of schizophrenia, and many other disorders of the cortex of our brain. So, what are the ethical issues involved? Why is there so much controversy about embryonic stem cell research? Well, on the one hand, it's important to know that human embryonic stem cells are cultured from fertilized eggs. And a religious argument would be against the culturing of fertilized eggs for anything other than, basically, making a human being. The counterpoint, however, is that human blastocysts and embryonic stem cells can be created with only egg and sperm. And they can be created in culture, with no womb required. Additionally, the technology that I've just described to you involves the exact same steps as an in vitro fertilization. So, I think these types of technologies are technologies that have made great advances. That said, there is the concern, the ethical concern, by some, that... that... that the use of fertilized eggs for in vitro fertilization is one thing, but the use for research is another. So, embryonic... embryos from in vitro fertilization are indeed often now discarded or frozen or unused. And so, again, this issue is, well, the embryos could be implanted into a woman who would like to have a child, and therefore it would be unethical to use those embryos for anything else. On the other hand, the counterpoint to this argument is that it's still that many embryos from in vitro fertilization are discarded, are unused. And the other aspect is that they could be used for scientists to be able to learn how to generate neurons, pancreatic islet cells, muscle cells, for treating human disorders and injuries. So, what are the hurdles -- beyond the ethical concerns -- of what scientists would need to do to be able to adapt this technology and take it one step further to the clinics? Well, the first big step is that embryonic stem cells that are used to generate neurons and then implanted into, for instance, an Alzheimer's patient would basically be rejected from the body. Because the immune system of your body recognizes foreign cells, and it eliminates them. And so, what were the next steps that scientists came up with to generate stem cells that would overcome the problem of immune rejection? In this particular technology, this is the technology known as nuclear transfer, in which case you take an unfertilized oocyte, now... in the previous version we were taking fertilized oocytes... fertilized oocytes, or eggs. Here, we're taking an unfertilized oocyte, removing and discarding the nucleus of an oocyte, and then replacing it with a nucleus of an adult somatic cell, such as a skin cell. This technology was actually technology that was performed all the way back in 1962 by John Gurdon, who recently won the Nobel Prize for these pioneering studies. He used Xenopus eggs and basically modified the eggs in this way, using nuclear transfer, in order to produce a hybrid cell, now -- a cell with the unfertilized oocyte from one cell and the nucleus from another cell -- and used that in order to be able to develop a normal tadpole. In my laboratory, back about a decade ago, now, we used this type of technology in order to take a skin stem cell, and basically used nuclear transfer in order to put that into an unfertilized mouse oocyte. And using that technology, we were able to clone, effectively, and create healthy mice. So, that technology exists, and it's been successful. And this is the gold standard of having a normal, healthy mouse that is produced from one of these somatic cell nuclear transfer experiments. The experiments, however, are fraught with some problems. Not every mouse is normal. And the efficiency is still leaving something to be desired. But I think it's remarkable that that technology is actually successful. So, how would one adapt this, then, to nuclear transfer to human research. Obviously, we wouldn't want to use this for something like cloning at the level of human. But there are more important types of approaches that could be used in this case. We go to the first steps, only, in this case, an unfertilized human oocyte, remove and discard its nucleus, and now replace it with the nucleus of an adult somatic cell, such as a skin cell. And now, if we go to those hybrid cells, effectively, and culture them to the level of the blastocyst, and then create inner cell mass cells, from which we can culture those cells, we can generate embryonic stem cells. Only now, the nucleus is basically the nucleus of the person who had the skin cells. And in this case, that could be the patient. And so, in this type of technology, now, the cell that is derived... such as a neuron, derived from somatic cell nuclear transfer, would not be recognized as foreign, and would be accepted by the person. So, this gets us over the hurdle. What happened at the biological level? What about the biology? Well, the biology that I've just described to you is the biology of something called epigenetics. We all know that all of the cells of our body have the same genes. Every cell of our body has the same genes. And yet during development, the genes are modified so that some genes are turned off in one cell, turned on in another cell, and other genes are turned off in that cell, turned on in another cell. And that's what gives every cell of our body its own identity. That's why our skin cell is a skin cell, a nerve cell is a neuron, epidermal stem cells, muscle cells, liver cells, hair cells. All of the cells of our body have their identity. And they get it from the same DNA, but through epigenetics -- turning on some genes and turning off others. So, if you start with a skin cell that's turned off all sorts of genes that were expressed in the embryonic stem cells, that modification of, this is my identity as skin cell, has to be erased in order for the cell to do something else, like become an embryonic-like stem cell. This phenomenon is called epigenetic reprogramming -- the ability to change the state of the genes within a nucleus to... to be able to do something else, to turn on some genes and to turn off others. So, the experiment that I just described to you in somatic cell nuclear transfer basically tells us that it's the cytoplasm of that unfertilized egg that must favor the unspecified state. It's producing all sorts of different chromatin reprogramming factors that basically erase the realization of that skin stem... of that skin... skin nuc... nucleus that it ever was a skin cell. There aren't any divisions that are required for the reprogramming event in somatic nuclear transfer, so the experiment that I described to you is the reprogramming of the skin nucleus just by the cytoplasmic factors from the unfertilized egg. But... and this is, again, the religious ethical issue... is that reprogramming by somatic nuclear transfer still involves an egg, in this case, an unfertilized, single embryonic cell. So, how can we get around the problem of using human embryonic cells in order to be able to create cells that exhibit an embryonic-like state that could be used for the kinds of therapies that I've just mentioned? So, let's go to the pioneering work of Shinya Yamanaka, who back in 2006, using mouse adult cells, was able to reprogram those adult skin cells into becoming an embryonic-like cell without the use of the oocyte cytoplasm, or without the use of the unfertilized egg. So, the technology that Yamanaka did, and his coworkers, was to take an adult skin cell and look at the differences between the adult skin cell and what kinds of transcription factors the adult skin cell was making versus the embryonic stem cell and what kinds of transcription factors the embryonic stem cell was making. Effectively, what are the differences that give those two cells their identity? And taking advantage of that, they then introduced a cohort of transcription factor genes that expressed the embryonic stem cell-like transcription factor profile into the adult stem cell... adult skin cell. And then they began, one-by-one, taking out -- reducing the complexity of the transcription factor profile -- until they found just four transcription factors that turned out to be expressed by embryonic stem cells, not by adult skin cells, but could conform or reprogram the skin cell to behave as if it was an embryonic stem cell. In this case, they used retroviruses that harbored the genes encoding KLF4, Oct4, Sox2, and cMyc. And they put those into the adult skin cell. And what they were able to do was to find cells within their culture that are called induced pluripotent stem cells. And these are cells that basically, for all practical purposes, are looking like an embryonic stem cell, only now there's no oocyte -- fertilized or unfertilized -- that's required for this technology. So, what are the differences between genetic and epigenetic reprogramming? Genetic reprogramming forces the switch by ectopically inducing active forms of KLF4, OCT4, SOX2, and cMyc. Epigenetic programming achieves the switch by changing the nuclear environment in a way that causes its endogenous KLF4, OCT4, SOX2, and cMyc genes to be turned on. Remember, all the cells of our body have the same genetic constitution. Our skin cells have these genes. It's just that our skin cells turn off these genes. And so, by epigenetic programming, as I described to you in somatic cell nuclear transfer, those genes are turned on again. In this case, by genetic reprogramming, as I just described to you in the pioneering studies of Shinya Yamanaka and his coworkers, those genes were actively... an active form of those genes were provided to the skin cell. So, during the conversion of a somatic to an induced pluripotent cell, the endogenous loci for the SOX2, OCT4, KLF4, and cMyc genes are turned on after about 10 days of sustained ectopic expression of these transcription factors. And this was a remarkable breakthrough to realize this. So, something about indu... introducing those four genes in an active state into the adult skin cell resulted in the activation of those four genes within this skin cell zone chromatin that were normally silent. That was remarkable, and we now are understanding quite a bit about that process, which I won't have time to tell you about in detail today. Another remarkable change that occurred in the conversion of the skin cell to a somatic iPS cell... to an induced pluripotent stem cell... was the silencing of the X chromosome. Normally in our body, during development only one X chromosome ends up being active, and the other X chromosome turns out to be silenced. But at the early embryonic state, both X chromosomes are activated, and in the induction... introduction to an iPS cell, basically this change effectively reverted. Another amazing thing is that the ectopically-introduced genes encoding SOX2, OCT4, KLF4, and cMYC turned out to be silenced after several days of sustained ectopic expression, and after the endogenous SOX2, OCT4, KLF4, and cMYC genes were turned on. So, only the transient ectopic expression of sustained activated transcription factors that were expressed in embryonic stem cells turned out to be necessary in order to effectively reset the undifferentiated clock of the adult skin cell. Truly remarkable from a molecular biology understanding, and truly transformative in terms of what induced pluripotent stem cells could do. So, let's take a look at where we are in 2018 with regards to the current methods in induced pluripotent stem cell, or iPS, reprogramming. I've talked about genetic changes, the integration of the DNA of the active genes, the active forms of the transcription factors, into the DNA of the skin cell. That's a genetic reprogramming event. And I've also talked to you about epigenetic targeting, no permanent gene changes. And here, scientists have used chemicals, they've used small molecules, they've used RNA in order to be able to epigenetically reprogram the adult skin cell without the need to introduce foreign genes or ectopic expression of genes into these cells. Remarkable advances, now, that just by putting a skin cell in a petri dish that... using the right cocktail of chemicals, that it's possible now to convert that cell, as if it was an embryonic-like cell. So, if we now look at summarizing the progress on the somatic cell nuclear transfer and induced pluripotent stem cell front, that it was back in 2007 when monkey embryonic stem cells were cultured using skin nuclear transfer. In 2013, human embryonic stem cells were cultured using adult skin nuclear transfer. In 2007, I talked about the pioneering work of Shinya Yamanaka with regards to producing human induced pluripotent stem cells by retroviral transfection of adult skin cells with these four transcription factors. I talked about... well, I actually didn't talk about, but I'll do so now... how we can dispense with cMyc, oncogenic cMyc, because Myc is a potential oncogene. It's an essential component of that cocktail that Shinya Yamanaka used in 2007. But now, just a mere one to two years later, scientists figured out that a different transcription factor, Nanog, which isn't oncogenic, basically could replace cMyc. And so the early mice that scientists were creating using embryonic stem cell-like technology, only with induced pluripotent stem cells... those early mice developed tumors. Now, the mice... avoiding Myc as one of the four transcription factors, has basically alleviated or eliminated that danger. Between 2008 and 2013, scientists initially used adenoviral delivery instead of retroviral delivery. Retroviruses integrate into the human stem cell... skin cell chromatin. Adenoviruses don't. They are transferred as episomes, and so eventually they're diluted out and lost. Then scientists started to provide direct delivery of recombinant transcription factor proteins, avoiding the genetic manipulation of the skin cell. And then it was a few years later when scientists began to introduce modified stable RNA for the pluripotency factors. And then finally, scientists have been using small molecules and chemicals to epigenetically switch on the embryonic-like gene expression program. And then fast forward... what do we do with those induced pluripotent stem cells? Now scientists have been making, initially, neurons, but now many different types of cells of the body, basically by starting with these reprogrammed adult skin cells, and effectively producing all the different types of cells in the body with induced pluripotent stem cell technology. So, I'll show you an example, this one again from my colleague Lorenz Studer at the Sloan Kettering Cancer Institute. And here I'm showing you iPS cell-derived peripheral sensory neurons, a whole dish of peripheral sensory neurons, that are generated from these iPS cells. Remarkably, now... it's now possible to be able to study these peripheral sensory neurons, pain sensitive, to check out to see... use various different probes that might allow them to see how pain might be received, and how it might be generated, how it might be signaled back to, effectively, the brain. So, are there clinical applications yet for iPS technology? Well, the very first technology-driven therapy was for macular degeneration. It was possible to take human iPS cells and differentiate them to make sheets of these beautiful human retinal pigment epithelial cells. And those are the cells that are degenerated in macular degeneration. And so the very first clinical studies that were done with iPS cell technology were those of Masayo Takahashi at the RIKEN Institute in Kobe, Japan. Those studies were conducted back in September of 2014, the first-ever clinical trial involving iPS cell technology. Unfortunately, those studies were halted. After the first two patients were treated, they started to show signs of improvement in their vision. But the problem was that when the scientists tested the DNA from the retinal pigment epithelial cells what they found were several genetic abnormalities that might have been deleterious. And so they stopped the studies and basically halted them. So, it's illustrating that, despite the promise, there's still work to be done. And scientists -- these scientists and others -- are now actively trying to figure out how to be able to maintain and propagate these retinal pigment epithelial cells under conditions where they don't acquire additional genetic alterations. So, this work, then, is currently tabled until genetic stability can be further evaluated. And ultimately, however, it should be a matter of time before such hurdles start to be overcome. So, what about stem cell therapy for type I diabetes. My colleague up at Harvard University, Doug Melton, has been working on this problem for more than twenty years, since his son was first diagnosed with type I diabetes. He changed the research that he was doing in order to focus on this problem, and has made really remarkable advances in the course of these last twenty years. So, what has he done? He started with human embryonic stem cells. But as researchers began to produce induced pluripotent stem cells, he's also used induced pluripotent stem cells. And now, remarkably, by understanding enough about pancreatic development, he could then coax these cells through individual steps, one by one, to be able to produce pancreatic beta cells, the cells that are degenerating in type I diabetes. And remarkably, when introduced into a diabetic mouse, he was able to show that these induced pluripotent stem cell-derived beta islet cells not only could survive, but basically also had the ability to properly regulate glucose. Remarkable breakthroughs. But we're still left with some problems and challenges in diabetes, because as... as scientists were working on developing more and more pancreatic beta islet cells in culture, other scientists were beginning to realize that pancreatic type I diabetes is largely rooted as an autoimmune disease, and that the autoimmune cells... that the immune cells of the patient are basically attacking their own beta cells. So, even if we generate buckets of beta islet cells, unless we overcome the problem of the autoimmune attack, those cells will still be subject to the same types of attack that the patient with type I diabetes has. So, there are new hurdles to be overcome. But I think these advances begin to illustrate for you just how many steps are involved in the process, and just what advances scientists have made, and hopefully you'll begin to understand just why advancing these types of technologies to the use of medicine turns out to have so many steps involved, and are seemingly, for the public, so slow. So, what can be done in the meantime as scientists are improving upon these various different techniques and as safe induced pluripotent stem cell therapies are being developed? Well, there's lots of work to be done. Developing therapeutics for genetic degenerative diseases in vitro. Parkinson's disease. Huntington's disease. Cardiomyopathies. Sudden... sudden death syndrome. Alzheimer's disease. Diabetes. Muscular dystrophy. The ability to culture induced pluripotent stem cells and manipulate them genetically to resemble these types of diseases all of a sudden gives scientists the first ability, now, to really study these types of diseases in a petri dish in a way that for sure is going to lead to new and improved therapies in the future. So, once scientists can derive the right type of cell from induced pluripotent stem cells, the cells, then, can be used in drug screens. And effectively, in this case, Lorenz Studer, my colleague, has again been able to derive cortical neurons, as I showed you. Only now, instead of testing them for their activity in mouse brain, they can also be used for activity screens for developing new drugs for the treatments of various different degenerative disorders and genetic disorders. So, these are advances for the future. And... and I think a really exciting time for basic science and for translational science.
