WEBVTT 00:00:01.000 --> 00:00:04.000 Good morning, class. Nice to see you here. 00:00:04.000 --> 00:00:09.000 Yesterday morning I was in Australia and today I'm here. 00:00:09.000 --> 00:00:14.000 And it's nice to be back. It's a long way home. 00:00:14.000 --> 00:00:19.000 We're talking today about the issues of growth and differentiation 00:00:19.000 --> 00:00:23.000 about normal cells becoming specialized into different types of 00:00:23.000 --> 00:00:28.000 cells throughout the body and how that leads ultimately to the issues 00:00:28.000 --> 00:00:33.000 of organismic cloning. And, to give you an overall 00:00:33.000 --> 00:00:37.000 background about this, I want to show you the way that a 00:00:37.000 --> 00:00:42.000 worm is put together. This is the worm C. elegans. 00:00:42.000 --> 00:00:46.000 I don't know how well this overhead will come out. 00:00:46.000 --> 00:00:50.000 You can see it reasonably well. So, this is the worm Caenorhabditis 00:00:50.000 --> 00:00:55.000 elegans. It's been an object of much study here. 00:00:55.000 --> 00:00:59.000 Bob Horowitz in our own department just got the Nobel Prize 00:00:59.000 --> 00:01:07.000 for his work on this. 00:01:07.000 --> 00:01:11.000 And the reason it's been the object of so much study is, 00:01:11.000 --> 00:01:16.000 in fact, that it's a relatively simple organism. 00:01:16.000 --> 00:01:20.000 It's the way that you and I looked about 600 million years ago when 00:01:20.000 --> 00:01:25.000 Metazoa, remember Metazoa means multicellular organisms, 00:01:25.000 --> 00:01:29.000 first arose on the face of the planet. Let's see if I have too 00:01:29.000 --> 00:01:34.000 much overlap here. Well, that's about the way it should 00:01:34.000 --> 00:01:38.000 look. Now, what's remarkable about this organism is it has something 00:01:38.000 --> 00:01:42.000 like 965 cells in the adult. The cells all descend from a 00:01:42.000 --> 00:01:47.000 fertilized egg. It happens to be that this worm is 00:01:47.000 --> 00:01:51.000 a hermaphrodite. So, you probably know 00:01:51.000 --> 00:01:55.000 hermaphrodites are both male and female and it can fertilize itself 00:01:55.000 --> 00:02:00.000 or two worms can get together and fertilize each other. 00:02:00.000 --> 00:02:04.000 Because, as you can see, it makes both eggs and it makes 00:02:04.000 --> 00:02:08.000 sperm. And what's most interesting is the finite number of cells in the 00:02:08.000 --> 00:02:13.000 body of the adult. As I said, it's 965, 00:02:13.000 --> 00:02:17.000 whose lineage can be traced through a pedigree that's given right up 00:02:17.000 --> 00:02:22.000 here. So, one can plot out, with great precision, how all the 00:02:22.000 --> 00:02:26.000 cells, how the egg cell and the fertilized egg divides in two, 00:02:26.000 --> 00:02:31.000 each of those cells divides again in two and how ultimately one has a 00:02:31.000 --> 00:02:35.000 whole series of different descendents from different branches 00:02:35.000 --> 00:02:40.000 of this very elaborate family tree. Now, one of the issues that we want 00:02:40.000 --> 00:02:44.000 to pursue is the fact that the genomes, in principle, 00:02:44.000 --> 00:02:48.000 all the cells in this organism are the same. That is to say whenever 00:02:48.000 --> 00:02:52.000 the cell goes through a cycle of growth and division the cell is 00:02:52.000 --> 00:02:56.000 genetically identical, yet phenotypically, yet the behavior 00:02:56.000 --> 00:03:00.000 reach of these cells becomes increasingly different. 00:03:00.000 --> 00:03:04.000 And here you can see the different lineage of cells. 00:03:04.000 --> 00:03:08.000 This organism devotes a disproportionately large amount of 00:03:08.000 --> 00:03:12.000 its anatomy to reproduction, much more even than we do. Here, 00:03:12.000 --> 00:03:16.000 this is the cuticle, the outer coat. Here's the vulva, the female organ. 00:03:16.000 --> 00:03:20.000 This is the male organ and a variety of other cells types. 00:03:20.000 --> 00:03:24.000 Here's the pharynx. And yet all the cells in all these organisms, 00:03:24.000 --> 00:03:28.000 with the exception of the gametes, remember gametes means sperm and egg 00:03:28.000 --> 00:03:33.000 are genetically identical. The gametes have only half the 00:03:33.000 --> 00:03:37.000 genetic content. Everybody else is identical, 00:03:37.000 --> 00:03:42.000 has a diploid genome, that is to say two copies of each gene. 00:03:42.000 --> 00:03:46.000 And one can trace this all out. And this represents one of the 00:03:46.000 --> 00:03:50.000 great mysteries of developmental biology, which is to say how are 00:03:50.000 --> 00:03:55.000 cells that genetically identical to one another genotypically identical 00:03:55.000 --> 00:03:59.000 to one another, phenotypically quite different one 00:03:59.000 --> 00:04:04.000 from the other? What makes them so different? 00:04:04.000 --> 00:04:08.000 In fact, this image that we have here, which in itself represents a 00:04:08.000 --> 00:04:13.000 stunning achievement, that is being able to trace the 00:04:13.000 --> 00:04:18.000 pedigree of each cell in the adult body is very different from our own 00:04:18.000 --> 00:04:22.000 lineage because, as I may have mentioned to you in 00:04:22.000 --> 00:04:27.000 the past, each of us goes through ten to the sixth mitoses in a 00:04:27.000 --> 00:04:32.000 lifetime, you and I. That's ten to the sixteen cell 00:04:32.000 --> 00:04:36.000 divisions. And at any one time we have roughly three times ten to the 00:04:36.000 --> 00:04:40.000 thirteenth cells in our body. So, that means, if you think about 00:04:40.000 --> 00:04:44.000 that carefully, the ratio between these two suggests 00:04:44.000 --> 00:04:48.000 there's roughly a hundred times more here than here, 00:04:48.000 --> 00:04:52.000 three hundred times more It means, roughly speaking, that our body 00:04:52.000 --> 00:04:56.000 turns over roughly a hundred times in our lifetime. 00:04:56.000 --> 00:04:59.000 That is to say the cells turnover. Not all of the cells, 00:04:59.000 --> 00:05:03.000 but that there's a continuing replacement of existing cells with 00:05:03.000 --> 00:05:06.000 new cells. After all, if, in fact, there were no such 00:05:06.000 --> 00:05:10.000 replacement than we might, as an adult, be formed of this many 00:05:10.000 --> 00:05:13.000 cells, and we would stay with the exact same number of cells 00:05:13.000 --> 00:05:17.000 throughout our lives. But this, in fact, is the number of 00:05:17.000 --> 00:05:21.000 cell divisions. And so, there's an enormous turnover 00:05:21.000 --> 00:05:24.000 which, right away on its surface, independent of the fact that it's an 00:05:24.000 --> 00:05:28.000 enormously large number, precludes one from really being able 00:05:28.000 --> 00:05:31.000 to draw out a pedigree like this, prevents anyone from really 00:05:31.000 --> 00:05:35.000 understanding how each particular cell can trace its line of decent 00:05:35.000 --> 00:05:39.000 back to the fertilized egg. So, what we really want to explore, 00:05:39.000 --> 00:05:44.000 in this lecture and the next one, is this major puzzle that one starts 00:05:44.000 --> 00:05:48.000 out with a fertilized egg. Let's say this is a fertilized egg. 00:05:48.000 --> 00:05:53.000 It divides in two. It divides in four. And through subsequent cycles 00:05:53.000 --> 00:05:58.000 of growth and division we ultimately end up with the adult. 00:05:58.000 --> 00:06:03.000 Already at the four-cell stage, here in a vertebrate embryo, these 00:06:03.000 --> 00:06:08.000 cells have begun to take different phenotypic paths. 00:06:08.000 --> 00:06:13.000 That is to say cells have begun to commit themselves into entering into 00:06:13.000 --> 00:06:18.000 one or another differentiation lineage. And when I say a 00:06:18.000 --> 00:06:23.000 differentiating lineage, I mean a group of cells which has 00:06:23.000 --> 00:06:29.000 already made the decision to become blood cells, to become gut, 00:06:29.000 --> 00:06:34.000 to become nerve cells and so forth. And these commitments already start 00:06:34.000 --> 00:06:38.000 here at the four-cell stage, and they continue to play themselves 00:06:38.000 --> 00:06:42.000 out until one reaches a newborn, and then thereafter one just grows 00:06:42.000 --> 00:06:46.000 bigger. One other important thing to show here is that this pedigree 00:06:46.000 --> 00:06:50.000 that I showed you here is not simply the result of exponential expansion 00:06:50.000 --> 00:06:54.000 of all the cells, because many cells, 00:06:54.000 --> 00:06:58.000 during the course of development, are actually weeded out from 00:06:58.000 --> 00:07:02.000 embryonic tissue. And this happens even in our own 00:07:02.000 --> 00:07:07.000 development. For example, here, if we look at our fingers, 00:07:07.000 --> 00:07:11.000 I remember now I talked about fingers about two weeks ago and got 00:07:11.000 --> 00:07:16.000 myself into some hot water. If we look at our fingers, you'll 00:07:16.000 --> 00:07:21.000 see here we have five fingers, God willing, and but early in 00:07:21.000 --> 00:07:26.000 embryogenesis our hand looks like a solid flap of tissue. 00:07:26.000 --> 00:07:30.000 And what happens, during the course of vertebra development, 00:07:30.000 --> 00:07:35.000 is that the tissue in between the beginning fingers is eliminated 00:07:35.000 --> 00:07:40.000 through the process of apoptosis. Apoptosis means programmed cell 00:07:40.000 --> 00:07:46.000 death. Apoptosis is equivalent to cell suicide. And what I mean by 00:07:46.000 --> 00:07:52.000 that is to say that development involves not only the exponential 00:07:52.000 --> 00:07:58.000 proliferation of cells but it involves the selective elimination 00:07:58.000 --> 00:08:04.000 of cells here in a very obvious anatomical way. 00:08:04.000 --> 00:08:07.000 It sometimes can be defective, in which case individuals are born 00:08:07.000 --> 00:08:11.000 with large webs between their fingers. And this is part of normal 00:08:11.000 --> 00:08:15.000 development. And the same can be said here. If there were no 00:08:15.000 --> 00:08:18.000 apoptosis during the development of this worm embryo then there would be 00:08:18.000 --> 00:08:22.000 vastly larger numbers of cells. We mentioned implicitly apoptosis 00:08:22.000 --> 00:08:26.000 during the development of the immune system, because recall there that 00:08:26.000 --> 00:08:29.000 bee cells, which are destine to produce antibodies, 00:08:29.000 --> 00:08:33.000 if they produce inappropriate kinds of antibodies, 00:08:33.000 --> 00:08:37.000 if they produce antibodies that are self-reactive, 00:08:37.000 --> 00:08:41.000 i.e., recognize some of the body's own proteins, those cells are 00:08:41.000 --> 00:08:44.000 eliminated by apoptosis. If they produce defective antibody 00:08:44.000 --> 00:08:48.000 molecules, they're eliminated by apoptosis. And, 00:08:48.000 --> 00:08:51.000 therefore, differentiation, which is what we're talking about 00:08:51.000 --> 00:08:55.000 here, involves not only the commitment of cells to a certain 00:08:55.000 --> 00:08:58.000 lineage, but the purpose of elimination of cells in certain 00:08:58.000 --> 00:09:02.000 parts of the organism in order to carve and sculpt out properly 00:09:02.000 --> 00:09:05.000 shaped tissue. Again, our fingers are one dramatic 00:09:05.000 --> 00:09:09.000 example of that. It turns out we can learn an awful 00:09:09.000 --> 00:09:13.000 lot about this process by studying one specialized adult tissue, 00:09:13.000 --> 00:09:17.000 which is to say the organs of hematopoiesis. 00:09:17.000 --> 00:09:20.000 And they'll teach us a lot about some of the lessons we need to learn 00:09:20.000 --> 00:09:24.000 about organismic development and differentiation. 00:09:24.000 --> 00:09:28.000 And when I use the word hematopoiesis, 00:09:28.000 --> 00:09:32.000 the term hematopoiesis, or the adjective hematopoietic 00:09:32.000 --> 00:09:36.000 refers to the creation, the formation of different kinds of 00:09:36.000 --> 00:09:40.000 blood cells. In fact, we know that all the cells 00:09:40.000 --> 00:09:45.000 in the blood descend in the organism from a common progenitor. 00:09:45.000 --> 00:09:50.000 And this progenitor is called a pluripotent stem cell. 00:09:50.000 --> 00:09:55.000 Pluripotent means that this stem cell, and we'll define a stem cell 00:09:55.000 --> 00:10:00.000 momentarily, this stem cell is able to create descendents which can 00:10:00.000 --> 00:10:05.000 commit themselves in a number of distinct directions. 00:10:05.000 --> 00:10:09.000 They can differentiate in a number of distinct directions. 00:10:09.000 --> 00:10:14.000 In this case, we see all these various kinds of white and red blood 00:10:14.000 --> 00:10:19.000 cells which descend from this pluripotent stem cell. 00:10:19.000 --> 00:10:24.000 And, as a consequence, we call it pluripotent because it 00:10:24.000 --> 00:10:29.000 has these multiple distinct types of differentiation lineages. 00:10:29.000 --> 00:10:34.000 So, here we talk about pluripotent. Later on I'll talk about totipotent 00:10:34.000 --> 00:10:38.000 cells. Totipotent are cells that can do everything. 00:10:38.000 --> 00:10:42.000 Therefore, in fact, what's a totipotent cell? 00:10:42.000 --> 00:10:46.000 Well, a cell in the early embryo, including a fertilized egg is 00:10:46.000 --> 00:10:50.000 totipotent in that it can direct its descendents into all of the 00:10:50.000 --> 00:10:54.000 differentiation lineages in the body. Here we have a cell that's already 00:10:54.000 --> 00:10:58.000 more limited. It's only pluripotent, pluri in the sense of multiple 00:10:58.000 --> 00:11:03.000 but not total. Totipotent obviously means it can do 00:11:03.000 --> 00:11:07.000 everything. And here we see the different kinds of derivative white 00:11:07.000 --> 00:11:11.000 blood cells that exists in the bone marrow and in the circulation, 00:11:11.000 --> 00:11:15.000 and there's a whole series of different ones of them. 00:11:15.000 --> 00:11:19.000 We'll talk about some of them shortly, but we've already 00:11:19.000 --> 00:11:23.000 encountered some of them up here in the form of T cells and B cells. 00:11:23.000 --> 00:11:27.000 When we use the word stem cell the essence of the definition 00:11:27.000 --> 00:11:33.000 is as follows. A stem cell is a cell that can 00:11:33.000 --> 00:11:39.000 self-renew and it can also have a differentiated daughter. 00:11:39.000 --> 00:11:46.000 So, here's the way one can diagram a stem cell. Here's a stem cell 00:11:46.000 --> 00:11:53.000 that has two daughter cells. One daughter cell is exactly like 00:11:53.000 --> 00:11:59.000 Mom and the other daughter cell has undertaken a program of becoming 00:11:59.000 --> 00:12:04.000 differentiated. So, here we have an asymmetric cell 00:12:04.000 --> 00:12:08.000 division on the part of this stem cell up here. 00:12:08.000 --> 00:12:14.000 We'll prove later on that these two 00:12:14.000 --> 00:12:18.000 cells are genetically identical, but clearly they're reading out 00:12:18.000 --> 00:12:21.000 their genes in quite different ways. This cell is absolutely the same as 00:12:21.000 --> 00:12:24.000 the mother cell. This cell has already committed 00:12:24.000 --> 00:12:28.000 itself. It's made the commitment to differentiate in one 00:12:28.000 --> 00:12:32.000 or another lineage. And another way of noting this 00:12:32.000 --> 00:12:36.000 graphically is the following. We can draw a picture like this, 00:12:36.000 --> 00:12:41.000 as we did before, and here we'll have a second arrow that goes around 00:12:41.000 --> 00:12:45.000 like this. It loops around back on itself, and that implies the whole 00:12:45.000 --> 00:12:49.000 program of self-renewal. Now, the whole concept of 00:12:49.000 --> 00:12:54.000 self-renewal is a simple one. If a stem cell can self-renew, 00:12:54.000 --> 00:12:58.000 that implies that the process of growth and division does not deplete 00:12:58.000 --> 00:13:03.000 the pool of stem cells in the body or in a particular tissue. 00:13:03.000 --> 00:13:08.000 So, let's imagine what we're looking at here. Here we have a stem cell. 00:13:08.000 --> 00:13:13.000 It has one cell that is just like Mom. This is a differentiated cell. 00:13:13.000 --> 00:13:18.000 Once again, you can have a growth and division. This is, 00:13:18.000 --> 00:13:24.000 once again, a cell like Mom. This is a differentiated cell and 00:13:24.000 --> 00:13:29.000 so forth. And what you notice here in this arrangement is that the stem 00:13:29.000 --> 00:13:34.000 cells perpetuate themselves. They are self-renewing. 00:13:34.000 --> 00:13:38.000 And, as a consequence, the pool of stem cells is never depleted in the 00:13:38.000 --> 00:13:42.000 best of all possible worlds. It turns out that in most of the 00:13:42.000 --> 00:13:46.000 tissues of our body there is self-renewing stem cells going on, 00:13:46.000 --> 00:13:51.000 because most of the differentiated cells in our body have a finite 00:13:51.000 --> 00:13:55.000 lifetime. Not all of them but most of them. And when I say finite, 00:13:55.000 --> 00:13:59.000 I mean it can be measured in a matter of days or weeks 00:13:59.000 --> 00:14:03.000 or months. In the case of the brain, 00:14:03.000 --> 00:14:07.000 things turnover very slowly. Even in the case of our bones, 00:14:07.000 --> 00:14:11.000 our bones actually turn over roughly once, 10% a year. 00:14:11.000 --> 00:14:15.000 10% of the matter in the bone is actually turnover in every year. 00:14:15.000 --> 00:14:19.000 So, almost all tissues in the body are in a process of continuing 00:14:19.000 --> 00:14:23.000 self-renewal and repair. And that self-renewal and repair is 00:14:23.000 --> 00:14:27.000 maintained by this stem cell compartment, as is indicated here. 00:14:27.000 --> 00:14:32.000 This has certain kinds of great advantages, and one of the 00:14:32.000 --> 00:14:38.000 advantages is indicated by the following. Let's imagine that we 00:14:38.000 --> 00:14:43.000 draw a picture, just for the sake of argument, 00:14:43.000 --> 00:14:49.000 of one of the most highly proliferative tissues in the body, 00:14:49.000 --> 00:14:54.000 which is to say the lining of the colon or of the duodenum. 00:14:54.000 --> 00:15:00.000 So, here we have, let me draw it slightly differently, 00:15:00.000 --> 00:15:06.000 here's the way the lining of the small intestine looks like. 00:15:06.000 --> 00:15:10.000 Out here are the contents of the small intestine. 00:15:10.000 --> 00:15:20.000 So, let's say here is the lumen of 00:15:20.000 --> 00:15:24.000 the small intestine. And here we have, protruding into 00:15:24.000 --> 00:15:28.000 the lumen, when I talk about a lumen I'm talking about the bore or the 00:15:28.000 --> 00:15:32.000 channel of a cylindrical or tube like organism. 00:15:32.000 --> 00:15:36.000 So, here is the lumen of the small intestine. Here are these 00:15:36.000 --> 00:15:41.000 fingerlike projections, they're called villi, that protrude 00:15:41.000 --> 00:15:45.000 into the lumen of the small intestine. And down here at the 00:15:45.000 --> 00:15:50.000 bottom of this are these cavities that are called crypts, 00:15:50.000 --> 00:15:54.000 C-R-Y-P-T-S. These are the crypts. Now, what's important to realize is 00:15:54.000 --> 00:15:59.000 that what goes through our intestines is not that pleasant. 00:15:59.000 --> 00:16:03.000 It's pretty corrosive stuff. I probably told you this already, 00:16:03.000 --> 00:16:07.000 more bacteria living in there than we have in our entire cells in our 00:16:07.000 --> 00:16:11.000 entire body. There are all kinds of digestive juices. 00:16:11.000 --> 00:16:15.000 And so the cells out here at the tips of these villi are continually 00:16:15.000 --> 00:16:19.000 exposed to all kinds of corrosive material, including the junk that we 00:16:19.000 --> 00:16:23.000 eat everyday which is flowing by like this. And this indicates how 00:16:23.000 --> 00:16:27.000 critically important it is that we have self-renewal, 00:16:27.000 --> 00:16:31.000 because the cells out here, being continually exposed to the 00:16:31.000 --> 00:16:35.000 most corrosive kinds of influences, are rapidly damages. 00:16:35.000 --> 00:16:39.000 And, therefore, the cells out here have a lifetime 00:16:39.000 --> 00:16:44.000 of only three or four days and are then induced to jump off the end of 00:16:44.000 --> 00:16:49.000 a gangplank and commit apoptosis. So, the cells at the tip of the 00:16:49.000 --> 00:16:54.000 villis are continually jumping off and dying. And what's happening is 00:16:54.000 --> 00:16:59.000 that down here in the bottom of the crypts we have stem cells. 00:16:59.000 --> 00:17:06.000 The stem cells are continually 00:17:06.000 --> 00:17:09.000 producing progeny that have committed themselves to 00:17:09.000 --> 00:17:13.000 differentiate. And the progeny, 00:17:13.000 --> 00:17:16.000 as you might guess from what I've just said, are continually migrating 00:17:16.000 --> 00:17:19.000 up the sides of the villis up to the end here. And this whole migration 00:17:19.000 --> 00:17:23.000 takes four or five days, and by the time they get to the tip 00:17:23.000 --> 00:17:26.000 and have stuck their heads into the contents of the lumen of the 00:17:26.000 --> 00:17:30.000 intestine for that period of three or four days. 00:17:30.000 --> 00:17:33.000 Finally, they're eliminated and they jump off into the abyss. 00:17:33.000 --> 00:17:37.000 So, there's a continuing action going on here. 00:17:37.000 --> 00:17:40.000 The stem cells are continually dividing. And what advantages does 00:17:40.000 --> 00:17:44.000 this have for us? Well, it means that cells that are 00:17:44.000 --> 00:17:47.000 damaged are not allowed to hang around for a very long period of 00:17:47.000 --> 00:17:51.000 time, i.e., cells up here in the top that are exposed to, 00:17:51.000 --> 00:17:55.000 for example, potential mutagenic influences are rapidly eliminated. 00:17:55.000 --> 00:17:58.000 Why is that good? Because the mutagenic influences up here could 00:17:58.000 --> 00:18:02.000 well create a mutant cell that, in principle, is able to become 00:18:02.000 --> 00:18:05.000 cancerous. And the body says, 00:18:05.000 --> 00:18:09.000 well, I don't mind if that happens because these cells up here are 00:18:09.000 --> 00:18:12.000 going to be eliminated anyhow. They're going to be pushed off the 00:18:12.000 --> 00:18:16.000 end of the diving board or the gangplank into the abyss, 00:18:16.000 --> 00:18:19.000 so they are continually undergoing apoptosis, not as a pathological 00:18:19.000 --> 00:18:23.000 process. As a normal process. They're continually being pushed 00:18:23.000 --> 00:18:27.000 out here. And what that means is that the 00:18:27.000 --> 00:18:31.000 cells down here, in the bottom of the crypt, 00:18:31.000 --> 00:18:36.000 are actually physically protected from the contents of the lumen of 00:18:36.000 --> 00:18:41.000 the small intestine because some of the cells in this crypt are 00:18:41.000 --> 00:18:45.000 continually secreting a kind of mucus in this area right here. 00:18:45.000 --> 00:18:50.000 It's called a mucin. And this mucin here creates a physical 00:18:50.000 --> 00:18:55.000 barrier, so the cells that are in the bottom of the crypt are never 00:18:55.000 --> 00:18:59.000 directly exposed to the contents of what's flowing by in the 00:18:59.000 --> 00:19:03.000 small intestine. And that is extremely important 00:19:03.000 --> 00:19:07.000 because, in fact, it means that these cells down here 00:19:07.000 --> 00:19:11.000 are shielded from the mutagenic influences of what might be present 00:19:11.000 --> 00:19:15.000 in the lumen of the small intestine. In theory, one might be able to 00:19:15.000 --> 00:19:19.000 evolve cells that don't mind being up here in the lumen of the small 00:19:19.000 --> 00:19:23.000 intestine. But, in fact, that's never been possible. 00:19:23.000 --> 00:19:27.000 That is to say evolution has just said, well, we can't really evolve 00:19:27.000 --> 00:19:31.000 cells that are resistant to the corrosive influences of what happens 00:19:31.000 --> 00:19:34.000 in the small intestine. And, therefore, 00:19:34.000 --> 00:19:38.000 we're just going to use these cells for a very short period of time and 00:19:38.000 --> 00:19:41.000 then get rid of them. What that also means is the 00:19:41.000 --> 00:19:44.000 following. The stem cells down here stay within that crypt. 00:19:44.000 --> 00:19:48.000 They don't migrate out. They stay there in that shielded 00:19:48.000 --> 00:19:51.000 site. And, in fact, if you think all this through, 00:19:51.000 --> 00:19:54.000 it's very important to protect these cells from becoming mutated because 00:19:54.000 --> 00:19:58.000 if they do become mutated they could become the precursors 00:19:58.000 --> 00:20:02.000 of cancer cells. If these cells become mutated out 00:20:02.000 --> 00:20:06.000 here, it doesn't matter because they're going to die anyhow. 00:20:06.000 --> 00:20:11.000 And so, we now have the following kind of dynamic. 00:20:11.000 --> 00:20:15.000 Here's the stem cell. I'll draw it again. I'll just 00:20:15.000 --> 00:20:20.000 abbreviate it stem cell. And one of the ways by which we 00:20:20.000 --> 00:20:24.000 want to preserve the genetic integrity of the stem cell is to 00:20:24.000 --> 00:20:29.000 insure that the stem cell divides as infrequently as possible. Why? 00:20:29.000 --> 00:20:32.000 Because the whole process of cell division is itself a fallible 00:20:32.000 --> 00:20:36.000 process. Every time a cell grows and divides, as we learned from the 00:20:36.000 --> 00:20:40.000 cell cycle, there's the possibility of different kinds of genetic 00:20:40.000 --> 00:20:44.000 disasters happening in which case we might end up with a mutant stem cell. 00:20:44.000 --> 00:20:47.000 And that mutant stem cell could in turn, I argue, 00:20:47.000 --> 00:20:51.000 become involved in creating a tumor. So, we have the following kind of 00:20:51.000 --> 00:20:55.000 arrangement. Here is the stem cell. It has one daughter that has 00:20:55.000 --> 00:20:59.000 committed herself to differentiate and the other that remains 00:20:59.000 --> 00:21:03.000 a stem cell. Well, you're saying this enormous 00:21:03.000 --> 00:21:08.000 amount of activity must involve a frenetic amount of cell division on 00:21:08.000 --> 00:21:13.000 the part of the stem cell. But that's not really the way it 00:21:13.000 --> 00:21:19.000 happens because this daughter cell undergoes a series of exponential 00:21:19.000 --> 00:21:24.000 divisions, I can't fit them all on the blackboard here, 00:21:24.000 --> 00:21:29.000 and might even yield a hundred descendents which then become the 00:21:29.000 --> 00:21:34.000 ultimate differentiated cell. This daughter cell becomes one stem 00:21:34.000 --> 00:21:38.000 cell. This daughter cell undergoes these exponential expansions in a 00:21:38.000 --> 00:21:42.000 process of creating a population of cells that are called transit 00:21:42.000 --> 00:21:53.000 amplifying cells. 00:21:53.000 --> 00:21:56.000 And at the bottom of this hierarchy, I can't draw all hundred, there may 00:21:56.000 --> 00:21:59.000 be a hundred of these cells, ten to the second, and these cells 00:21:59.000 --> 00:22:06.000 then differentiate. 00:22:06.000 --> 00:22:10.000 At the bottom, the hundred cells at the bottom, 00:22:10.000 --> 00:22:14.000 they go into the last stage of differentiation. 00:22:14.000 --> 00:22:18.000 They become the specialized cells that line the tips of the villis. 00:22:18.000 --> 00:22:22.000 Now, why is there this arrangement? Well, look at what the advantage of 00:22:22.000 --> 00:22:26.000 it is. The stem cell has just divided once, but this cell has 00:22:26.000 --> 00:22:30.000 generated a hundred progeny. And that means that the stem cell 00:22:30.000 --> 00:22:34.000 doesn't have to divide that often. The stem cell can divide once. 00:22:34.000 --> 00:22:38.000 Every time the villis needs a hundred new cells it needs to divide 00:22:38.000 --> 00:22:42.000 only once. And, therefore, the stem cell actually is 00:22:42.000 --> 00:22:46.000 one of the most slowly dividing cells in the entire gut because it 00:22:46.000 --> 00:22:50.000 only needs to divide episodically. Each time it divides it generates 00:22:50.000 --> 00:22:54.000 this enormous array of progeny. One other aspect of differentiation, 00:22:54.000 --> 00:22:58.000 and when I talk about differentiation here, 00:22:58.000 --> 00:23:02.000 I mean the acquisition by these cells of all of the traits they need 00:23:02.000 --> 00:23:06.000 to line the colon. When I talk about differentiation in 00:23:06.000 --> 00:23:12.000 the skin, I talk about the ability, the acquisition of the cells of 00:23:12.000 --> 00:23:17.000 becoming fully competent, fully functional skin cells. 00:23:17.000 --> 00:23:23.000 The same thing with neurons in the brain. And one additional important 00:23:23.000 --> 00:23:29.000 concept I'd like to introduce is that when cells differentiate they 00:23:29.000 --> 00:23:34.000 often become post-mitotic. Post-mitotic means that these cells 00:23:34.000 --> 00:23:38.000 give up the option of ever dividing again. In other words, 00:23:38.000 --> 00:23:42.000 as they acquire more and more specialized traits they say to 00:23:42.000 --> 00:23:46.000 themselves now I'm a nerve cell, now I'm a cell in the tip of villis 00:23:46.000 --> 00:23:50.000 in the intestine, now I'm a muscle cell, 00:23:50.000 --> 00:23:54.000 I'm not going to divide ever again. And this is generally true. The 00:23:54.000 --> 00:23:58.000 most differentiated cells in the body in general end up losing the 00:23:58.000 --> 00:24:02.000 ability to divide. They become post-mitotic. 00:24:02.000 --> 00:24:06.000 They've exited irreversibly from the cell cycle. 00:24:06.000 --> 00:24:11.000 They can't go back in. As you recall, we talked about 00:24:11.000 --> 00:24:16.000 cells going from G0 back into the active G1 phase of the cell cycle. 00:24:16.000 --> 00:24:20.000 That's a reversible exit from the cell cycle. Post-mitotic cells are 00:24:20.000 --> 00:24:25.000 irreversibly committed never to divide again. And, 00:24:25.000 --> 00:24:30.000 again, that holds true for almost all cells of the body. 00:24:30.000 --> 00:24:34.000 One exception to that is, interesting enough, in the liver. 00:24:34.000 --> 00:24:38.000 Because what you can do with a mouse, or even with a human, 00:24:38.000 --> 00:24:43.000 is you can cut away a couple lobes of the liver, major league surgery. 00:24:43.000 --> 00:24:47.000 And when you do that, what happens is that all the remaining cells of 00:24:47.000 --> 00:24:52.000 the liver, and these remaining cells in the liver are called hepatocytes, 00:24:52.000 --> 00:24:56.000 these hepatocytes, which until that time had been highly specialized 00:24:56.000 --> 00:25:00.000 differentiated cells in the liver, many of them divide, they double 00:25:00.000 --> 00:25:04.000 again. And in short order one ends up with 00:25:04.000 --> 00:25:07.000 a liver which is exactly the same size as one had before. 00:25:07.000 --> 00:25:10.000 And that's actually quite remarkable because there are very 00:25:10.000 --> 00:25:12.000 few organs in the adult human being where that will happen. 00:25:12.000 --> 00:25:15.000 There is, by the way, an interesting puzzle here, 00:25:15.000 --> 00:25:18.000 and that is the following. Let's say you cut away half the 00:25:18.000 --> 00:25:21.000 liver and many of the hepatocytes, which were already highly 00:25:21.000 --> 00:25:24.000 differentiated, began to divide again, 00:25:24.000 --> 00:25:27.000 so they were not serious post-mitotic cells, 00:25:27.000 --> 00:25:30.000 they could reenter into proliferative phase. 00:25:30.000 --> 00:25:34.000 How do these cells know when to stop dividing so that they end up 00:25:34.000 --> 00:25:38.000 regenerating a liver of exactly the right size? People have been 00:25:38.000 --> 00:25:43.000 looking at that for 30 or 40 years. Nobody has any idea why. Why 00:25:43.000 --> 00:25:47.000 doesn't the liver when all these cells divide become one and a half 00:25:47.000 --> 00:25:51.000 times the size of its former diameter or half the size? 00:25:51.000 --> 00:25:56.000 Nobody really understands that. In any case, I just want to 00:25:56.000 --> 00:26:00.000 indicate that there is this dynamic between differentiation and 00:26:00.000 --> 00:26:05.000 proliferative capacity, one in opposition to the other. 00:26:05.000 --> 00:26:10.000 Well, how much hematopoiesis is taking place here in our bone marrow, 00:26:10.000 --> 00:26:16.000 for example, where a lot of this takes place? So, 00:26:16.000 --> 00:26:22.000 here are just some interesting numbers. There are roughly five 00:26:22.000 --> 00:26:28.000 times ten to the twelfth red blood cells -- 00:26:28.000 --> 00:26:34.000 -- per liter of blood. 00:26:34.000 --> 00:26:40.000 And red blood cells, you may recall, are called erythrocytes. 00:26:40.000 --> 00:26:45.000 Remember, it's never good to use a short Anglo-Saxon term if you can 00:26:45.000 --> 00:26:51.000 use a long complicated Greek one. And each of these red blood cells 00:26:51.000 --> 00:26:56.000 has roughly a lifetime of 120 days. That is to say after it's made it 00:26:56.000 --> 00:27:02.000 sits around in the blood for roughly 120 days. 00:27:02.000 --> 00:27:06.000 It gets warn out. It gets gobbled up by the cells in 00:27:06.000 --> 00:27:11.000 the spleen. Much of the contents are recycled. The pigment in our 00:27:11.000 --> 00:27:16.000 stool comes from the recycling of the hemoglobin, 00:27:16.000 --> 00:27:20.000 the iron and the hemoglobin, and we're constantly renewing that. 00:27:20.000 --> 00:27:25.000 Well, the average human being, let's say, has roughly ten liters of 00:27:25.000 --> 00:27:32.000 blood per person. 00:27:32.000 --> 00:27:36.000 And if you put all those numbers together, I once calculated that the 00:27:36.000 --> 00:27:41.000 number of red blood cells that is being generated in the body is 00:27:41.000 --> 00:27:45.000 roughly ten to the tenth per hour. And you can go through the 00:27:45.000 --> 00:27:50.000 calculations if you want, it is fine with me, but take my word 00:27:50.000 --> 00:27:54.000 for it's roughly what's going on there. Ten to the tenth per hour. 00:27:54.000 --> 00:27:59.000 Each time I utter a word there's probably, I don't know, 00:27:59.000 --> 00:28:04.000 ten to the seventh new red blood cells being made in my bone marrow. 00:28:04.000 --> 00:28:07.000 So, this is not minor league proliferation. 00:28:07.000 --> 00:28:11.000 Now we begin to understand why there are ten to the sixteenth cell 00:28:11.000 --> 00:28:15.000 divisions in the lifetime of a human being. In fact, 00:28:15.000 --> 00:28:19.000 a great majority of them, to be fair, are occurring in the 00:28:19.000 --> 00:28:22.000 bone marrow and in the intestine. And if you have an individual who's 00:28:22.000 --> 00:28:26.000 being exposed to certain kinds of chemotherapy, chemotherapy, 00:28:26.000 --> 00:28:30.000 as you may know, is very toxic for dividing cells, 00:28:30.000 --> 00:28:34.000 and the side effect toxicity of anticancer chemotherapy is largely 00:28:34.000 --> 00:28:38.000 felt, first of all, in the bone marrow where individuals 00:28:38.000 --> 00:28:42.000 tend to become anemic. Anemic means they have lower than 00:28:42.000 --> 00:28:46.000 normal numbers of red blood cells and also they lose a lot of the 00:28:46.000 --> 00:28:51.000 lining of the intestine which creates all kinds of also 00:28:51.000 --> 00:28:55.000 unpleasantness as well. So, we're not talking about 00:28:55.000 --> 00:28:59.000 something that happens on rare occasion in the life 00:28:59.000 --> 00:29:04.000 an individual. This is a staggering amount of 00:29:04.000 --> 00:29:08.000 mitosis that's happening every day of our lives. Let's go back for a 00:29:08.000 --> 00:29:12.000 moment to this diagram here and realize that when I'm talking about 00:29:12.000 --> 00:29:16.000 erythrocytes, I'm only talking about one of the branches of this 00:29:16.000 --> 00:29:20.000 multi-branch pathway. And here we see some other 00:29:20.000 --> 00:29:24.000 interesting aspects of what's going on here, and I'll give you some 00:29:24.000 --> 00:29:28.000 proofs very shortly that this actually is what it says it is. 00:29:28.000 --> 00:29:32.000 It actually is organized this way. The pluripotent stem cell is capable 00:29:32.000 --> 00:29:36.000 of self-renewal, and it can spew off daughters which 00:29:36.000 --> 00:29:40.000 actually can go in two different directions. Its daughter may decide 00:29:40.000 --> 00:29:44.000 that it might become the precursor of the lymphoid cells in the blood 00:29:44.000 --> 00:29:48.000 or it might commit itself to becoming a myeloid precursor. 00:29:48.000 --> 00:29:52.000 So, that's already the beginning of a bifurcation. 00:29:52.000 --> 00:29:56.000 These cells are not yet differentiated. 00:29:56.000 --> 00:30:00.000 They've just made the commitment that each of them can, 00:30:00.000 --> 00:30:05.000 in principle, become the ancestor of highly differentiated cells. 00:30:05.000 --> 00:30:08.000 And these cells, we can imagine, are transient 00:30:08.000 --> 00:30:12.000 amplifying cells in the sense that even though they're committed to 00:30:12.000 --> 00:30:16.000 create progeny of one sort or another they themselves are not yet 00:30:16.000 --> 00:30:19.000 fully differentiated. Keep in mind in the context of the 00:30:19.000 --> 00:30:23.000 crypt these transient amplifying cells are on the way to becoming 00:30:23.000 --> 00:30:27.000 fully differentiated, but only at the bottom of this 00:30:27.000 --> 00:30:31.000 exponential expansion of cells do we have cells that are fully entered 00:30:31.000 --> 00:30:35.000 into a highly differentiated state. Here we see that these two cells are 00:30:35.000 --> 00:30:39.000 also stem cells in the sense that the can self-renew. 00:30:39.000 --> 00:30:44.000 They have a limited self-renewal capacity, but they can self-renew. 00:30:44.000 --> 00:30:48.000 And then they begin to create progeny which themselves can 00:30:48.000 --> 00:30:53.000 undertake several distinct alternative differentiation paths. 00:30:53.000 --> 00:30:57.000 So, the lymphoid cells can become the progenitors of the T lymphocytes 00:30:57.000 --> 00:31:02.000 and the B lymphocytes. And, in fact, if you recall our 00:31:02.000 --> 00:31:06.000 discussion of immunology, there's actually several different 00:31:06.000 --> 00:31:10.000 kinds of T lymphocytes and B lymphocytes. So, 00:31:10.000 --> 00:31:14.000 this pathway has further radiations further down. Here, 00:31:14.000 --> 00:31:18.000 alternatively, these are the myeloid cells. And myeloid refers to the 00:31:18.000 --> 00:31:22.000 bone marrow. And the myeloid cells can become these kinds of cells up 00:31:22.000 --> 00:31:26.000 here, eosinophils and basophils and neutrophils and monocytes, 00:31:26.000 --> 00:31:30.000 and this class of cells is largely involved in gobbling up infectious 00:31:30.000 --> 00:31:34.000 agents and as agents which are able to defend us largely against 00:31:34.000 --> 00:31:38.000 bacterial infections. Here's the macrophage. 00:31:38.000 --> 00:31:42.000 We talked about the macrophage. Remember the macrophage was this 00:31:42.000 --> 00:31:45.000 glutton, this pig which wandered around our tissues and gobbled up 00:31:45.000 --> 00:31:49.000 whatever kind of material it could find and presented it to the immune 00:31:49.000 --> 00:31:52.000 system. And here are several other of the lineages. 00:31:52.000 --> 00:31:56.000 Here is a megakaryocytic and this is an erythrocyte. 00:31:56.000 --> 00:32:00.000 What's a megakariocyte? Well, it has a very large nucleus. 00:32:00.000 --> 00:32:04.000 That's what the term implies. And what happens to the 00:32:04.000 --> 00:32:09.000 megakariocyte is it buds off little chunks of cytoplasm lacking nuclei. 00:32:09.000 --> 00:32:13.000 And these little chunks of cytoplasm become the blood platelets. 00:32:13.000 --> 00:32:18.000 Blood platelets lack nuclei. They're enucleate because they're 00:32:18.000 --> 00:32:22.000 just little bags of material which are sent out into the circulation. 00:32:22.000 --> 00:32:27.000 And, as we said also earlier in the semester, once the platelets are in 00:32:27.000 --> 00:32:31.000 the circulation they are there ready to help should there be any kind of 00:32:31.000 --> 00:32:36.000 wounding, any kind of hemorrhaging occurring. 00:32:36.000 --> 00:32:39.000 And the platelets can release upon being activated in a site of wound 00:32:39.000 --> 00:32:43.000 coagulation factors and growth factors for the regeneration and 00:32:43.000 --> 00:32:47.000 reconstruction of wound sites. And, finally, here are our friends 00:32:47.000 --> 00:32:51.000 the erythrocytes. So, here we have a whole sequence 00:32:51.000 --> 00:32:55.000 of different kinds of differentiation commitments which 00:32:55.000 --> 00:32:59.000 are going on at an enormous rate. How do we know that there actually 00:32:59.000 --> 00:33:03.000 is a pluripotent stem cell? What evidence can I provide you that 00:33:03.000 --> 00:33:07.000 this actually exists or it's just a figment of my normally florid 00:33:07.000 --> 00:33:12.000 imagination? And the most direct demonstration of that is, 00:33:12.000 --> 00:33:16.000 in fact, the use of bone marrow transplantation. 00:33:16.000 --> 00:33:21.000 So, when we talk about a bone marrow transplantation, 00:33:21.000 --> 00:33:26.000 or BMT as it's called in the trade, -- 00:33:26.000 --> 00:33:33.000 -- one can do a relatively simple 00:33:33.000 --> 00:33:37.000 experiment. You can take a mouse or even a human and you can irradiate 00:33:37.000 --> 00:33:41.000 it rather heavily. And if you irradiate it under the 00:33:41.000 --> 00:33:44.000 right conditions you'll actually be able to kill off all the cells in 00:33:44.000 --> 00:33:48.000 the bone marrow without killing off the mouse or the human being. 00:33:48.000 --> 00:33:52.000 In fact, there are drugs you can also use in human beings to 00:33:52.000 --> 00:33:55.000 eliminate virtually all the cells in the bone marrow. 00:33:55.000 --> 00:33:59.000 And then what you can do is you can take bone marrow from another 00:33:59.000 --> 00:34:03.000 organism, from another mouse or another human, 00:34:03.000 --> 00:34:07.000 and you inject it into the blood of the irradiated mouse or human. 00:34:07.000 --> 00:34:10.000 And the bone marrow cells, many of them will home to the bone 00:34:10.000 --> 00:34:14.000 marrow. In other words, you're injecting the bone marrow 00:34:14.000 --> 00:34:17.000 cells in the general circulation, but within a couple hours they'll 00:34:17.000 --> 00:34:21.000 all end up in the bone marrow, in the space in the middle of the 00:34:21.000 --> 00:34:25.000 bone because there are many kinds of cells which have this homing 00:34:25.000 --> 00:34:29.000 capacity. They go to the right place in the body. 00:34:29.000 --> 00:34:33.000 So, they can home. The injected cells can home to bone 00:34:33.000 --> 00:34:37.000 marrow. And then, if things are going well, 00:34:37.000 --> 00:34:41.000 these injected bone marrow cells will begin to proliferate and they 00:34:41.000 --> 00:34:45.000 will ultimately regenerate this entire cascade of differentiation 00:34:45.000 --> 00:34:49.000 decisions, as is indicated here. And, therefore, that individual or 00:34:49.000 --> 00:34:53.000 that mouse will actually be rescued. Because in the absence of such a 00:34:53.000 --> 00:34:58.000 rescue an individual will rapidly die. 00:34:58.000 --> 00:35:01.000 You can't live for very long in the absence of an active bone marrow 00:35:01.000 --> 00:35:05.000 because these cells here are rapidly depleted. They turn over with some 00:35:05.000 --> 00:35:08.000 speed. The red blood cells hang around for 120 days we said and, 00:35:08.000 --> 00:35:12.000 therefore, you don't need to make them immediately because there's a 00:35:12.000 --> 00:35:15.000 whole bunch around that have a rather slow turnover. 00:35:15.000 --> 00:35:19.000 But the platelets only have a lifetime of several days before 00:35:19.000 --> 00:35:22.000 they're lost, they're turned over. And if you don't have platelets 00:35:22.000 --> 00:35:26.000 you're in very bad shape because you start hemorrhaging all over the body 00:35:26.000 --> 00:35:29.000 because, remember, the platelets are there to stop up 00:35:29.000 --> 00:35:33.000 all the holes in the dike to prevent bleeding. 00:35:33.000 --> 00:35:37.000 These cells here are very important, the eosinophils, basophils, 00:35:37.000 --> 00:35:41.000 neutrophils, and even macrophages in preventing bacterial infections. 00:35:41.000 --> 00:35:45.000 And in the absence of having these on site one can readily succumb to 00:35:45.000 --> 00:35:49.000 overwhelming infections. Keep in mind that the reason why 00:35:49.000 --> 00:35:53.000 we're not constantly dying from bacterial infections is not because 00:35:53.000 --> 00:35:57.000 each of us takes an antibiotic pill every day, it's because these cells 00:35:57.000 --> 00:36:01.000 are on watch to kill any bacteria that happen to be in the wrong place 00:36:01.000 --> 00:36:05.000 in the body outside of the lumen of the gut. 00:36:05.000 --> 00:36:09.000 And consequently the question is always can one rescue a mouse or a 00:36:09.000 --> 00:36:14.000 human rapidly enough? Can one replace its bone marrow 00:36:14.000 --> 00:36:19.000 rapidly enough so that this disaster from losing all ones bone marrow 00:36:19.000 --> 00:36:23.000 doesn't overtake one and the organism dies before the bone marrow 00:36:23.000 --> 00:36:28.000 has had a chance to become reconstituted, regenerated, 00:36:28.000 --> 00:36:32.000 reconstructed. Still how do we know from all this 00:36:32.000 --> 00:36:36.000 that, in fact, there is a pluripotent stem cell? 00:36:36.000 --> 00:36:39.000 If you listened to everything I said correctly you could say, 00:36:39.000 --> 00:36:43.000 well, there isn't such a thing as a pluripotent stem cell. 00:36:43.000 --> 00:36:46.000 There are these other kinds of stem cells, this one and this one, 00:36:46.000 --> 00:36:50.000 or these might all be stem cells. And when I'm injecting the bone 00:36:50.000 --> 00:36:54.000 marrow of a donor animal into the recipient, I'm injecting a whole 00:36:54.000 --> 00:36:57.000 mixture of different kinds of stem cells here each of which then goes 00:36:57.000 --> 00:37:01.000 on and populates a specialized compartment in the bone marrow 00:37:01.000 --> 00:37:05.000 or in the blood. So how do we know there's one 00:37:05.000 --> 00:37:09.000 pluripotent stem cell? One way to prove this is the 00:37:09.000 --> 00:37:14.000 following. Let's say we take the bone marrow from the donor, 00:37:14.000 --> 00:37:18.000 that is the bone marrow that we're going to inject into the irradiated 00:37:18.000 --> 00:37:23.000 recipient, and we irradiate that bone marrow very lightly, 00:37:23.000 --> 00:37:28.000 not to kill the bone marrow cells but to introduce random chromosomal 00:37:28.000 --> 00:37:32.000 breaks, a very small number of random chromosomal breaks in the 00:37:32.000 --> 00:37:37.000 donor bone marrow. So, the purpose now of irradiation 00:37:37.000 --> 00:37:41.000 is quite different from what I said before. Before we wanted to give a 00:37:41.000 --> 00:37:45.000 heavy dose of radiation to wipe out the recipient bone marrow. 00:37:45.000 --> 00:37:49.000 Now we're going to just give a wee bit of radiation to the donor bone 00:37:49.000 --> 00:37:53.000 marrow. What's the purpose of that? The purpose of that small amount of 00:37:53.000 --> 00:37:58.000 radiation is to create chromosomal abnormalities. 00:37:58.000 --> 00:38:03.000 So, for example, if here are two homologous 00:38:03.000 --> 00:38:09.000 chromosomes in the donor cells. Since the radiation, the very weak 00:38:09.000 --> 00:38:14.000 dose of radiation is acting randomly it will create all kinds of 00:38:14.000 --> 00:38:20.000 abnormalities including, for example, a very specific 00:38:20.000 --> 00:38:25.000 chromosomal translocation so that what might happen after this is that 00:38:25.000 --> 00:38:31.000 a whole chunk of this chromosome is translocated over to this chromosome 00:38:31.000 --> 00:38:37.000 here. This is called a chromosomal translocation. 00:38:37.000 --> 00:38:44.000 And now here's the donor, 00:38:44.000 --> 00:38:48.000 these are donor bone marrow cells. And keep in mind that every donor 00:38:48.000 --> 00:38:52.000 bone marrow cell that gets a little bit of this radiation will get its 00:38:52.000 --> 00:38:56.000 own very specific randomly occurring translocation just because radiation 00:38:56.000 --> 00:39:00.000 is able to break chromosomes and then they will rejoin in 00:39:00.000 --> 00:39:04.000 unpredictable ways. What that means is that if we take 00:39:04.000 --> 00:39:08.000 the donor bone marrow and irradiate it very lightly so that we don't 00:39:08.000 --> 00:39:12.000 kill the cells but we do induce these translocations, 00:39:12.000 --> 00:39:17.000 one donor cell will have this translocation and another donor cell 00:39:17.000 --> 00:39:21.000 over here will have a totally different translocation from a 00:39:21.000 --> 00:39:25.000 different chromosome also induced randomly by these stochastic 00:39:25.000 --> 00:39:30.000 processes. So the karyotype which is the whole 00:39:30.000 --> 00:39:34.000 array of chromosomes of a cell, which can be viewed at the metaphase 00:39:34.000 --> 00:39:39.000 of mitosis when all the chromosomes condense, the karyotype of each of 00:39:39.000 --> 00:39:43.000 these donor bone marrow cells will be messed up slightly. 00:39:43.000 --> 00:39:48.000 And it will have recognizable abnormalities, 00:39:48.000 --> 00:39:53.000 but they're all different, one after the other. And after 00:39:53.000 --> 00:39:57.000 we've done this, after we've marked millions of bone 00:39:57.000 --> 00:40:02.000 marrow cells in the donor with these random low-dose radiations, 00:40:02.000 --> 00:40:06.000 we can then inject a small number of bone marrow cells into 00:40:06.000 --> 00:40:11.000 the recipient. And what we can sometimes find on 00:40:11.000 --> 00:40:15.000 occasion is if we look at the recipient after that recipient has 00:40:15.000 --> 00:40:19.000 been rescued, i. ., after the donor bone marrow has 00:40:19.000 --> 00:40:23.000 established itself within the recipient, is that the donor bone 00:40:23.000 --> 00:40:27.000 marrow is established in the recipient and populates all of these 00:40:27.000 --> 00:40:31.000 different lineages. And if we're able to look at the 00:40:31.000 --> 00:40:35.000 karyotype of these different kinds of cells in the recipient organism, 00:40:35.000 --> 00:40:39.000 we can find that in some mice all of these cells have the same very 00:40:39.000 --> 00:40:43.000 peculiar translocation. They have either this one or this 00:40:43.000 --> 00:40:46.000 one or they have yet a third translocation, 00:40:46.000 --> 00:40:50.000 any one of a whole series of randomly occurring mutations, 00:40:50.000 --> 00:40:54.000 a very peculiar idiosyncratic unusual translocation induced by the 00:40:54.000 --> 00:40:58.000 low-dose radiation. They all have it. 00:40:58.000 --> 00:41:02.000 The T cells and the B cells and the monocytes and the basophiles, 00:41:02.000 --> 00:41:06.000 they all have exactly the same translocation. 00:41:06.000 --> 00:41:10.000 Obviously, we can't do that experiment with the platelets. 00:41:10.000 --> 00:41:14.000 Why? Because they don't have nuclei. And we can't do that with 00:41:14.000 --> 00:41:18.000 the erythrocytes either, the red blood cells. Why can't we 00:41:18.000 --> 00:41:22.000 do that? Because in mammals, when the red blood cells are formed, 00:41:22.000 --> 00:41:26.000 the nuclei are spit out. Our red blood cells don't have nuclei in 00:41:26.000 --> 00:41:30.000 them anymore. They've become enucleate or, to put it another way, 00:41:30.000 --> 00:41:34.000 they have been enucleated. That is to say they've been deprived 00:41:34.000 --> 00:41:38.000 of their nuclei. Why? Because they're post-mitotic. 00:41:38.000 --> 00:41:43.000 Obviously, a cell which lacks nuclei is by definition post-mitotic. 00:41:43.000 --> 00:41:47.000 And our cells don't really need, red blood cells don't need nuclei. 00:41:47.000 --> 00:41:52.000 We know that ancestral organisms, for instance chickens, 00:41:52.000 --> 00:41:56.000 their red blood cells are nucleated, but our red blood cells are not 00:41:56.000 --> 00:42:02.000 because they're just not necessary. 00:42:02.000 --> 00:42:06.000 How is translocation different from crossing over? 00:42:06.000 --> 00:42:11.000 Crossing over occurs between two homologous chromosomes. 00:42:11.000 --> 00:42:15.000 So if we have a chromosome here, here's chromosome 13 and here's 00:42:15.000 --> 00:42:20.000 another chromosome 13, they're both chromosomes 13. 00:42:20.000 --> 00:42:25.000 This one came from Ma. This one came from Pa. All right? 00:42:25.000 --> 00:42:30.000 Each of us has a pair of homologous chromosomes. 00:42:30.000 --> 00:42:33.000 Here's the maternal one. Here's the paternal one. 00:42:33.000 --> 00:42:37.000 When we talk about crossing over we're talking about a process of 00:42:37.000 --> 00:42:47.000 homologous recombination. 00:42:47.000 --> 00:42:50.000 And when that happens we have a situation like this. 00:42:50.000 --> 00:42:53.000 This chunk is exchanged with this chunk over here. 00:42:53.000 --> 00:42:56.000 It's an equal exchange, absolutely equal down to the 00:42:56.000 --> 00:43:00.000 nucleotide, so that after this flipping has occurred we have two 00:43:00.000 --> 00:43:04.000 fully intact chromosomes. It's just that both of these 00:43:04.000 --> 00:43:08.000 chromosomes are fully normal. It's just that there's been a 00:43:08.000 --> 00:43:12.000 switching, an exchange between the two homologous chromosomes, 00:43:12.000 --> 00:43:16.000 the two paired chromosomes 13. When we talk about translocation 00:43:16.000 --> 00:43:20.000 there a chunk of chromosome 15 can go onto chromosome 7 or a chunk of 00:43:20.000 --> 00:43:24.000 chromosome 2 can go on chromosome 8. It's totally random, it's 00:43:24.000 --> 00:43:28.000 non-homologous, and it creates aberrant chromosomes. 00:43:28.000 --> 00:43:32.000 Neither of these recombined chromosomes is abnormal. 00:43:32.000 --> 00:43:36.000 It's just changed it allelic configuration. 00:43:36.000 --> 00:43:40.000 So, it's an important distinction. And the fact is you can pick out 00:43:40.000 --> 00:43:45.000 these translocated chromosomes because one even has specific dyes 00:43:45.000 --> 00:43:49.000 that can be able to tell you which chromosome this came from and which 00:43:49.000 --> 00:43:53.000 chromosome this came from. So, there is a profound difference. 00:43:53.000 --> 00:43:57.000 Translocations are, invariably, pathologic. When I say pathologic, 00:43:57.000 --> 00:44:02.000 I mean they're really sick. They're not the proper course of 00:44:02.000 --> 00:44:06.000 things that happens in a healthy cell. The fact, 00:44:06.000 --> 00:44:11.000 the very fact that we're able to generate an entire array of cells in 00:44:11.000 --> 00:44:15.000 the blood indicated, by necessity, that if all these 00:44:15.000 --> 00:44:20.000 cells have the same chromosomal translocation that they descend from 00:44:20.000 --> 00:44:24.000 a donor cell that originally was lightly irradiated and happened to 00:44:24.000 --> 00:44:28.000 receive that translocation. If we never get this array of common 00:44:28.000 --> 00:44:32.000 translocations in all the cells in a bone marrow recipient then we can't 00:44:32.000 --> 00:44:36.000 prove this, but the fact is this has been proven time and again over the 00:44:36.000 --> 00:44:40.000 years. And this indicates to us that this cell which is genetically 00:44:40.000 --> 00:44:44.000 slightly altered can, therefore, generate all these other 00:44:44.000 --> 00:44:48.000 cells in the body. Again, keep in mind that the 00:44:48.000 --> 00:44:52.000 irradiation of the donor marrow is simply to create these chromosomal 00:44:52.000 --> 00:44:56.000 markings. They're not necessarily good for the organism, 00:44:56.000 --> 00:45:00.000 but they don't compromise the viability of the cell. 00:45:00.000 --> 00:45:04.000 They just reshuffle the chromosomal structure. Now, 00:45:04.000 --> 00:45:09.000 in principle the levels of each one of these kinds of end-stage cells 00:45:09.000 --> 00:45:13.000 need to be carefully regulated. And, by the way, let me just note 00:45:13.000 --> 00:45:18.000 here, you see the T cells have the arrow going back on themselves, 00:45:18.000 --> 00:45:23.000 as do the B cells. That indicates that they have not become 00:45:23.000 --> 00:45:27.000 post-mitotic. Remember we talked about these embraces between helper 00:45:27.000 --> 00:45:32.000 T cells and B cells where they're walking down the allies and they get 00:45:32.000 --> 00:45:36.000 excited and they start multiplying? The fact that the T cells and the B 00:45:36.000 --> 00:45:40.000 cells are able to proliferate in response to certain antigenic 00:45:40.000 --> 00:45:43.000 stimuli implies that they're not post-mitotic. They still have the 00:45:43.000 --> 00:45:47.000 ability to proliferate, and that retained ability to 00:45:47.000 --> 00:45:50.000 proliferate like that of hepatocytes is indicated by these arrows that 00:45:50.000 --> 00:45:54.000 are looping back on themselves. Conversely, these cells are all 00:45:54.000 --> 00:45:57.000 essentially, as I've said before, post-mitotic. So how do we insure 00:45:57.000 --> 00:46:01.000 that's there proper concentrations of all of these different 00:46:01.000 --> 00:46:05.000 cells in the blood? And the fact is the concentrations 00:46:05.000 --> 00:46:10.000 of many of these cells in the blood are maintained to concentrations of 00:46:10.000 --> 00:46:14.000 plus or minus 10%. And this is, itself, 00:46:14.000 --> 00:46:19.000 a stunning testimonial to the successes of human physiology. 00:46:19.000 --> 00:46:24.000 We're talking here about a process which is sometimes called 00:46:24.000 --> 00:46:28.000 homeostasis. Homeostasis means that somehow there is a balance, 00:46:28.000 --> 00:46:33.000 an equilibrium that is achieved, and that there aren't profound 00:46:33.000 --> 00:46:38.000 fluctuations so that we always have roughly the equal level of red blood 00:46:38.000 --> 00:46:43.000 cells, a proper level of lymphocytes in our blood. 00:46:43.000 --> 00:46:47.000 And I want to get into the homeostasis which results in the 00:46:47.000 --> 00:46:52.000 formation of red blood cells, the RBCs, the erythrocytes in the 00:46:52.000 --> 00:46:56.000 blood. In fact, it happens to be the case that the 00:46:56.000 --> 00:47:01.000 red blood cells are one of the cell types that could actually vary quite 00:47:01.000 --> 00:47:06.000 profoundly in response to environment. 00:47:06.000 --> 00:47:09.000 To the extent that lymphocytes change, they go up and down, 00:47:09.000 --> 00:47:12.000 that might be due in response to an infection. So, 00:47:12.000 --> 00:47:15.000 if we have a serious bacterial infection we might have increased in 00:47:15.000 --> 00:47:19.000 the lymphocytes in the blood that have been mobilized in order to 00:47:19.000 --> 00:47:22.000 attack the infecting bacteria. But what about the RBCs? What 00:47:22.000 --> 00:47:25.000 about the red blood cells? What causes them to change? 00:47:25.000 --> 00:47:29.000 Well, if you move from here to Denver, Colorado or you go up skiing 00:47:29.000 --> 00:47:32.000 in the Rockies, you're going up to ten or twelve 00:47:32.000 --> 00:47:36.000 thousand feet. And within a matter of three or four 00:47:36.000 --> 00:47:40.000 days the concentration of your red blood cells increases very 00:47:40.000 --> 00:47:45.000 substantially. Why? Because obviously the oxygen 00:47:45.000 --> 00:47:50.000 tension at high altitude is down. And in order that your peripheral 00:47:50.000 --> 00:47:54.000 tissues become adequately oxygenated, the oxygen carrying capacity of the 00:47:54.000 --> 00:47:59.000 blood must be increased. And the way that is increased is in 00:47:59.000 --> 00:48:04.000 part to increase the concentration of red blood cells. 00:48:04.000 --> 00:48:08.000 So how does that happen? How is it possible that we can 00:48:08.000 --> 00:48:13.000 rapidly modulate the concentration of red blood cells? 00:48:13.000 --> 00:48:17.000 And the way we can do that is in part through a hormone called 00:48:17.000 --> 00:48:22.000 erythropoietin, EPO. We're going to talk about 00:48:22.000 --> 00:48:27.000 erythropoietin at the beginning of the lecture next time, 00:48:27.000 --> 00:48:31.000 but the homeostasis which maintains the appropriate number of red blood 00:48:31.000 --> 00:48:36.000 cells in our circulation is dictated in no small part by the levels of 00:48:36.000 --> 00:48:41.000 EPO that are in their blood. To anticipate some of the things 00:48:41.000 --> 00:48:45.000 we're going to say next time, when you are in a low-oxygen 00:48:45.000 --> 00:48:49.000 environment the levels of erythropoietin shoot up. 00:48:49.000 --> 00:48:53.000 And when they shoot up they insure that there is shortly thereafter a 00:48:53.000 --> 00:48:57.000 rapid increase in the level of circulating red blood cells which in 00:48:57.000 --> 00:49:01.000 turn enables the oxygen coming into your lungs to be transported more 00:49:01.000 --> 00:49:06.000 efficiently, more effectively into the peripheral tissues. 00:49:06.000 --> 00:49:10.000 You've heard about athletes perhaps who are able to dope themselves with 00:49:10.000 --> 00:49:14.000 erythropoietin. This is a rather devious strategy 00:49:14.000 --> 00:49:18.000 because it means that if they do so, they inject themselves with a little 00:49:18.000 --> 00:49:22.000 erythropoietin, the oxygen carrying capacity of 00:49:22.000 --> 00:49:26.000 their blood is temporarily increased and as a consequence they might be 00:49:26.000 --> 00:49:29.000 able to run further or jump higher. This, by the way, 00:49:29.000 --> 00:49:33.000 has its dangers. Because if you're injecting erythropoietin not in 00:49:33.000 --> 00:49:37.000 response to certain physiologic signals but just because you want to 00:49:37.000 --> 00:49:41.000 win a marathon or something, you're violating the normal 00:49:41.000 --> 00:49:45.000 physiologic mechanisms in the body which very carefully control the 00:49:45.000 --> 00:49:49.000 levels of erythropoietin. And if you inject too much 00:49:49.000 --> 00:49:53.000 erythropoietin you get in a very bad situation because the bone marrow 00:49:53.000 --> 00:49:57.000 makes more and more red blood cells. And then what happens? You start 00:49:57.000 --> 00:50:01.000 clotting up everywhere all over the body, and this isn't good. 00:50:01.000 --> 00:50:05.000 In fact, you can die. So this is not a warning against 00:50:05.000 --> 00:50:10.000 erythropoietin in the way that I warned you against cigarettes. 00:50:10.000 --> 00:50:14.000 This is just to tell you these kinds of drugs, 00:50:14.000 --> 00:50:19.000 or these kinds of growth factors, which they are, are maintained at 00:50:19.000 --> 00:50:23.000 very precise levels as we'll discuss in more detail next time. 00:50:23.000 --> 00:50:28.000 See you on Wednesday.