Skin Stem Cells: Their Biology and Promise for Medicine
Transcript of Part 2: Tapping the Potential of Adult Stem Cells
00:00:14.25 I'm Elaine Fuchs. 00:00:16.10 I'm a professor at The Rockefeller University in New York City. 00:00:21.04 I'm also an investigator of the Howard Hughes Medical Institute. 00:00:25.20 And today I'd like to tell you a little bit about tapping the potential of adult stem cells. 00:00:32.12 Every tissue of our body has to replenish dying cells, dead cells, 00:00:39.01 that through wear and tear are lost from our tissues. 00:00:43.18 50-70 billion cells die every day in our body, and they have to be replaced. 00:00:50.28 How does that happen? 00:00:52.09 Our body must repair its tissues, as well, when damaged. 00:00:56.26 How does that happen? 00:01:00.13 Every tissue of our body has reservoirs of stem cells for homeostasis and wound repair. 00:01:07.05 So, it's the stem cells that replace these 50-70 billion di... billion cells that die 00:01:13.13 every day in our body, and that also repair our wounds. 00:01:20.11 Some tissues turnover rapidly and have many stem cells. 00:01:24.24 Those are... such as hematopoietic stem cells, which regenerate the red blood cells and immune cells. 00:01:30.28 Or skin stem cells, which regenerate the epidermis and the hair follicles and our sweat glands. 00:01:37.02 Or intestinal stem cells, that rejuvenate the food adsorptive cells of our body. 00:01:44.00 Other tissues have very little regenerative capacity. 00:01:47.25 Our central and peripheral nervous system, for instance, pancreatic tissue, or cardiac tissue. 00:01:55.24 So, what are the characteristics of tissue stem cells that exist in adult tissues? 00:02:02.04 These cells have the capacity to divide, to produce more stem cells 00:02:06.21 -- that's a process known as self-renewal -- 00:02:10.02 and they produce a differentiated tissue, such as the epidermis or the hair. 00:02:15.04 And they do so long-term. 00:02:19.09 These cells are relatively immature compared to their progeny, compared to the tissue cells. 00:02:27.19 They're typically multipotent; they're able to produce a subset of cell lineages. 00:02:33.14 Examples: hematopoietic stem cells can make the blood and immune cells; 00:02:38.13 skin stem cells can make the epidermis and hair. 00:02:43.23 It's only the fertilized egg that has unlimited potential, that can make all the cells of our body. 00:02:51.15 Tissue stem cells tend to be more restricted in what they can do. 00:02:55.28 And to understand why, we need to study the biology of adult tissue stem cells. 00:03:02.14 So, my laboratory studies the skin and skin stem cells. 00:03:06.25 And I've always thought that nature has clearly had a lot more fun and fancy in creating body surfaces 00:03:11.15 than she has in creating any of the ugly organs that most scientists work on. 00:03:18.13 My group works on the stem cells of the skin. 00:03:23.16 So, where do adult skin stem cells come from? 00:03:27.14 They began in early embryonic development, shortly after gastrulation, when the embryo 00:03:37.20 effectively turns inside-out and begins to develop into the animal. 00:03:44.10 The skin begins as a single layer of cells that are on the surface of the embryo. 00:03:51.03 Those are the cells that are going to give rise to the stratified epidermis that you see here. 00:03:58.15 They'll also generate the hair follicles. 00:04:01.04 They'll also generate the corneal surface of our eye, the sweat glands, and the mammary glands. 00:04:07.15 All of these cells come from this single layer of multipotent progenitor cells, 00:04:15.10 that are the origins of the stem cells of our adult skin. 00:04:20.07 So, skin stem cells provide the nearly endless supply of cells that replenish the epidermis 00:04:28.04 and the hairs of our body. 00:04:30.21 Can we isolate them? 00:04:32.02 Can we coax them to become hair as well as epidermis? 00:04:37.21 What value do they have in regenerative medicine? 00:04:42.12 Back when I was a graduate student, I was working on bacteria. 00:04:45.26 And I heard a lecture by Howard Green, who at the time was at MIT as a research scientist. 00:04:53.20 And Howard had developed a method where he could take a piece of human skin, 00:04:58.11 and put the cells into culture, and be able to propagate them endlessly, without losing their ability 00:05:04.20 to make skin. 00:05:06.08 I was just fascinated by his lecture. 00:05:09.18 And I knew then that I wanted to become a stem cell biologist. 00:05:18.09 So, here are examples, then, of the stem cells that are growing in culture. 00:05:24.20 And you can see by the dark chromosomes that these cells are actively dividing. 00:05:30.15 And they can be propagated, as I mentioned, endlessly. 00:05:34.20 Below, what you see is a three-dimensional, so-called organoid culture, or a tissue, 00:05:43.06 where we've reconstituted the epidermis starting from scratch using these cultured 00:05:48.05 human epidermal stem cells. 00:05:50.11 It looks pretty good compared to normal human skin epidermis. 00:05:57.04 So, Howard Green went on to develop this technology for the treatment of burn patients, 00:06:03.04 where he could take a small piece of the patient's good skin, propagate those cells in culture, 00:06:09.00 and make sheets -- hundreds of sheets of epidermal cells -- that then could be grafted 00:06:15.06 in the patient's bad skin. 00:06:16.27 So, the epidermal stem cells could be grown for months in a dish, and forever when grafted 00:06:23.16 onto a patient. 00:06:25.00 And this shows you an example of some of the grafted skin from one of these patients. 00:06:30.24 The patients that Howard Green treated successfully with these cultured human epidermal stem cells 00:06:37.05 from the patient covered up to 95% of the patient's burned skin, 00:06:43.16 with only 5% of the patient having unburned skin. 00:06:47.07 And from that stem cells could be cultured. 00:06:50.08 A remarkable advance. 00:06:53.17 After a year, the skin epidermis still looked like the normal skin epidermis would. 00:07:00.27 And it didn't show signs of cancer or deleterious effects that might give us an indication that 00:07:07.06 all of that culturing is a bad thing for these human epidermal stem cells. 00:07:13.04 Michele De Luca and Graziella Pellegrini, who were then studying in Howard Green's laboratory 00:07:22.06 and then started up their own labs in Italy, adapted the pioneering culturing procedures 00:07:28.23 that Howard Green had performed, in this case, to culture cornea or corneal epithelial cells 00:07:36.22 from a patient's stem cells of the cornea. 00:07:40.16 So, in this case, the patient had an industrial accident, where one of the eyes 00:07:46.12 -- the eye that you see here -- was blinded. 00:07:50.02 By taking stem cells from the healthy eye, culturing those cells, and producing a sheet 00:07:57.00 of corneal epithelial cells, it was possible, then, to graft it onto the blind eye. 00:08:02.25 And here is the result of the stem cell therapy: a restored corneal epithelium that effectively 00:08:11.00 restored the vision in this eye to this patient. 00:08:14.22 So, let's take a look at ten years later, after those experiments with the cornea were performed. 00:08:24.22 Just in the last month, there was a very successful report of whole body regenerative medicine 00:08:34.02 from disease-corrected epidermal stem cells. 00:08:38.01 And in this case, the patient, a seven-year-old boy, suffered from a genetic disorder known 00:08:45.15 as junctional epidermolysis bullosa. 00:08:49.05 This genetic disorder is due to recessive genetic mutations that can be either in the 00:08:55.18 laminin5, an extracellular matrix protein that's important for the epidermis 00:09:01.02 to adhere to the underlying dermis, or mutations in integrins alpha-6 or beta-4, 00:09:08.05 which are the integrins in the epidermal stem cells that are necessary to adhere the epidermis 00:09:14.16 to the underlying dermis. 00:09:16.15 And this particular patient had a disorder, or genetic lesion, in the laminin5 chain. 00:09:22.13 And as a consequence, the epidermis did not adhere, and effectively sluffed off 00:09:30.06 from the patient's body with the slightest of mechanical trauma. 00:09:34.10 The patient was near death at the point at which Michele De Luca, my colleague in Rome, 00:09:42.15 took on this task of working with German clinicians in order to be able to take a small sample 00:09:51.01 of the patient's cells, junctional epidermolysis bullosa-defective keratinocytes; 00:09:58.25 used gene therapy to be able to put back the normal laminin chain and to be able to excise 00:10:05.00 the genetic mutation of the patient; and effectively, at that point, then, generated healthy 00:10:12.20 human epidermal stem cells that could be maintained and propagated for generating sheets of epidermis 00:10:19.23 that were then grafted onto this patient. 00:10:23.11 So, this gene therapy of epidermal stem cells required only a very few number of stem cells 00:10:34.09 in order to be able to replenish, or repopulate, the entire epidermis of this patient 00:10:40.13 using whole-body regenerative medicine from disease-corrected epidermal stem cells. 00:10:45.18 And here is the boy today, basically able to go out and play in a way that he was 00:10:54.23 barely able to survive at the time of the therapy. 00:11:00.08 So, even a year ago, scientists did not think, or were concerned, that it might not be possible 00:11:10.15 to be able to produce normal, healthy epidermal stem cells from gene editing of the mutation 00:11:21.11 out of the stem cells. 00:11:24.02 And it also was doubtful that whole-body regenerative medicine would be possible to correct 00:11:31.04 the genetic basis of this disease, or to correct this genetic disease. 00:11:35.20 The patient's other cells still harbor that gene defect, but what the important aspect 00:11:42.26 of this is is that the patient -- the patient's skin epidermis -- is now corrected. 00:11:50.05 And this boy may be able to at least live a relatively normal life. 00:12:03.01 So, what are, then, the various different associated therapies that have followed 00:12:14.11 the culturing of epidermal stem cells that were first performed in 1975? 00:12:22.10 And now we have the correction of third-degree burns with epidermal stem cells, and the correction 00:12:29.24 of junctional epidermolysis bullosa with epidermal stem cells that have been gene edited. 00:12:36.04 In 1985, we had corneal epithelial stem cells to correct blindness from burns and from chemicals. 00:12:45.03 In 1992, Peterson and Bissell, scientists at Lawrence Berkeley Laboratory, and also 00:12:53.10 in Denmark, were able to produce milk-producing glands from human mammary epithelial stem cells 00:13:02.21 cultured in the laboratory. 00:13:05.01 And these have been enormously helpful, not only to study mammalian mammary biology, 00:13:11.11 but also to study breast cancer. 00:13:14.08 In 2013, Sato and Clevers made the impressive advance to be able to produce organoid cultures 00:13:25.11 from simple epithelia, from intestinal epithelial, lung epithelial, stomach, and liver stem cells. 00:13:32.09 These were, again, advances that took years for scientists to make with, again, the realization 00:13:39.20 that what's important is to understand enough about the biology of the stem cell to be able 00:13:45.18 to know about their environment and basically recreate the environment that that stem cell 00:13:51.22 normally has in its normal tissue, and recreate that in a culture dish. 00:13:56.27 It was determined early on, by Howard Green and others, for epidermal stem cells. 00:14:02.16 Many years later, it's now become possible to culture many different types of adult stem cells. 00:14:09.06 And so, here, by the use of the ability to culture, for instance, lung stem cells, 00:14:15.08 it was also possible in... again, in just the last year in the Netherlands, to be able to 00:14:21.02 treat a patient with cystic fibrosis by gene editing mini-lung organoid cultures grown 00:14:29.22 from the patient, and... and basically allow the patient to... to again be able to breathe properly, 00:14:41.05 and... and promise for the future. 00:14:47.17 So, where are we in 2018 with our knowledge about the biology of tissue stem cells? 00:14:56.13 Because it's the biology that is going to drive future advances in the clinics, 00:15:02.24 and also in the pharmaceutical and biotechnology industry. 00:15:06.11 So, what we know is that stem cells of adult tissues typically reside in niches, in defined 00:15:14.02 microenvironments. 00:15:15.07 That's true for the hair follicle, for the intestine, and for the hematopoietic system, 00:15:22.00 the three stem cells for which we know most about the stem cells and their niches. 00:15:30.23 When does a tissue stem cell decide, am I gonna make tissue, or am I going to be dormant, 00:15:38.22 or quiescent? 00:15:40.06 It's the interactions between the stem cell and its microenvironment, or the niche stem-cell interactions, 00:15:46.04 that make that decision. 00:15:48.27 When a stem cell is not making tissue and exists, for instance, in the skin, it basically 00:15:54.24 is receiving inhibitory signals from the microenvironment, 00:15:59.01 telling it to stay cool, be quiet, be dormant, and not make tissue. 00:16:05.21 But if a stem cell needs to make tissue, now there have to be interacting cues that are 00:16:12.26 positive-acting cues that are now going to override those inhibitory cues. 00:16:18.22 And at that point, those activated stem cells then go on to make short-lived progenitors, 00:16:24.11 which then go on to produce the bulk of the tissue. 00:16:27.13 And in the case of the skin, it would be, for instance, the epidermis or the hair follicle. 00:16:33.07 How many and how often stem cells are active depends upon the needs of the particular tissue. 00:16:40.22 So, we know a lot about the skin stem cells from my laboratory and other laboratories' research 00:16:47.01 over the last several decades now. 00:16:51.22 And we know that the tissue stem cells reside at the interface between the epidermis 00:16:57.09 and the dermis. 00:17:00.03 But they're defined in their task and also their program of gene expression 00:17:05.24 by their local niches. 00:17:06.25 So, the epidermal stem cells that reside in the innermost layer of the epidermis are in 00:17:12.14 a niche microenvironment that's very different from that of the hair follicle stem cells 00:17:17.23 that are located at the very base of the non-cycling portion of the hair follicle. 00:17:25.04 And that stem cell-niche interactions are what defines the stem cells in each of these 00:17:33.07 niches... niches, and tells the stem cells of the epidermis to make epidermis 00:17:38.24 -- produce our barrier -- whereas stem cells from the hair follicle to make hair. 00:17:45.23 So, my laboratory has studied the hair follicle for a number of years now. 00:17:51.13 It's really the perfect system to understand a basic biological problem. 00:17:56.07 And that is how stem cells transition between quiescence and tissue regeneration. 00:18:02.02 This is a problem that's faced by all the stem cells of our adult tissues. 00:18:06.26 And we've learned a lot about the various different interactions that are necessary. 00:18:11.23 We know that these red arrows are basically inter... inter-niche cells that are 00:18:17.15 sending out a signal called BMP that tells these stem cells, in purple, to be quiescent. 00:18:25.12 We know that at the base of the stem cell niche, there are signals -- new signals, activating signals -- 00:18:30.14 called Wnts, which are produced by the hair follicle stem cells themselves, 00:18:36.25 and BMP inhibitors produced by the underlying green cell, 00:18:41.13 basically a pocket of specialized mesenchymal cells called dermal papilla cells. 00:18:45.27 And these are the pro-activating signals that are necessary to take the stem cells 00:18:51.24 out of their quiescent state and coax them to make hair. 00:18:55.18 So, when the threshold levels of activating signals overcome the inhibitory BMP signal, 00:19:02.26 then, the stem cells are basically activated. 00:19:06.25 They start to make those short-lived progeny. 00:19:09.16 And now the short-lived progeny may get a new signal, this one called sonic hedgehog. 00:19:15.02 Sonic hedgehog then acts in two ways, which we learned from conditional knockout 00:19:23.09 of both the receptor and the ligand. 00:19:26.02 What we learned is that sonic hedgehog first acts to stimulate the specialized mesenchymal cells, 00:19:32.17 the dermal papilla cells, to produce more BMP inhibitors and also pro-activating FGFs. 00:19:40.08 Then, several days later, sonic hedgehog acts on the quiescent stem cells, those purple cells, 00:19:48.23 to be able to restock the stem cells utilized in the course of tissue regeneration, 00:19:55.05 but also to produce the shaft that then grows downward, pushing the signaling center 00:20:02.21 away from the stem cell niche. 00:20:04.18 And now the stem cells return to quiescence, but the short-lived progeny produce hair 00:20:11.23 and the channel that the hair follicle sits in. 00:20:15.06 And so, in this way, we understand very well about how the hair cycle works because 00:20:23.28 once the hair cycle is begun and gets going, it's essentially the short-lived progenitors 00:20:30.26 that then do the bulk of making tissue, in this case, the hair in its channel. 00:20:35.19 So, the short-lived progenitors are short-lived. 00:20:38.10 Eventually, they lose their proliferative capacity, and undergo apoptosis and differentiation. 00:20:46.02 And now the system returns back to normal again, to reset the hair cycle. 00:20:52.15 So, what does it take to make a hair follicle? 00:20:56.24 I talked about the importance of the Wnt signal, but many years ago my laboratory activated 00:21:04.17 the Wnt signal... at the time we didn't even know it was a Wnt signal... 00:21:10.06 we activated its interacting partner known as beta-catenin. 00:21:15.04 And when we activated beta-catenin in the mouse skin, what we got is this super furry mouse, 00:21:20.11 relative to its brother next to it. 00:21:24.03 These mice ended up producing far more hair follicles than normal. 00:21:29.13 Initially, we were very excited by that. 00:21:32.19 But the downside of this is is that those mice develop tumors over time. 00:21:38.02 Obviously, not yet ready for primetime. 00:21:41.24 But it told us a very important thing, and that is that Wnt signaling is important in 00:21:47.06 dictating these unspecified progenitors to make a hair follicle, not to make epidermis. 00:21:54.14 So, when taken from their niche and cultured, the green stem cells that you see here, 00:22:01.05 of the hair follicle, turn out to acquire plasticity. 00:22:05.00 We can culture these stem cells much the same way that Howard Green cultured epidermal stem cells 00:22:11.03 many years ago. 00:22:13.01 And when we culture those cells, we can take a colony, or a clone, derived from one of 00:22:19.09 the single stem cells, and we can engraft it onto a mouse that doesn't have hair, 00:22:25.14 and those mice now can produce hair. 00:22:28.06 They also produce epidermis and sebaceous glands. 00:22:31.25 And that's something that they normally don't do. 00:22:35.01 Normally, they just make hair. 00:22:38.14 They will, however, make epidermis and sebaceous glands when the skin is wounded, 00:22:44.25 and when the epidermis and the sebaceous glands are missing. 00:22:47.16 So, the stem cells receive signals, in these case... in this case, wound signals, 00:22:54.04 that stimulate those stem cells to be able to make hair, but also to be able to make epidermis 00:22:59.21 and sebaceous glands, to replenish the tissues that were missing 00:23:04.13 as a consequence of the wound. 00:23:07.05 And when you take the stem cells out of context, out of the hair follicle niche, 00:23:11.23 and you put them into culture, now the stem cells kind of forget what they were supposed to do 00:23:18.13 when they're grafted back onto the skin. 00:23:20.20 And now they basically act like they're in a wound state, and effectively will also make 00:23:26.21 epidermis and sebaceous glands. 00:23:29.00 We call this phenomenon stem cell plasticity, and it's something that we see in 00:23:35.10 a variety of different stem cell... stem cells from tissues. 00:23:40.24 It's a phenomenon that basically allows a tissue to receive, essentially, a danger call 00:23:49.18 in a wound that tells the stem cells nearby to be able to repair and replenish the tissue 00:23:58.14 at all costs, and as fast as possible. 00:24:00.25 So, it's always the closest stem cells, then, to the wound that can repair it. 00:24:06.03 Unfortunately, these stem cells will not automatically make neurons or make other tissues 00:24:13.05 of our body without some manipulation. 00:24:15.27 But these stem cells will, however, naturally make not only hair follicles but also epidermis 00:24:24.16 and sebaceous glands. 00:24:26.26 So, we've studied these stem cells over the course of many years, now. 00:24:33.16 And we know that these stem cells are defined by their transcriptional profile, effectively, 00:24:41.16 the gene expression pattern that these stem cells make. 00:24:44.28 And we know a good deal about this. 00:24:46.20 We know that there are the transcription factors in purple, a variety of different transcription factors 00:24:52.19 that are made by these hair follicle stem cells and not by other stem cells, 00:24:59.03 at least as a cohort. 00:25:00.17 We know that there are some factors, like LGR5, which are made by a number of different stem cells: 00:25:05.27 intestinal epithelial stem cells, hair follicle stem cells, and many other 00:25:11.15 types of stem cells. 00:25:12.28 We know that integrins are a characteristic of many different types of stem cells, 00:25:18.22 and they're expressed at very high levels in the hair follicle stem cells. 00:25:23.11 And then for a quiescent stem cell, the niche signals basically produce a gene expression profile 00:25:31.09 by the stem cells that reflect reduced levels of Wnt signaling and reduced levels 00:25:37.04 of sonic hedgehog signaling. 00:25:39.00 Those are the pro-activating cues for the stem cells. 00:25:42.08 Whereas BMP signals and FGF18 signals, the inhibitory cues that tell those stem cells 00:25:48.19 to remain in quiescence, those signals are upregulated. 00:25:53.13 And then differentiation, whether it be differentiation for the epidermis or the sebaceous gland 00:25:58.27 or the hair follicle, all of those programs are suppressed by the stem cells. 00:26:04.13 So, their transcriptional program reflects the behavior of these stem cells. 00:26:11.10 We characterized the transcriptional program by taking the stem cells directly out of the tissue, 00:26:17.28 using fluorescence activated cell sorting to purify those cells, 00:26:21.26 and basically taking those RNAs and directly profiling them. 00:26:26.24 We also used a technology known as conditional knockout technology to be able to excise 00:26:34.19 the transcription factors from the skin of the animal. 00:26:39.06 When we excise TCF3 and TCF4 -- these are DNA binding proteins which can also bind beta-catenin, 00:26:46.15 which is a downstream Wnt effector -- we found that the loss of TCF3 and 4 basically no longer... 00:26:53.19 the stem cells can no longer maintain the quies... their... their activity. 00:26:59.20 We learned that the loss of Lhx2 is required for maintaining the stem cells. 00:27:05.11 And similarly, the loss of Sox9 is required for maintaining the stem cells. 00:27:11.06 Surprisingly, when we knocked out beta-catenin, what we found is no hair follicles made. 00:27:17.15 So, with no Wnt signaling there were no hair follicles made. 00:27:21.28 Interestingly, when we knocked out TCF3 and 4, even though it was necessary 00:27:27.20 to make a hair follicle, what we found to maintain the hair follicles stem cells... what we found 00:27:32.23 is a burst of hair growth. 00:27:35.02 So in this way, we learned that TCF3 and 4 antagonize what Wnt signaling can do, 00:27:41.10 and that keeps the stem cells in quiescence, because Wnt is typically a proliferation-associated factor. 00:27:49.04 Lhx2, in the absence, basically produces, where the stem cell niche was, 00:27:56.01 now a niche full of sebaceous gland. 00:27:58.05 So, Lhx2 is necessary to repress the differentiation program of the sebaceous gland while 00:28:06.12 the stem cells are stem cells in their native niche. 00:28:09.12 Sox9 represses the epidermal stem cell differentiation program. 00:28:14.26 And without Sox9, the hair follicle stem cell niche turns into an epidermal cyst. 00:28:19.28 So, we understand how these different factors are working and how these different transcription factors... 00:28:25.27 what they're necessary for. 00:28:28.13 So, let's put this all together now. 00:28:31.14 And I'll give you one example, and that is from BMP signaling, which I told you is important 00:28:37.12 to control stem cell quiescence. 00:28:40.23 So, we've learned, over the course of our studies, that the BMP interacting with 00:28:47.09 the BMP receptor on the stem cells is sufficient to activate the canonical transcription factor 00:28:55.18 called phospho-SMAD1, along with phospho... along with SMAD4, which then control, downstream, 00:29:02.03 a group of transcription factors that I've shown you here. 00:29:06.17 Together, these factors are necessary to control the quiescence of the stem cells. 00:29:13.11 So, we then looked at what happens in an aging mouse. 00:29:18.11 And what we found was that in the first hair cycle of the mouse, that happens relatively quickly. 00:29:26.08 Only a couple days separates the time at which one hair follicle has completed its cycle 00:29:32.11 and the next hair cycle begins. 00:29:34.05 But now, as the animals get older, there's longer and longer times between when our hairs grow 00:29:40.20 -- or when the animal's hair grows -- and when the hair, basically... when the stem cells 00:29:46.20 sit in quiescence. 00:29:48.05 They spend far longer sitting in quiescence than they do in the younger mice. 00:29:54.23 And when we traced this, what we found is that the dermis and the old fat, 00:30:01.26 or the old adipose tissue, of these older mice turned out to produce very high levels of BMP. 00:30:08.15 And that's what maintained these stem cells in their quiescent state. 00:30:12.15 We could treat the old mice with a drug called VIVIT which is an inhibitor of Nfatc1, 00:30:21.14 one of the transcription factors downstream of the BMP signal. 00:30:25.23 And we could regrow... reactivate those dormant hair follicle stem cells in order to make 00:30:32.05 a new round of hair growth. 00:30:34.07 And so, at this point in time in our studies, what we learned is that actually the stem cell numbers 00:30:39.16 still remain high in the older animal, but it's their activity that wanes with age. 00:30:46.10 So, hair grain is also a phenomenon that... that happens with age. 00:30:53.26 And in this study, what others, May... 00:30:57.11 Mayumi Ito and her coworkers, learned was that the melanocyte stem cells and 00:31:04.21 the hair follicle stem cells reside in the same niche, and they respond to similar signals. 00:31:09.24 And this was also the work of Emi Nishimura and her coworkers in Japan. 00:31:17.19 And so hair follicle and melanocytes stem cells are hanging out in the same niche. 00:31:22.18 They respond to the same signals. 00:31:25.02 And the importance of that is that the hair follicle stem cells and melanocytes stem cells 00:31:32.18 will get activated at the same time. 00:31:35.08 They'll differentiate so that the melanocyte cells will now begin producing pigment, 00:31:42.06 and the hair follicle cells will begin producing hair. 00:31:45.27 And at that time, the differentiated melanocytes can provide that pigment to the differentiating 00:31:53.00 hair cells. 00:31:54.04 And that's what gives our hair its color. 00:31:57.03 And so, when the stem cells of the hair follicle are unable to be able to produce hair, 00:32:04.09 so too, typically, the melanocytes stem cells end up being unable to be activated, 00:32:12.04 because they reside in the same niche. 00:32:14.05 So, one of my graduate students, Kenneth Lay, had the remarkable brilliant idea that perhaps 00:32:20.28 if he simply deleted these transcription factors that are important in controlling quiescence 00:32:29.01 downstream of the BMP pathway, that he might be able to accelerate the differences, then, 00:32:34.20 that exist between the younger mice and the older mice 00:32:38.06 with regards to the length of their hair cycle. 00:32:41.21 Could he reduce the length of time that stem cells spend in quiescence? 00:32:47.23 And the answer to that experiment was quite remarkable. 00:32:51.09 And in fact, now, the mice just kept on cranking out hair. 00:32:54.21 Their stem cells were almost constantly activated. 00:32:58.11 A new hair would grow, the process would go through its cycle, and then the cycle would 00:33:03.22 reinitiate again, over and over and over again. 00:33:07.13 So, this was pretty exciting for us. 00:33:12.20 And we began to wonder whether BMP inhibition could be the Fountain of Youth 00:33:17.26 for endless hair growth. 00:33:19.16 Well, unfortunately in science, things aren't always so simple. 00:33:24.14 And what happened was that the animals precociously became grey, sooner than did their counterparts. 00:33:32.14 And it turned out that when the transcription factor was mutated, and lacking, 00:33:39.05 while the hairs kept on being cranked out eventually the number of stem cells declined. 00:33:45.03 And that then led to a precocious graying and a precocious thinning of the hair. 00:33:52.07 So, we're not yet there. 00:33:54.06 But we have some predictions. 00:33:56.15 And the predictions might be that the key to the Fountain of Youth with regard to hair growth 00:34:00.17 is a well-balanced BMP. 00:34:03.01 Clearly, by cranking out and activating those stem cells continuously.... was deleterious 00:34:09.12 for the mouse's ability to produce hair endlessly. 00:34:14.21 But perhaps with a balance tempering those numbers of hair cycles, one might be able 00:34:21.14 to achieve the goal of being able to extend the ability of... time that individuals 00:34:27.27 could grow their hair. 00:34:29.17 So, BMP also plays an important role in development and development of the skin. 00:34:37.08 And here we were fascinated by the fact that the dermis instructs the fate of what 00:34:44.02 the epidermal bud would do. 00:34:46.06 So, in embryonic development, the skin begins as that single layer of epithelium and epidermal buds 00:34:54.09 begin to appear. 00:34:56.06 And those buds can become a mammary gland, they can become a hair bud, they can become 00:35:02.02 a sweat gland bud. 00:35:04.14 It's depending upon what the dermis is instructing them to do. 00:35:09.14 And so regionally, we end up getting these different appendages of the epithelium, 00:35:17.26 generated as a consequence of the different types of mesenchyme or the derm... dermis that we have. 00:35:23.10 It turns out that in most mammals there's a BMP signal called BMP5 which is 00:35:29.27 regionally spiking only in the paw skin of the animals. 00:35:33.00 And so what happens is that most of the body region of the animals has hair, and there's 00:35:37.20 very few sweat glands. 00:35:39.02 There's only sweat glands, or eccrine sweat glands, on their paws. 00:35:43.04 And eccrine sweat glands are what allows us, what allows our body, to thermoregulate. 00:35:48.20 It's what allows us to go out there and run a marathon as we sweat three liters of sweat 00:35:54.18 in so doing. 00:35:56.04 But most mammals can't do that, and they can't do it because they have fur instead of 00:36:02.17 sweat glands over their body surface. 00:36:05.13 And so we began to study this process. 00:36:09.07 And as we discovered, the BMP5 regionally spikes in the paw dermis. 00:36:15.22 It doesn't spike in the body dermis, and therefore the animals get hair exclusively. 00:36:21.11 But then when we looked at human embryonic development, what we discovered is that BMP5 00:36:28.22 seems to be deregulated in a different way in humans than it is even in higher primates. 00:36:35.15 And in this case, BMP5 spikes broadly and temporally in the body dermis, so that 00:36:41.14 the very first few waves of epidermal bud development are forming hair in humans, 00:36:50.08 but then that last wave, when BMP5 spikes over the body, basically we end up getting hair... 00:36:57.04 getting sweat glands over our body. 00:36:59.17 And that's what gives us those wonderful properties to be able to run marathons, and to be able 00:37:07.03 to sweat and survive in extreme climates relative to other... other animals. 00:37:15.02 So, what else can we do with these adult skin cells? 00:37:19.26 Well, in part one of my discussion, I already told you about the ability to change the behavior 00:37:29.05 of an adult skin cell, and turn it into an induced pluripotent stem cell, by the activity 00:37:37.28 of four transcription factors that are produced by, or expressed by, embryonic stem cells. 00:37:43.08 And that was a remarkable change and a remarkable advance because it allowed us, now, 00:37:48.24 to be able to culture neurons, pancreatic beta cells, heart cells, etc. in vitro, starting with 00:37:56.20 skin cells. 00:37:58.23 So, can we reprogram in smaller steps? 00:38:02.17 Well, our ability to be able to culture, for instance, hair follicle stem cells... 00:38:07.22 we now know that if we simply switch off LHX2, we can make sebocytes in culture. 00:38:13.25 If we switch off the transcription factor SOX9, we can make epidermis rather than hair. 00:38:21.10 And if we switch off the regulatory factors for NFATc1 or Foxc1, we can basically halt tissue... 00:38:28.02 we can... if we switch them on, we can halt tissue regeneration. 00:38:33.24 If we switch them off, we can favor hair follicle tissue regeneration and hair regeneration. 00:38:40.04 And finally, an interesting aspect is that if we simply give the hair follicle stem cell 00:38:47.04 a transcriptional regulatory factor called PAX, a particular type of the PAX transcription factor, 00:38:52.07 then we can instruct hair follicle stem cells to make corn... 00:38:56.18 to become corneal stem cells. 00:38:59.02 And when you think about cases where patients have both eyes that are blinded by some industrial burn, 00:39:05.05 then this type of potential reprogramming in smaller steps might offer the potential 00:39:12.02 for future therapy. 00:39:14.08 So, I'd like to switch, now, to a problem of, what do I mean by switching on genes and 00:39:21.14 switching off genes? 00:39:23.18 And two of my graduate students, Rene Adam and Hanseul Yang had the interest of being able 00:39:31.04 to examine the chromatin landscape, the gene expression landscape, of the... of the 00:39:40.03 hair follicle stem cells. 00:39:41.26 And what they learned from that is that there are about 350 genes that are expressed by 00:39:48.04 the hair follicle stem cells, that are regulated by very large enhancers 00:39:53.00 -- Rick Young has coined them "super enhancers" -- 00:39:57.22 that bind all of the different transcription factors, 00:40:00.20 as we showed, that are expressed by the hair follicle stem cells. 00:40:04.28 So, Tcf3, Tcf4. 00:40:07.21 We talked about Sox9. 00:40:09.06 We talked about Lhx2 and Nfatc1. 00:40:12.20 And they all bind to relatively short stretches of DNA that are contained within these large 00:40:18.27 "super enhancers". 00:40:21.09 So, those short... short stretches of DNA seem to have the... not only the binding of 00:40:29.22 all these transcription factors, but they also have the sequence motifs to which 00:40:35.06 those transcription factors bind. 00:40:38.00 So, these genes turn out to be also the genes that are most important in maintaining 00:40:45.26 the hair follicle stem cells in their quiescent, undifferentiated state. 00:40:50.01 If we take a look at a handful of the 350 genes, then, that are these special genes 00:40:55.27 regulated by super enhancers, what we find is that they include the Sox9 gene, the Lhx2 gene, 00:41:03.13 the Nfatc1 gene, and DNA... the BMP downstream gene... transcription factor activators. 00:41:12.11 They also include the BMP inhibitor and the BMP receptor. 00:41:16.24 They include a Wnt inhibitor, Dkk3, and the Wnt receptor, or Frizzled. 00:41:22.17 They also include the integrin genes that I emphasized as being important for maintaining 00:41:28.22 hair follicle stem cells. 00:41:30.23 Effectively, this short list of super enhancer-regulated genes contains all of the critical information 00:41:38.14 that is necessary for these genes to be able to essentially be a hair follicle stem cell. 00:41:45.21 I also point out that many of the genes on this list have already been associated 00:41:51.00 with human genetic disorders of the hair and the skin. 00:41:56.15 So, what I've told you is that there's a master group of transcription factors for the skin 00:42:03.21 stem cells, but it also turns out that there's a master group of transcription factors 00:42:08.04 for every stem cell type. 00:42:10.11 And they're different for different stem cell types. 00:42:13.09 So, the first studies that were done were on embryonic stem cells. 00:42:18.15 And Rick Young and his coworkers showed that the super enhancers are regulated by 00:42:24.09 the transcription factors that are necessary to maintain embryonic stem cells. 00:42:30.13 What I've just showed you is that in an adult tissue stem cell, the hair follicle stem cell, 00:42:36.02 that there's a different cohort of transcription factors that are necessary to maintain 00:42:41.00 these stem cells in being hair follicle stem cells. 00:42:44.11 So, we're starting to dig deeper into understanding, what are the differences in embryonic versus 00:42:49.26 adult stem cells? 00:42:51.26 Another interesting aspect is that all of these transcription factors are only expressed 00:42:58.03 as a cohort in hair follicle stem cells. 00:43:01.04 So, you might see Tcf3, for instance, in brain stem cells. 00:43:05.17 You'll see Tcf4 in intestinal stem cells, Lhx2 in hematopoietic stem cells, 00:43:10.20 Sox9 in bone stem cells. 00:43:14.08 But the only place you see them all together at one place in time are 00:43:17.25 the hair follicle stem cells. 00:43:19.21 And it turns out that we can take advantage of that. 00:43:22.15 We can take one of these elements that contain the binding sites for all of these 00:43:29.10 different transcription factors, and we can use that to drive the expression of a fluorescent green protein 00:43:34.20 in mice. 00:43:36.22 And now, the fluorescent green protein is only expressed by the hair follicle stem cells 00:43:42.05 -- far more specificity than we've ever achieved simply by taking a chunk of chromatin or 00:43:49.24 a promoter region, an enhancer region, and using that to drive expression in mice. 00:43:56.09 So, this type of information is really important for researchers to be able to dig deeper into 00:44:02.23 the biology of stem cells, in this case the hair follicle stem cells. 00:44:08.05 We also are learning that stem cells change their gene expression program outside of their niche. 00:44:14.05 I already showed you that a stem cell outside of its niche, when grafted back onto the... 00:44:19.18 to an animal, can generate hair follicles, epidermis, and sebaceous glands. 00:44:24.25 But now, if we take a wound induced enhancer, if we characterize the chromatin state of 00:44:31.21 the stem cell while it's in the act of repairing a wound, and now identify those regulatory elements, 00:44:37.25 and use those to drive eGFP in a mice... in a mouse, now what we find is that 00:44:45.00 in the unwounded skin there's no activity. 00:44:48.14 And now we wound the skin, and the activity turns on. 00:44:52.00 So, these regulatory elements contain the information necessary to not only 00:44:58.27 maintain this stem cell, but in this case to tell the stem cell what to do, and to change 00:45:04.16 its program of gene expression if it's going to repair a wound versus if it's just going to replace 00:45:10.27 a cell that is dying in the tissue. 00:45:14.00 So, we're also looking at how skin inflammation elicits changes in gene expression in epidermal stem cells. 00:45:22.19 So, when stem cells receive an inflammatory assault, how do they respond? 00:45:29.05 And what we learned from our studies is that they respond, and they activate new genes. 00:45:36.11 And by taking the regulatory elements from those genes, we can use those to drive 00:45:42.01 the expression of, in this case, a reporter GFP gene. 00:45:45.13 So, here we've generated mice that express these particular reporter elements, 00:45:52.21 but they only do so when the skin has been subjected to inflammation. 00:45:57.24 So, in the naive skin, we see no expression of the eGFP protein. 00:46:05.03 In the presence of inflammation -- if we give the skin an inflammatory assault -- 00:46:09.26 now we see the activation of these enhancer elements. 00:46:14.27 So we call these elements inflammation sensors. 00:46:18.03 And what we've found is that there are about 2,000 inflammation-sensing chromatin domains 00:46:23.17 that remain open in skin stem cells long after the inflammation has resolved. 00:46:30.20 These sensors rapidly, then, become activated again with... and activate their associated genes 00:46:37.20 when the skin sees inflammation again. 00:46:40.19 And that's a remarkable property, because, basically, this confers to the epidermal stem cells 00:46:47.03 a memory, an inflammatory memory, that it's seen the inflammation before. 00:46:52.28 And even though the gene expression program is normal in... after the information has resolved, 00:47:00.01 there are these hidden memory open chromatin domains that basically allow 00:47:05.24 the genes to be poised so that the genes can now be activated again, and activated quicker, 00:47:12.21 the next time inflammation occurs. 00:47:16.24 And why do we think that that's important or relevant? 00:47:20.17 Skin inflammation elicits marks on epidermal stem cell chromatin. 00:47:25.09 Remember that the epidermal stem cells are there for the long haul. 00:47:28.05 They see the inflammation; they remain in their active state, as stem cells, 00:47:34.16 long after the inflammation has resolved. 00:47:37.10 But they have that memory that they've seen the inflammation before. 00:47:41.24 That memory resides in the virtue of epigenetic marks. 00:47:47.26 And here are some examples. 00:47:49.08 There are columns, now, of the epigenetic marks that are shown, in this case... in control chromatin, 00:47:56.12 shown in gray, there's no epigenetic marks. 00:48:00.22 Now, there's inflammation, the skin experiences inflammation, and we see two epigenetic marks 00:48:08.16 appear: here in this gene on the right. 00:48:11.25 And now, 30 days later, what we find is those epigenetic marks are still there. 00:48:19.02 And even 6 months later, there's still traces of those epigenetic marks residing within 00:48:26.15 these... this chromatin. 00:48:29.16 So, the epidermal stem cells have a memory, harbored in chromatin, that they've seen 00:48:37.07 the inflammation before. 00:48:39.10 And what's the importance of that? 00:48:40.21 Well, let's think about psoriasis or atopic dermatitis or contact dermatitis infections. 00:48:47.24 The infections typically come and go. 00:48:53.26 And when they come back again, they typically come back to the same places. 00:48:58.26 We're now starting to understand why that is, because, effectively, the epidermal stem cells 00:49:04.14 have a memory. 00:49:06.05 They've seen the inflammation before so that, if they see it again, they respond faster 00:49:11.14 the next time. 00:49:12.20 So, what I've told you is that adult stem cells replenish dying cells of a tissue, 00:49:19.10 and that they repair wounds. 00:49:20.27 I've told you that they reside in niches whose crosstalk dictates their behavior. 00:49:26.15 I've told you that key stem cell genes are regulated by super enhancers that bind stem cell 00:49:32.13 transcription factors, as well as transcription factors that are induced by the niche microenvironment. 00:49:38.13 For infla... inflammation, for instance, it might be a phosphorylated, 00:49:43.11 active version of STAT3, along with the epidermal stem cell factors. 00:49:48.24 I've also told you that trained immunity, the memory of inflammation, is not a phenomenon 00:49:58.14 that's restricted to the immune system, but occurs in epidermal, and we think likely 00:50:03.26 other epithelial, stem cells that bear the brunt of inflammation. 00:50:09.05 Inflammatory bowel disease, for in... for instance, or certain types of lung inflammation. 00:50:14.12 Somehow the tissue remembers that it's seen the inflammation before. 00:50:18.11 So, for immune cells, trained immunity enhances the ability of the immune cells 00:50:26.04 to eliminate the infection if they see it again. 00:50:29.13 For epidermal stem cells, trained immunity enhances their ability to restore 00:50:34.22 the skin barrier when breached and to aid in recruiting the immune system. 00:50:39.22 And remember that the most important thing that the skin epithelium does is 00:50:45.13 basically to produce that barrier that keeps harmful microbes out, and essential bodily fluids in. 00:50:51.24 And whether we're wounding the skin, or whether we end up with a hyper... 00:50:56.02 hyperinflammatory response, the barrier is at risk. 00:51:01.04 And the epithelium, the epidermal stem cells, respond by trying to repair that barrier 00:51:06.16 as fast as they can. 00:51:08.06 Sometimes they're not very effective at doing it, and that then can lead to chronic wound-healing 00:51:13.28 or chronic inflammation. 00:51:15.25 And those are obviously areas that we'd like to be able to adapt our understanding for 00:51:21.26 to make progress in treating those diseases in the future.