Skin Stem Cells: Their Biology and Promise for Medicine
Transcript of Part 1: Skin Stem Cells: Their Biology and Promise for Regenerative Medicine
00:00:14.12 Hello. 00:00:15.12 My name is Elaine Fuchs. 00:00:17.02 I'm a professor at The Rockefeller University in New York City, 00:00:22.05 and I'm an investigator of the Howard Hughes Medical Institute. 00:00:26.16 And today I'd like to tell you about stem cells, about their biology and about 00:00:31.13 their promise for regenerative medicine. 00:00:36.18 So, let's go to the very beginnings of stem cell biology. 00:00:41.00 The word stem cell itself is a relatively recent word. 00:00:44.20 It was only in 1877 that Ernst Haeckel, a German scientist, coined the word stammzelle, or stem cell, 00:00:55.06 a fertilized egg that gives rise to all the cells in the body. 00:00:59.25 That was the initial definition of stem cells. 00:01:04.19 And then the word stem cell became popularized by EB Wilson, a famous cell biologist in the late 1800s, 00:01:11.25 who wrote about it in one of his books. 00:01:16.02 So then, it was about 30 years later when this person, Alexander Maximow, was a... 00:01:21.08 he was a Russian scientist. 00:01:23.26 He fled the Russian Revolution and joined the ranks of the University of Chicago. 00:01:28.28 He was a cytologist. 00:01:30.20 And just looking underneath the microscope at bone marrow cells, he saw some cells 00:01:36.19 that seemed undifferentiated. 00:01:38.24 And he predicted, at that time, that perhaps the bone marrow contained a cell that 00:01:46.01 can give rise to all the different hematopoietic cell lineages. 00:01:51.05 And that was the first notion that stem cells might not simply be restricted to 00:01:58.16 embryonic stem cells, the cells that can give rise to all the cells of our body, but actually that 00:02:02.26 different tissues might actually contain stem cells, adult tissue stem cells that had 00:02:09.22 a more restricted potential, being, in this case, able to give rise to the hematopoietic lineages. 00:02:16.18 But that was just a postulate. 00:02:20.02 And we now have to fast-forward some 60 years later, when these two individuals, 00:02:27.06 Ernest McCullough and James Till, back in 1961, did the remarkable experiment that demonstrated 00:02:33.20 the existence of adult tissue stem cells. 00:02:38.03 They took a laboratory animal, and they irradiated its bone marrow. 00:02:43.24 And then they took a healthy bone marrow, and one by one, put in the cells from 00:02:48.24 the healthy bone marrow until they found a single cell that could reconstitute the 00:02:55.13 entire hematopoietic cell lineages of the bone marrow. 00:02:59.04 And that was the first demonstration that tissue stem cells existed within adult... 00:03:08.06 the adult. 00:03:09.11 And that really opened the door for some really exciting biology to come. 00:03:15.02 So, let's fast-forward now, again, to... another 15 years or so, after the pioneering work 00:03:23.04 of Till and McCullough, and now it wasn't until the mid 1970s when this person, 00:03:30.24 Howard Green, who recently passed away... he was then at MIT, and he took a piece of 00:03:37.23 human skin, and was able to culture and passage cells from the piece of human skin, 00:03:45.22 and he could passage them endlessly under conditions where the cells could still make tissue. 00:03:52.25 This established the existence of the ability to culture adult tissue stem cells, 00:03:59.18 in this case stem cells coming from the skin. 00:04:02.13 So, let's take a look at some of the experiments that were done in those early days. 00:04:07.20 It was possible to effectively culture the embryo, in this case the adult skin stem cells, 00:04:17.00 and passage those cells. 00:04:18.03 And you see in the middle panel the chromosomes, the dark chromosomes within the cells. 00:04:24.15 These cells were rapidly dividing in culture. 00:04:28.06 And if you look at the bottom panel, what you see is actually reconstituted skin, 00:04:34.15 in this case reconstituted epidermis, that was generated entirely from these single stem cells 00:04:41.06 that were cultured. 00:04:42.17 In fact, we didn't call them stem cells at the time. 00:04:46.12 We called them keratinocytes, epidermal keratinocytes. 00:04:49.27 But effectively, these were tissue stem cells. 00:04:54.07 So, Howard Green very soon recognized the power of the technology that he had just developed 00:05:01.27 for the treatment, in this case, of burn patients. 00:05:05.01 He took skin from the unaffected area of a burn patient. 00:05:10.06 And then cultured those cells, grew sheets of epidermis, and then grafted those cells 00:05:16.27 onto the burned area of the patient. 00:05:21.00 Same patient, just taking good skin and expanding the cells in culture. 00:05:25.20 And the reason why that worked is that you could use growth factors and nutrients 00:05:30.09 that allowed the cells to grow fast, generate sheets of skin, in order to be able to be used 00:05:36.06 for burn therapy. 00:05:37.27 In those early days, Dr. Green was able to effectively cure or treat patients 00:05:47.24 that had burns up to 95% of the total surface of their skin. 00:05:53.04 Remarkable. 00:05:54.04 All generated from a few stem cells that were endlessly passaged in skin. 00:06:00.13 And we've learned a very important lesson from this. 00:06:03.08 Effectively, now, we have 35 years of success of the use of stem cells in a clinical setting. 00:06:10.09 And I think what one of the most remarkable aspects of that is that those patients 00:06:15.17 whose skin was almost entirely reconstituted from stem cell therapy, if you will, 00:06:23.16 never showed signs of abnormalities, never showed signs of genetic alterations in the skin, 00:06:31.01 or of cancer, that might give us an idea or a notion that culturing those cells long-term 00:06:38.11 might be deleterious for the cells in some way. 00:06:42.25 Remarkably, the skin of those patients still produced healthy epidermis. 00:06:50.13 This ability to culture stem cells in the... in the laboratory turned out to be the foundations 00:07:00.20 for embryonic stem cell research, which was going to come another five years after 00:07:07.00 Howard Green's pioneering work. 00:07:09.07 And there, the breakthrough was really the realization that stem cells cannot survive 00:07:15.22 on their own. 00:07:16.23 They require other cells for their sustenance. 00:07:20.24 And what Howard Green did in his breakthrough work to culture embryonic... to culture human 00:07:25.28 skin stem cells was basically the realization that epidermal stem cells rely upon 00:07:32.21 dermal cells in order for them to grow and be maintained. 00:07:36.06 So, he used what is called a fibroblast feeder layer, an irradiated layer of fibroblasts, 00:07:43.15 in order to be able to grow the human epidermal stem cells on top of it. 00:07:49.06 And that research then served as, also, the foundation for embryonic stem cell culture, 00:07:55.06 which also succeeded because of the addition of the fibroblast feeder layer. 00:08:01.14 So, what are stem cells, then? 00:08:04.13 Let's get down to the basics and the definition. 00:08:07.17 Basically, stem cells are cells that are able to make an animal, if we're talking about 00:08:15.07 embryonic stem cells. 00:08:17.02 The embryonic stem cells have all the power to reconstitute an entire animal. 00:08:24.11 Stem cells have the ability to make and replenish a tissue, if we're talking about adult stem cells. 00:08:30.28 So, adult stem cells replenish the tissue. 00:08:34.24 Embryonic stem cells have the capacity to make the animal. 00:08:40.07 And effectively, what we know about stem cells -- and particularly stem cells of adult tissues -- 00:08:45.10 is that they have the ability to be able to generate not only self 00:08:49.25 -- so, that means that they can self-renew -- 00:08:53.16 but they also have the ability to produce short-lived progenitors. 00:08:57.03 These are often rapidly proliferating progenitors that then go on to make the differentiated 00:09:02.16 cells of a tissue. 00:09:04.14 So, in this case, it would be the epidermis. 00:09:07.28 And it's often, then, the short-lived progenitor cells, or these transit-amplifying cells, 00:09:13.28 that have the ability to generate the bulk of the tissue. 00:09:17.21 The stem cells only divide relatively sparingly in most situations. 00:09:23.22 So, what are the differences, then, between embryonic and adult stem cells? 00:09:29.17 Embryonic stem cells are pluripotent, and they can generate all of the cells of the animal 00:09:35.12 except for the support cells or the placenta. 00:09:41.20 In the case of adult stem cells, these are usually multipotent, and can generate 00:09:47.08 several different tissues of our body. 00:09:49.00 Sometimes, they're unipotent, and can only generate a single tissue 00:09:54.09 or differentiated cell of our body. 00:09:56.25 So, in the first part of this series of lectures, I'd like to concentrate on embryonic stem cells, 00:10:05.15 on induced pluripotent stem cells, and on their applications to basic science, 00:10:11.11 medicine, and the pharmaceutical industry. 00:10:14.13 So, where do embryonic stem cells come from? 00:10:19.08 If we go to the early developing embryo, the blastocyst, that's just a few days old, 00:10:25.20 it consists of several hundred cells at the most. 00:10:29.28 And at this point, the blastocyst has a group of cells which are called inner cell mass cells. 00:10:40.06 And those are the cells that develop into the fetus, or the embryo. 00:10:50.07 The surrounding cells -- this single layer of cells that surround the inner cell mass -- 00:10:55.20 are called the trophectoderm. 00:10:58.10 The trophectoderm is going to develop into the fetal support tissue for the embryo. 00:11:04.16 So, those cells do not give rise to any part of the embryo, but basically they're necessary 00:11:11.21 in the womb to be able to develop the fetus. 00:11:16.23 So, scientists learned that it's possible to take the inner cell mass cells, then, 00:11:23.05 and put those cells into a petri dish on a fibroblast feeder layer, or with the appropriate nutrients 00:11:32.08 that support the inner cell mass cells. 00:11:34.09 And now they can culture those cells. 00:11:38.15 That's the culture that are called cultures of embryonic stem cells. 00:11:44.17 That's where embryonic stem cells come from. 00:11:47.00 And thereafter, those embryonic stem cells can be passaged effectively endlessly, 00:11:54.06 just like, as I mentioned to you, epidermal stem cells were initially. 00:11:59.00 And these cells, now, though, have much more power than the cells of the skin. 00:12:05.06 So, why is this important? 00:12:07.26 Well, let's think about the future for regenerative medicine. 00:12:11.15 There are many different cell types for which we don't have very much information, 00:12:16.26 and for which there are many different types of human genetic diseases. 00:12:21.06 For nerve cells, we could generate, potentially, nerve cells, possibly for the treatment of 00:12:26.03 Parkinson's disease or Alzheimer's disease. 00:12:30.11 Scientists are excited about the ability to generate nerve cells for the treatment of 00:12:34.18 spinal cord injuries. 00:12:37.11 Scientists are excited about the ability to culture pancreatic islet cells for 00:12:44.04 the possible treatment of diabetes. 00:12:47.07 Muscle cells for muscular dystrophy, and sudden death heart syndrome. 00:12:51.17 Immune cells for immunodeficiency disorders. 00:12:56.04 Or, thinking about the treatment of cancers, it's possible to generate the stem cells 00:13:05.01 that derive or produce the cancers, and that's also possible in culture. 00:13:10.26 And scientists are very interested in utilizing these types of technologies in order to 00:13:17.08 be able to understand more about the biology. 00:13:20.02 So, it's not just the treatment of patients, but it's really understanding the biology 00:13:26.07 of how these cells develop and what gives them their properties, to be able to ultimately 00:13:33.10 come up with new cures and treatments for various different types of diseases. 00:13:38.27 And that involves not only the clinician and the scientists, but also 00:13:44.02 the pharmaceutical industry or the biotechnology industry. 00:13:46.26 So, let's take a look at a couple of examples of what embryonic stem cells can do. 00:13:54.07 In this case, human embryonic stem cells were treated with particular growth factors 00:14:00.04 that allowed these cells to differentiate into muscle cells. 00:14:06.00 And now, looking at the embryoid bodies -- and this is the work of Gordon Keller and his 00:14:12.00 colleagues at the University of Toronto -- you can see that the cells undergo this beating feature. 00:14:21.24 And you can see a few examples of that, of the cells basically in the culture dish, now, 00:14:27.23 undergoing the contractions that heart muscle cells do. 00:14:31.09 And interestingly, they undergo them in a synchronized fashion, in the way that 00:14:36.03 our heart cells do in our body. 00:14:39.01 So, in one of the very earliest experiments, done, now, a decade ago, scientists took embryonic stem cells 00:14:50.21 and differentiated them into neurons, and used them to treat paralysis of a rat, 00:14:57.21 in this case. 00:14:59.22 And here was the rat before the injection of embryonic stem cell... stem cells differentiated 00:15:07.28 into spinal cord neurons. 00:15:11.12 And here is the rat after the treatment. 00:15:15.13 Now, these were early studies. 00:15:17.17 Scientists have gotten much better at these sorts of treatments. 00:15:21.12 But again, these kinds of treatments are what ultimately will be necessary in order to 00:15:27.08 move the field forward, and in order to think about therapies for the future. 00:15:32.04 So, let's now go to 2017. 00:15:37.18 What are scientists doing now? 00:15:39.10 Well, now it's possible to differentiate the embryonic stem cells not only into a neuron 00:15:47.13 but into specific types of neurons. 00:15:49.26 There are hundreds of different types of neurons in the body. 00:15:53.08 In this case, they took human embryonic stem cell-derived cortical neurons and 00:16:01.00 grafted them into a mouse cortex. 00:16:03.22 So, the importance of this work I'll describe in a moment. 00:16:07.09 But these are the studies of Lorenz Studer, a scientist and one of my colleagues 00:16:12.19 across the street at Sloan Kettering Cancer Institute. 00:16:17.03 So in this case, the cells that you see in white, now, are human cortical neurons. 00:16:24.11 And the slice of tissue that you're looking at is a mouse brain. 00:16:29.11 Three months after the graft. 00:16:33.24 And even later after the graft. 00:16:37.02 Six months after the graft, there are still human cortical neurons that are existing 00:16:43.23 in the mouse brain. 00:16:44.26 And scientists are currently trying to understand, are they working in the right way? 00:16:50.03 Are they behaving as cortical neurons? 00:16:54.00 And this is important for the treatment of a whole number of different disorders. 00:17:03.20 And it allows scientists to learn more about the biology of Alzheimer's, of autism, 00:17:10.23 of schizophrenia, and many other disorders of the cortex of our brain. 00:17:16.28 So, what are the ethical issues involved? 00:17:21.19 Why is there so much controversy about embryonic stem cell research? 00:17:25.24 Well, on the one hand, it's important to know that human embryonic stem cells are cultured 00:17:33.24 from fertilized eggs. 00:17:36.06 And a religious argument would be against the culturing of fertilized eggs for anything 00:17:44.09 other than, basically, making a human being. 00:17:48.23 The counterpoint, however, is that human blastocysts and embryonic stem cells can be created with 00:17:56.24 only egg and sperm. 00:17:58.27 And they can be created in culture, with no womb required. 00:18:05.10 Additionally, the technology that I've just described to you involves the exact same steps 00:18:15.09 as an in vitro fertilization. 00:18:17.25 So, I think these types of technologies are technologies that have made great advances. 00:18:26.26 That said, there is the concern, the ethical concern, by some, that... that... that the 00:18:35.17 use of fertilized eggs for in vitro fertilization is one thing, but the use for research is 00:18:42.03 another. 00:18:43.21 So, embryonic... embryos from in vitro fertilization are indeed often now discarded or frozen or 00:18:53.22 unused. 00:18:55.17 And so, again, this issue is, well, the embryos could be implanted into a woman who 00:19:02.20 would like to have a child, and therefore it would be unethical to use those embryos for anything else. 00:19:09.04 On the other hand, the counterpoint to this argument is that it's still that many embryos 00:19:15.14 from in vitro fertilization are discarded, are unused. 00:19:20.01 And the other aspect is that they could be used for scientists to be able to learn 00:19:25.04 how to generate neurons, pancreatic islet cells, muscle cells, for treating human disorders and injuries. 00:19:33.04 So, what are the hurdles -- beyond the ethical concerns -- of what scientists would 00:19:39.21 need to do to be able to adapt this technology and take it one step further to the clinics? 00:19:45.13 Well, the first big step is that embryonic stem cells that are used to generate neurons 00:19:53.20 and then implanted into, for instance, an Alzheimer's patient would basically be rejected 00:20:02.00 from the body. 00:20:03.07 Because the immune system of your body recognizes foreign cells, and it eliminates them. 00:20:10.13 And so, what were the next steps that scientists came up with to generate stem cells that would 00:20:16.07 overcome the problem of immune rejection? 00:20:21.11 In this particular technology, this is the technology known as nuclear transfer, 00:20:27.00 in which case you take an unfertilized oocyte, now... in the previous version we were taking fertilized oocytes... 00:20:33.04 fertilized oocytes, or eggs. 00:20:36.09 Here, we're taking an unfertilized oocyte, removing and discarding the nucleus of an oocyte, 00:20:41.17 and then replacing it with a nucleus of an adult somatic cell, such as a skin cell. 00:20:49.01 This technology was actually technology that was performed all the way back in 1962 00:20:55.00 by John Gurdon, who recently won the Nobel Prize for these pioneering studies. 00:21:00.12 He used Xenopus eggs and basically modified the eggs in this way, using nuclear transfer, 00:21:07.09 in order to produce a hybrid cell, now -- a cell with the unfertilized oocyte from one cell 00:21:14.20 and the nucleus from another cell -- and used that in order to be able to develop 00:21:20.04 a normal tadpole. 00:21:23.14 In my laboratory, back about a decade ago, now, we used this type of technology 00:21:29.17 in order to take a skin stem cell, and basically used nuclear transfer in order to put that into 00:21:37.22 an unfertilized mouse oocyte. 00:21:39.26 And using that technology, we were able to clone, effectively, and create healthy mice. 00:21:47.27 So, that technology exists, and it's been successful. 00:21:52.27 And this is the gold standard of having a normal, healthy mouse that is produced 00:21:59.00 from one of these somatic cell nuclear transfer experiments. 00:22:02.23 The experiments, however, are fraught with some problems. 00:22:06.14 Not every mouse is normal. 00:22:08.28 And the efficiency is still leaving something to be desired. 00:22:13.25 But I think it's remarkable that that technology is actually successful. 00:22:18.03 So, how would one adapt this, then, to nuclear transfer to human research. 00:22:23.22 Obviously, we wouldn't want to use this for something like cloning at the level of human. 00:22:29.23 But there are more important types of approaches that could be used in this case. 00:22:34.19 We go to the first steps, only, in this case, an unfertilized human oocyte, remove and discard 00:22:40.24 its nucleus, and now replace it with the nucleus of an adult somatic cell, such as a skin cell. 00:22:47.18 And now, if we go to those hybrid cells, effectively, and culture them to the level of the blastocyst, 00:22:57.09 and then create inner cell mass cells, from which we can culture those cells, we can generate 00:23:03.10 embryonic stem cells. 00:23:05.03 Only now, the nucleus is basically the nucleus of the person who had the skin cells. 00:23:11.22 And in this case, that could be the patient. 00:23:15.16 And so, in this type of technology, now, the cell that is derived... such as a neuron, 00:23:23.12 derived from somatic cell nuclear transfer, would not be recognized as foreign, 00:23:29.09 and would be accepted by the person. 00:23:32.23 So, this gets us over the hurdle. 00:23:37.02 What happened at the biological level? 00:23:39.14 What about the biology? 00:23:40.22 Well, the biology that I've just described to you is the biology of something called 00:23:46.06 epigenetics. 00:23:49.04 We all know that all of the cells of our body have the same genes. 00:23:54.11 Every cell of our body has the same genes. 00:23:57.20 And yet during development, the genes are modified so that some genes are turned off in one cell, 00:24:03.22 turned on in another cell, and other genes are turned off in that cell, 00:24:08.17 turned on in another cell. 00:24:10.10 And that's what gives every cell of our body its own identity. 00:24:14.25 That's why our skin cell is a skin cell, a nerve cell is a neuron, epidermal stem cells, 00:24:21.18 muscle cells, liver cells, hair cells. 00:24:24.01 All of the cells of our body have their identity. 00:24:26.09 And they get it from the same DNA, but through epigenetics 00:24:31.19 -- turning on some genes and turning off others. 00:24:34.07 So, if you start with a skin cell that's turned off all sorts of genes that were expressed 00:24:40.07 in the embryonic stem cells, that modification of, this is my identity as skin cell, 00:24:47.01 has to be erased in order for the cell to do something else, like become an embryonic-like stem cell. 00:24:54.22 This phenomenon is called epigenetic reprogramming -- the ability to change the state of the 00:25:01.15 genes within a nucleus to... to be able to do something else, to turn on some genes and 00:25:08.13 to turn off others. 00:25:10.08 So, the experiment that I just described to you in somatic cell nuclear transfer basically 00:25:16.22 tells us that it's the cytoplasm of that unfertilized egg that must favor the unspecified state. 00:25:24.27 It's producing all sorts of different chromatin reprogramming factors that basically erase 00:25:31.19 the realization of that skin stem... of that skin... skin nuc... nucleus that it ever was 00:25:39.20 a skin cell. 00:25:41.27 There aren't any divisions that are required for the reprogramming event in somatic nuclear transfer, 00:25:47.15 so the experiment that I described to you is the reprogramming of the skin nucleus 00:25:55.14 just by the cytoplasmic factors from the unfertilized egg. 00:26:00.19 But... and this is, again, the religious ethical issue... is that reprogramming by 00:26:08.11 somatic nuclear transfer still involves an egg, in this case, an unfertilized, single embryonic cell. 00:26:15.22 So, how can we get around the problem of using human embryonic cells in order to be able 00:26:23.27 to create cells that exhibit an embryonic-like state that could be used for the kinds of 00:26:31.05 therapies that I've just mentioned? 00:26:33.16 So, let's go to the pioneering work of Shinya Yamanaka, who back in 2006, using mouse adult cells, 00:26:43.21 was able to reprogram those adult skin cells into becoming an embryonic-like cell 00:26:55.05 without the use of the oocyte cytoplasm, or without the use of the unfertilized egg. 00:27:01.18 So, the technology that Yamanaka did, and his coworkers, was to take an adult skin cell 00:27:11.06 and look at the differences between the adult skin cell and what kinds of 00:27:16.25 transcription factors the adult skin cell was making versus the embryonic stem cell and what kinds of 00:27:23.06 transcription factors the embryonic stem cell was making. 00:27:26.26 Effectively, what are the differences that give those two cells their identity? 00:27:32.18 And taking advantage of that, they then introduced a cohort of transcription factor genes 00:27:42.04 that expressed the embryonic stem cell-like transcription factor profile into the adult stem cell... 00:27:50.15 adult skin cell. 00:27:51.19 And then they began, one-by-one, taking out -- reducing the complexity of the transcription 00:27:58.24 factor profile -- until they found just four transcription factors that turned out to 00:28:04.24 be expressed by embryonic stem cells, not by adult skin cells, but could conform or reprogram 00:28:12.22 the skin cell to behave as if it was an embryonic stem cell. 00:28:19.00 In this case, they used retroviruses that harbored the genes encoding KLF4, Oct4, Sox2, 00:28:26.22 and cMyc. 00:28:28.26 And they put those into the adult skin cell. 00:28:32.10 And what they were able to do was to find cells within their culture that are called 00:28:40.02 induced pluripotent stem cells. 00:28:42.15 And these are cells that basically, for all practical purposes, are looking like 00:28:47.21 an embryonic stem cell, only now there's no oocyte -- fertilized or unfertilized -- that's required for 00:28:55.00 this technology. 00:28:57.07 So, what are the differences between genetic and epigenetic reprogramming? 00:29:04.22 Genetic reprogramming forces the switch by ectopically inducing active forms of KLF4, 00:29:11.27 OCT4, SOX2, and cMyc. 00:29:17.17 Epigenetic programming achieves the switch by changing the nuclear environment in a way 00:29:23.15 that causes its endogenous KLF4, OCT4, SOX2, and cMyc genes to be turned on. 00:29:31.26 Remember, all the cells of our body have the same genetic constitution. 00:29:36.13 Our skin cells have these genes. 00:29:40.09 It's just that our skin cells turn off these genes. 00:29:44.03 And so, by epigenetic programming, as I described to you in somatic cell nuclear transfer, 00:29:51.12 those genes are turned on again. 00:29:54.06 In this case, by genetic reprogramming, as I just described to you in the 00:29:59.00 pioneering studies of Shinya Yamanaka and his coworkers, those genes were actively... 00:30:05.21 an active form of those genes were provided to the skin cell. 00:30:09.22 So, during the conversion of a somatic to an induced pluripotent cell, the endogenous loci 00:30:17.19 for the SOX2, OCT4, KLF4, and cMyc genes are turned on after about 10 days of 00:30:27.01 sustained ectopic expression of these transcription factors. 00:30:30.07 And this was a remarkable breakthrough to realize this. 00:30:33.26 So, something about indu... introducing those four genes in an active state into 00:30:40.17 the adult skin cell resulted in the activation of those four genes within this skin cell zone chromatin 00:30:49.00 that were normally silent. 00:30:51.17 That was remarkable, and we now are understanding quite a bit about that process, 00:30:57.13 which I won't have time to tell you about in detail today. 00:31:00.23 Another remarkable change that occurred in the conversion of the skin cell to 00:31:06.03 a somatic iPS cell... to an induced pluripotent stem cell... was the silencing of the X chromosome. 00:31:13.26 Normally in our body, during development only one X chromosome ends up being active, 00:31:20.12 and the other X chromosome turns out to be silenced. 00:31:23.23 But at the early embryonic state, both X chromosomes are activated, and in the induction... 00:31:31.03 introduction to an iPS cell, basically this change effectively reverted. 00:31:38.08 Another amazing thing is that the ectopically-introduced genes encoding SOX2, OCT4, KLF4, and cMYC 00:31:50.14 turned out to be silenced after several days of sustained ectopic expression, 00:31:58.17 and after the endogenous SOX2, OCT4, KLF4, and cMYC genes were turned on. 00:32:06.05 So, only the transient ectopic expression of sustained activated transcription factors 00:32:15.07 that were expressed in embryonic stem cells turned out to be necessary in order to effectively 00:32:20.18 reset the undifferentiated clock of the adult skin cell. 00:32:25.20 Truly remarkable from a molecular biology understanding, and truly transformative 00:32:33.28 in terms of what induced pluripotent stem cells could do. 00:32:37.28 So, let's take a look at where we are in 2018 with regards to the current methods in 00:32:44.07 induced pluripotent stem cell, or iPS, reprogramming. 00:32:48.04 I've talked about genetic changes, the integration of the DNA of the active genes, 00:32:55.06 the active forms of the transcription factors, into the DNA of the skin cell. 00:32:59.28 That's a genetic reprogramming event. 00:33:03.17 And I've also talked to you about epigenetic targeting, no permanent gene changes. 00:33:09.11 And here, scientists have used chemicals, they've used small molecules, 00:33:14.28 they've used RNA in order to be able to epigenetically reprogram the adult skin cell without 00:33:21.18 the need to introduce foreign genes or ectopic expression of genes into these cells. 00:33:28.06 Remarkable advances, now, that just by putting a skin cell in a petri dish that... 00:33:34.20 using the right cocktail of chemicals, that it's possible now to convert that cell, 00:33:41.01 as if it was an embryonic-like cell. 00:33:44.00 So, if we now look at summarizing the progress on the somatic cell nuclear transfer 00:33:50.19 and induced pluripotent stem cell front, that it was back in 2007 when monkey embryonic stem cells 00:34:00.04 were cultured using skin nuclear transfer. 00:34:05.15 In 2013, human embryonic stem cells were cultured using adult skin nuclear transfer. 00:34:12.25 In 2007, I talked about the pioneering work of Shinya Yamanaka with regards to 00:34:19.26 producing human induced pluripotent stem cells by retroviral transfection of adult skin cells with 00:34:27.17 these four transcription factors. 00:34:29.07 I talked about... well, I actually didn't talk about, but I'll do so now... how we can 00:34:34.25 dispense with cMyc, oncogenic cMyc, because Myc is a potential oncogene. 00:34:42.03 It's an essential component of that cocktail that Shinya Yamanaka used in 2007. 00:34:47.24 But now, just a mere one to two years later, scientists figured out that 00:34:53.28 a different transcription factor, Nanog, which isn't oncogenic, basically could replace cMyc. 00:35:00.25 And so the early mice that scientists were creating using embryonic stem cell-like technology, 00:35:09.11 only with induced pluripotent stem cells... those early mice developed tumors. 00:35:14.25 Now, the mice... avoiding Myc as one of the four transcription factors, has basically 00:35:21.18 alleviated or eliminated that danger. 00:35:28.04 Between 2008 and 2013, scientists initially used adenoviral delivery instead of retroviral delivery. 00:35:37.12 Retroviruses integrate into the human stem cell... skin cell chromatin. 00:35:45.01 Adenoviruses don't. 00:35:46.11 They are transferred as episomes, and so eventually they're diluted out and lost. 00:35:52.18 Then scientists started to provide direct delivery of recombinant transcription factor proteins, 00:35:58.22 avoiding the genetic manipulation of the skin cell. 00:36:04.08 And then it was a few years later when scientists began to introduce modified stable RNA 00:36:10.19 for the pluripotency factors. 00:36:12.20 And then finally, scientists have been using small molecules and chemicals to 00:36:18.21 epigenetically switch on the embryonic-like gene expression program. 00:36:26.27 And then fast forward... what do we do with those induced pluripotent stem cells? 00:36:32.27 Now scientists have been making, initially, neurons, but now many different types of cells 00:36:39.09 of the body, basically by starting with these reprogrammed adult skin cells, 00:36:47.10 and effectively producing all the different types of cells in the body with induced pluripotent 00:36:53.01 stem cell technology. 00:36:54.01 So, I'll show you an example, this one again from my colleague Lorenz Studer at 00:37:01.06 the Sloan Kettering Cancer Institute. 00:37:04.06 And here I'm showing you iPS cell-derived peripheral sensory neurons, a whole dish of 00:37:12.10 peripheral sensory neurons, that are generated from these iPS cells. 00:37:17.14 Remarkably, now... it's now possible to be able to study these peripheral sensory neurons, 00:37:24.14 pain sensitive, to check out to see... use various different probes that might 00:37:29.08 allow them to see how pain might be received, and how it might be generated, how it might 00:37:38.05 be signaled back to, effectively, the brain. 00:37:43.11 So, are there clinical applications yet for iPS technology? 00:37:49.22 Well, the very first technology-driven therapy was for macular degeneration. 00:37:58.25 It was possible to take human iPS cells and differentiate them to make sheets of 00:38:04.17 these beautiful human retinal pigment epithelial cells. 00:38:08.24 And those are the cells that are degenerated in macular degeneration. 00:38:14.06 And so the very first clinical studies that were done with iPS cell technology were 00:38:19.26 those of Masayo Takahashi at the RIKEN Institute in Kobe, Japan. 00:38:26.02 Those studies were conducted back in September of 2014, the first-ever clinical trial 00:38:35.16 involving iPS cell technology. 00:38:38.02 Unfortunately, those studies were halted. 00:38:41.11 After the first two patients were treated, they started to show signs of improvement 00:38:47.06 in their vision. 00:38:48.06 But the problem was that when the scientists tested the DNA from the retinal pigment epithelial cells 00:38:55.13 what they found were several genetic abnormalities that might have been deleterious. 00:39:03.16 And so they stopped the studies and basically halted them. 00:39:07.08 So, it's illustrating that, despite the promise, there's still work to be done. 00:39:13.08 And scientists -- these scientists and others -- are now actively trying to figure out 00:39:19.02 how to be able to maintain and propagate these retinal pigment epithelial cells under conditions 00:39:25.14 where they don't acquire additional genetic alterations. 00:39:29.13 So, this work, then, is currently tabled until genetic stability can be further evaluated. 00:39:38.28 And ultimately, however, it should be a matter of time before such hurdles start to be overcome. 00:39:47.07 So, what about stem cell therapy for type I diabetes. 00:39:51.08 My colleague up at Harvard University, Doug Melton, has been working on this problem 00:39:57.17 for more than twenty years, since his son was first diagnosed with type I diabetes. 00:40:03.13 He changed the research that he was doing in order to focus on this problem, 00:40:08.14 and has made really remarkable advances in the course of these last twenty years. 00:40:13.09 So, what has he done? 00:40:15.18 He started with human embryonic stem cells. 00:40:18.08 But as researchers began to produce induced pluripotent stem cells, he's also used 00:40:24.12 induced pluripotent stem cells. 00:40:26.04 And now, remarkably, by understanding enough about pancreatic development, he could then 00:40:32.18 coax these cells through individual steps, one by one, to be able to produce 00:40:38.23 pancreatic beta cells, the cells that are degenerating in type I diabetes. 00:40:44.05 And remarkably, when introduced into a diabetic mouse, he was able to show that these 00:40:53.15 induced pluripotent stem cell-derived beta islet cells not only could survive, but basically also 00:41:03.02 had the ability to properly regulate glucose. 00:41:06.09 Remarkable breakthroughs. 00:41:07.22 But we're still left with some problems and challenges in diabetes, because as... as scientists 00:41:15.02 were working on developing more and more pancreatic beta islet cells in culture, other scientists 00:41:23.20 were beginning to realize that pancreatic type I diabetes is largely rooted as an autoimmune disease, 00:41:32.23 and that the autoimmune cells... that the immune cells of the patient are 00:41:37.03 basically attacking their own beta cells. 00:41:39.03 So, even if we generate buckets of beta islet cells, unless we overcome the problem of 00:41:46.15 the autoimmune attack, those cells will still be subject to the same types of attack 00:41:52.26 that the patient with type I diabetes has. 00:41:55.20 So, there are new hurdles to be overcome. 00:41:59.08 But I think these advances begin to illustrate for you just how many steps are involved 00:42:05.24 in the process, and just what advances scientists have made, and hopefully you'll begin to understand 00:42:11.11 just why advancing these types of technologies to the use of medicine turns out to have 00:42:19.08 so many steps involved, and are seemingly, for the public, so slow. 00:42:25.06 So, what can be done in the meantime as scientists are improving upon these various different techniques 00:42:30.07 and as safe induced pluripotent stem cell therapies are being developed? 00:42:36.05 Well, there's lots of work to be done. 00:42:39.06 Developing therapeutics for genetic degenerative diseases in vitro. 00:42:44.05 Parkinson's disease. 00:42:45.09 Huntington's disease. 00:42:47.02 Cardiomyopathies. 00:42:48.02 Sudden... sudden death syndrome. 00:42:50.23 Alzheimer's disease. 00:42:51.20 Diabetes. 00:42:52.12 Muscular dystrophy. 00:42:54.04 The ability to culture induced pluripotent stem cells and manipulate them genetically 00:42:59.04 to resemble these types of diseases all of a sudden gives scientists the first ability, now, 00:43:04.24 to really study these types of diseases in a petri dish in a way that for sure 00:43:11.15 is going to lead to new and improved therapies in the future. 00:43:15.26 So, once scientists can derive the right type of cell from induced pluripotent stem cells, 00:43:22.21 the cells, then, can be used in drug screens. 00:43:26.12 And effectively, in this case, Lorenz Studer, my colleague, has again been able to derive 00:43:34.17 cortical neurons, as I showed you. 00:43:36.20 Only now, instead of testing them for their activity in mouse brain, they can also 00:43:43.04 be used for activity screens for developing new drugs for the treatments of various different 00:43:49.02 degenerative disorders and genetic disorders. 00:43:52.15 So, these are advances for the future. 00:43:55.12 And... and I think a really exciting time for basic science and for translational science.