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.