1 00:00:01,000 --> 00:00:04,000 Good morning, class. Nice to see you here. 2 00:00:04,000 --> 00:00:09,000 Yesterday morning I was in Australia and today I'm here. 3 00:00:09,000 --> 00:00:14,000 And it's nice to be back. It's a long way home. 4 00:00:14,000 --> 00:00:19,000 We're talking today about the issues of growth and differentiation 5 00:00:19,000 --> 00:00:23,000 about normal cells becoming specialized into different types of 6 00:00:23,000 --> 00:00:28,000 cells throughout the body and how that leads ultimately to the issues 7 00:00:28,000 --> 00:00:33,000 of organismic cloning. And, to give you an overall 8 00:00:33,000 --> 00:00:37,000 background about this, I want to show you the way that a 9 00:00:37,000 --> 00:00:42,000 worm is put together. This is the worm C. elegans. 10 00:00:42,000 --> 00:00:46,000 I don't know how well this overhead will come out. 11 00:00:46,000 --> 00:00:50,000 You can see it reasonably well. So, this is the worm Caenorhabditis 12 00:00:50,000 --> 00:00:55,000 elegans. It's been an object of much study here. 13 00:00:55,000 --> 00:00:59,000 Bob Horowitz in our own department just got the Nobel Prize 14 00:00:59,000 --> 00:01:07,000 for his work on this. 15 00:01:07,000 --> 00:01:11,000 And the reason it's been the object of so much study is, 16 00:01:11,000 --> 00:01:16,000 in fact, that it's a relatively simple organism. 17 00:01:16,000 --> 00:01:20,000 It's the way that you and I looked about 600 million years ago when 18 00:01:20,000 --> 00:01:25,000 Metazoa, remember Metazoa means multicellular organisms, 19 00:01:25,000 --> 00:01:29,000 first arose on the face of the planet. Let's see if I have too 20 00:01:29,000 --> 00:01:34,000 much overlap here. Well, that's about the way it should 21 00:01:34,000 --> 00:01:38,000 look. Now, what's remarkable about this organism is it has something 22 00:01:38,000 --> 00:01:42,000 like 965 cells in the adult. The cells all descend from a 23 00:01:42,000 --> 00:01:47,000 fertilized egg. It happens to be that this worm is 24 00:01:47,000 --> 00:01:51,000 a hermaphrodite. So, you probably know 25 00:01:51,000 --> 00:01:55,000 hermaphrodites are both male and female and it can fertilize itself 26 00:01:55,000 --> 00:02:00,000 or two worms can get together and fertilize each other. 27 00:02:00,000 --> 00:02:04,000 Because, as you can see, it makes both eggs and it makes 28 00:02:04,000 --> 00:02:08,000 sperm. And what's most interesting is the finite number of cells in the 29 00:02:08,000 --> 00:02:13,000 body of the adult. As I said, it's 965, 30 00:02:13,000 --> 00:02:17,000 whose lineage can be traced through a pedigree that's given right up 31 00:02:17,000 --> 00:02:22,000 here. So, one can plot out, with great precision, how all the 32 00:02:22,000 --> 00:02:26,000 cells, how the egg cell and the fertilized egg divides in two, 33 00:02:26,000 --> 00:02:31,000 each of those cells divides again in two and how ultimately one has a 34 00:02:31,000 --> 00:02:35,000 whole series of different descendents from different branches 35 00:02:35,000 --> 00:02:40,000 of this very elaborate family tree. Now, one of the issues that we want 36 00:02:40,000 --> 00:02:44,000 to pursue is the fact that the genomes, in principle, 37 00:02:44,000 --> 00:02:48,000 all the cells in this organism are the same. That is to say whenever 38 00:02:48,000 --> 00:02:52,000 the cell goes through a cycle of growth and division the cell is 39 00:02:52,000 --> 00:02:56,000 genetically identical, yet phenotypically, yet the behavior 40 00:02:56,000 --> 00:03:00,000 reach of these cells becomes increasingly different. 41 00:03:00,000 --> 00:03:04,000 And here you can see the different lineage of cells. 42 00:03:04,000 --> 00:03:08,000 This organism devotes a disproportionately large amount of 43 00:03:08,000 --> 00:03:12,000 its anatomy to reproduction, much more even than we do. Here, 44 00:03:12,000 --> 00:03:16,000 this is the cuticle, the outer coat. Here's the vulva, the female organ. 45 00:03:16,000 --> 00:03:20,000 This is the male organ and a variety of other cells types. 46 00:03:20,000 --> 00:03:24,000 Here's the pharynx. And yet all the cells in all these organisms, 47 00:03:24,000 --> 00:03:28,000 with the exception of the gametes, remember gametes means sperm and egg 48 00:03:28,000 --> 00:03:33,000 are genetically identical. The gametes have only half the 49 00:03:33,000 --> 00:03:37,000 genetic content. Everybody else is identical, 50 00:03:37,000 --> 00:03:42,000 has a diploid genome, that is to say two copies of each gene. 51 00:03:42,000 --> 00:03:46,000 And one can trace this all out. And this represents one of the 52 00:03:46,000 --> 00:03:50,000 great mysteries of developmental biology, which is to say how are 53 00:03:50,000 --> 00:03:55,000 cells that genetically identical to one another genotypically identical 54 00:03:55,000 --> 00:03:59,000 to one another, phenotypically quite different one 55 00:03:59,000 --> 00:04:04,000 from the other? What makes them so different? 56 00:04:04,000 --> 00:04:08,000 In fact, this image that we have here, which in itself represents a 57 00:04:08,000 --> 00:04:13,000 stunning achievement, that is being able to trace the 58 00:04:13,000 --> 00:04:18,000 pedigree of each cell in the adult body is very different from our own 59 00:04:18,000 --> 00:04:22,000 lineage because, as I may have mentioned to you in 60 00:04:22,000 --> 00:04:27,000 the past, each of us goes through ten to the sixth mitoses in a 61 00:04:27,000 --> 00:04:32,000 lifetime, you and I. That's ten to the sixteen cell 62 00:04:32,000 --> 00:04:36,000 divisions. And at any one time we have roughly three times ten to the 63 00:04:36,000 --> 00:04:40,000 thirteenth cells in our body. So, that means, if you think about 64 00:04:40,000 --> 00:04:44,000 that carefully, the ratio between these two suggests 65 00:04:44,000 --> 00:04:48,000 there's roughly a hundred times more here than here, 66 00:04:48,000 --> 00:04:52,000 three hundred times more It means, roughly speaking, that our body 67 00:04:52,000 --> 00:04:56,000 turns over roughly a hundred times in our lifetime. 68 00:04:56,000 --> 00:04:59,000 That is to say the cells turnover. Not all of the cells, 69 00:04:59,000 --> 00:05:03,000 but that there's a continuing replacement of existing cells with 70 00:05:03,000 --> 00:05:06,000 new cells. After all, if, in fact, there were no such 71 00:05:06,000 --> 00:05:10,000 replacement than we might, as an adult, be formed of this many 72 00:05:10,000 --> 00:05:13,000 cells, and we would stay with the exact same number of cells 73 00:05:13,000 --> 00:05:17,000 throughout our lives. But this, in fact, is the number of 74 00:05:17,000 --> 00:05:21,000 cell divisions. And so, there's an enormous turnover 75 00:05:21,000 --> 00:05:24,000 which, right away on its surface, independent of the fact that it's an 76 00:05:24,000 --> 00:05:28,000 enormously large number, precludes one from really being able 77 00:05:28,000 --> 00:05:31,000 to draw out a pedigree like this, prevents anyone from really 78 00:05:31,000 --> 00:05:35,000 understanding how each particular cell can trace its line of decent 79 00:05:35,000 --> 00:05:39,000 back to the fertilized egg. So, what we really want to explore, 80 00:05:39,000 --> 00:05:44,000 in this lecture and the next one, is this major puzzle that one starts 81 00:05:44,000 --> 00:05:48,000 out with a fertilized egg. Let's say this is a fertilized egg. 82 00:05:48,000 --> 00:05:53,000 It divides in two. It divides in four. And through subsequent cycles 83 00:05:53,000 --> 00:05:58,000 of growth and division we ultimately end up with the adult. 84 00:05:58,000 --> 00:06:03,000 Already at the four-cell stage, here in a vertebrate embryo, these 85 00:06:03,000 --> 00:06:08,000 cells have begun to take different phenotypic paths. 86 00:06:08,000 --> 00:06:13,000 That is to say cells have begun to commit themselves into entering into 87 00:06:13,000 --> 00:06:18,000 one or another differentiation lineage. And when I say a 88 00:06:18,000 --> 00:06:23,000 differentiating lineage, I mean a group of cells which has 89 00:06:23,000 --> 00:06:29,000 already made the decision to become blood cells, to become gut, 90 00:06:29,000 --> 00:06:34,000 to become nerve cells and so forth. And these commitments already start 91 00:06:34,000 --> 00:06:38,000 here at the four-cell stage, and they continue to play themselves 92 00:06:38,000 --> 00:06:42,000 out until one reaches a newborn, and then thereafter one just grows 93 00:06:42,000 --> 00:06:46,000 bigger. One other important thing to show here is that this pedigree 94 00:06:46,000 --> 00:06:50,000 that I showed you here is not simply the result of exponential expansion 95 00:06:50,000 --> 00:06:54,000 of all the cells, because many cells, 96 00:06:54,000 --> 00:06:58,000 during the course of development, are actually weeded out from 97 00:06:58,000 --> 00:07:02,000 embryonic tissue. And this happens even in our own 98 00:07:02,000 --> 00:07:07,000 development. For example, here, if we look at our fingers, 99 00:07:07,000 --> 00:07:11,000 I remember now I talked about fingers about two weeks ago and got 100 00:07:11,000 --> 00:07:16,000 myself into some hot water. If we look at our fingers, you'll 101 00:07:16,000 --> 00:07:21,000 see here we have five fingers, God willing, and but early in 102 00:07:21,000 --> 00:07:26,000 embryogenesis our hand looks like a solid flap of tissue. 103 00:07:26,000 --> 00:07:30,000 And what happens, during the course of vertebra development, 104 00:07:30,000 --> 00:07:35,000 is that the tissue in between the beginning fingers is eliminated 105 00:07:35,000 --> 00:07:40,000 through the process of apoptosis. Apoptosis means programmed cell 106 00:07:40,000 --> 00:07:46,000 death. Apoptosis is equivalent to cell suicide. And what I mean by 107 00:07:46,000 --> 00:07:52,000 that is to say that development involves not only the exponential 108 00:07:52,000 --> 00:07:58,000 proliferation of cells but it involves the selective elimination 109 00:07:58,000 --> 00:08:04,000 of cells here in a very obvious anatomical way. 110 00:08:04,000 --> 00:08:07,000 It sometimes can be defective, in which case individuals are born 111 00:08:07,000 --> 00:08:11,000 with large webs between their fingers. And this is part of normal 112 00:08:11,000 --> 00:08:15,000 development. And the same can be said here. If there were no 113 00:08:15,000 --> 00:08:18,000 apoptosis during the development of this worm embryo then there would be 114 00:08:18,000 --> 00:08:22,000 vastly larger numbers of cells. We mentioned implicitly apoptosis 115 00:08:22,000 --> 00:08:26,000 during the development of the immune system, because recall there that 116 00:08:26,000 --> 00:08:29,000 bee cells, which are destine to produce antibodies, 117 00:08:29,000 --> 00:08:33,000 if they produce inappropriate kinds of antibodies, 118 00:08:33,000 --> 00:08:37,000 if they produce antibodies that are self-reactive, 119 00:08:37,000 --> 00:08:41,000 i.e., recognize some of the body's own proteins, those cells are 120 00:08:41,000 --> 00:08:44,000 eliminated by apoptosis. If they produce defective antibody 121 00:08:44,000 --> 00:08:48,000 molecules, they're eliminated by apoptosis. And, 122 00:08:48,000 --> 00:08:51,000 therefore, differentiation, which is what we're talking about 123 00:08:51,000 --> 00:08:55,000 here, involves not only the commitment of cells to a certain 124 00:08:55,000 --> 00:08:58,000 lineage, but the purpose of elimination of cells in certain 125 00:08:58,000 --> 00:09:02,000 parts of the organism in order to carve and sculpt out properly 126 00:09:02,000 --> 00:09:05,000 shaped tissue. Again, our fingers are one dramatic 127 00:09:05,000 --> 00:09:09,000 example of that. It turns out we can learn an awful 128 00:09:09,000 --> 00:09:13,000 lot about this process by studying one specialized adult tissue, 129 00:09:13,000 --> 00:09:17,000 which is to say the organs of hematopoiesis. 130 00:09:17,000 --> 00:09:20,000 And they'll teach us a lot about some of the lessons we need to learn 131 00:09:20,000 --> 00:09:24,000 about organismic development and differentiation. 132 00:09:24,000 --> 00:09:28,000 And when I use the word hematopoiesis, 133 00:09:28,000 --> 00:09:32,000 the term hematopoiesis, or the adjective hematopoietic 134 00:09:32,000 --> 00:09:36,000 refers to the creation, the formation of different kinds of 135 00:09:36,000 --> 00:09:40,000 blood cells. In fact, we know that all the cells 136 00:09:40,000 --> 00:09:45,000 in the blood descend in the organism from a common progenitor. 137 00:09:45,000 --> 00:09:50,000 And this progenitor is called a pluripotent stem cell. 138 00:09:50,000 --> 00:09:55,000 Pluripotent means that this stem cell, and we'll define a stem cell 139 00:09:55,000 --> 00:10:00,000 momentarily, this stem cell is able to create descendents which can 140 00:10:00,000 --> 00:10:05,000 commit themselves in a number of distinct directions. 141 00:10:05,000 --> 00:10:09,000 They can differentiate in a number of distinct directions. 142 00:10:09,000 --> 00:10:14,000 In this case, we see all these various kinds of white and red blood 143 00:10:14,000 --> 00:10:19,000 cells which descend from this pluripotent stem cell. 144 00:10:19,000 --> 00:10:24,000 And, as a consequence, we call it pluripotent because it 145 00:10:24,000 --> 00:10:29,000 has these multiple distinct types of differentiation lineages. 146 00:10:29,000 --> 00:10:34,000 So, here we talk about pluripotent. Later on I'll talk about totipotent 147 00:10:34,000 --> 00:10:38,000 cells. Totipotent are cells that can do everything. 148 00:10:38,000 --> 00:10:42,000 Therefore, in fact, what's a totipotent cell? 149 00:10:42,000 --> 00:10:46,000 Well, a cell in the early embryo, including a fertilized egg is 150 00:10:46,000 --> 00:10:50,000 totipotent in that it can direct its descendents into all of the 151 00:10:50,000 --> 00:10:54,000 differentiation lineages in the body. Here we have a cell that's already 152 00:10:54,000 --> 00:10:58,000 more limited. It's only pluripotent, pluri in the sense of multiple 153 00:10:58,000 --> 00:11:03,000 but not total. Totipotent obviously means it can do 154 00:11:03,000 --> 00:11:07,000 everything. And here we see the different kinds of derivative white 155 00:11:07,000 --> 00:11:11,000 blood cells that exists in the bone marrow and in the circulation, 156 00:11:11,000 --> 00:11:15,000 and there's a whole series of different ones of them. 157 00:11:15,000 --> 00:11:19,000 We'll talk about some of them shortly, but we've already 158 00:11:19,000 --> 00:11:23,000 encountered some of them up here in the form of T cells and B cells. 159 00:11:23,000 --> 00:11:27,000 When we use the word stem cell the essence of the definition 160 00:11:27,000 --> 00:11:33,000 is as follows. A stem cell is a cell that can 161 00:11:33,000 --> 00:11:39,000 self-renew and it can also have a differentiated daughter. 162 00:11:39,000 --> 00:11:46,000 So, here's the way one can diagram a stem cell. Here's a stem cell 163 00:11:46,000 --> 00:11:53,000 that has two daughter cells. One daughter cell is exactly like 164 00:11:53,000 --> 00:11:59,000 Mom and the other daughter cell has undertaken a program of becoming 165 00:11:59,000 --> 00:12:04,000 differentiated. So, here we have an asymmetric cell 166 00:12:04,000 --> 00:12:08,000 division on the part of this stem cell up here. 167 00:12:08,000 --> 00:12:14,000 We'll prove later on that these two 168 00:12:14,000 --> 00:12:18,000 cells are genetically identical, but clearly they're reading out 169 00:12:18,000 --> 00:12:21,000 their genes in quite different ways. This cell is absolutely the same as 170 00:12:21,000 --> 00:12:24,000 the mother cell. This cell has already committed 171 00:12:24,000 --> 00:12:28,000 itself. It's made the commitment to differentiate in one 172 00:12:28,000 --> 00:12:32,000 or another lineage. And another way of noting this 173 00:12:32,000 --> 00:12:36,000 graphically is the following. We can draw a picture like this, 174 00:12:36,000 --> 00:12:41,000 as we did before, and here we'll have a second arrow that goes around 175 00:12:41,000 --> 00:12:45,000 like this. It loops around back on itself, and that implies the whole 176 00:12:45,000 --> 00:12:49,000 program of self-renewal. Now, the whole concept of 177 00:12:49,000 --> 00:12:54,000 self-renewal is a simple one. If a stem cell can self-renew, 178 00:12:54,000 --> 00:12:58,000 that implies that the process of growth and division does not deplete 179 00:12:58,000 --> 00:13:03,000 the pool of stem cells in the body or in a particular tissue. 180 00:13:03,000 --> 00:13:08,000 So, let's imagine what we're looking at here. Here we have a stem cell. 181 00:13:08,000 --> 00:13:13,000 It has one cell that is just like Mom. This is a differentiated cell. 182 00:13:13,000 --> 00:13:18,000 Once again, you can have a growth and division. This is, 183 00:13:18,000 --> 00:13:24,000 once again, a cell like Mom. This is a differentiated cell and 184 00:13:24,000 --> 00:13:29,000 so forth. And what you notice here in this arrangement is that the stem 185 00:13:29,000 --> 00:13:34,000 cells perpetuate themselves. They are self-renewing. 186 00:13:34,000 --> 00:13:38,000 And, as a consequence, the pool of stem cells is never depleted in the 187 00:13:38,000 --> 00:13:42,000 best of all possible worlds. It turns out that in most of the 188 00:13:42,000 --> 00:13:46,000 tissues of our body there is self-renewing stem cells going on, 189 00:13:46,000 --> 00:13:51,000 because most of the differentiated cells in our body have a finite 190 00:13:51,000 --> 00:13:55,000 lifetime. Not all of them but most of them. And when I say finite, 191 00:13:55,000 --> 00:13:59,000 I mean it can be measured in a matter of days or weeks 192 00:13:59,000 --> 00:14:03,000 or months. In the case of the brain, 193 00:14:03,000 --> 00:14:07,000 things turnover very slowly. Even in the case of our bones, 194 00:14:07,000 --> 00:14:11,000 our bones actually turn over roughly once, 10% a year. 195 00:14:11,000 --> 00:14:15,000 10% of the matter in the bone is actually turnover in every year. 196 00:14:15,000 --> 00:14:19,000 So, almost all tissues in the body are in a process of continuing 197 00:14:19,000 --> 00:14:23,000 self-renewal and repair. And that self-renewal and repair is 198 00:14:23,000 --> 00:14:27,000 maintained by this stem cell compartment, as is indicated here. 199 00:14:27,000 --> 00:14:32,000 This has certain kinds of great advantages, and one of the 200 00:14:32,000 --> 00:14:38,000 advantages is indicated by the following. Let's imagine that we 201 00:14:38,000 --> 00:14:43,000 draw a picture, just for the sake of argument, 202 00:14:43,000 --> 00:14:49,000 of one of the most highly proliferative tissues in the body, 203 00:14:49,000 --> 00:14:54,000 which is to say the lining of the colon or of the duodenum. 204 00:14:54,000 --> 00:15:00,000 So, here we have, let me draw it slightly differently, 205 00:15:00,000 --> 00:15:06,000 here's the way the lining of the small intestine looks like. 206 00:15:06,000 --> 00:15:10,000 Out here are the contents of the small intestine. 207 00:15:10,000 --> 00:15:20,000 So, let's say here is the lumen of 208 00:15:20,000 --> 00:15:24,000 the small intestine. And here we have, protruding into 209 00:15:24,000 --> 00:15:28,000 the lumen, when I talk about a lumen I'm talking about the bore or the 210 00:15:28,000 --> 00:15:32,000 channel of a cylindrical or tube like organism. 211 00:15:32,000 --> 00:15:36,000 So, here is the lumen of the small intestine. Here are these 212 00:15:36,000 --> 00:15:41,000 fingerlike projections, they're called villi, that protrude 213 00:15:41,000 --> 00:15:45,000 into the lumen of the small intestine. And down here at the 214 00:15:45,000 --> 00:15:50,000 bottom of this are these cavities that are called crypts, 215 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 216 00:15:54,000 --> 00:15:59,000 that what goes through our intestines is not that pleasant. 217 00:15:59,000 --> 00:16:03,000 It's pretty corrosive stuff. I probably told you this already, 218 00:16:03,000 --> 00:16:07,000 more bacteria living in there than we have in our entire cells in our 219 00:16:07,000 --> 00:16:11,000 entire body. There are all kinds of digestive juices. 220 00:16:11,000 --> 00:16:15,000 And so the cells out here at the tips of these villi are continually 221 00:16:15,000 --> 00:16:19,000 exposed to all kinds of corrosive material, including the junk that we 222 00:16:19,000 --> 00:16:23,000 eat everyday which is flowing by like this. And this indicates how 223 00:16:23,000 --> 00:16:27,000 critically important it is that we have self-renewal, 224 00:16:27,000 --> 00:16:31,000 because the cells out here, being continually exposed to the 225 00:16:31,000 --> 00:16:35,000 most corrosive kinds of influences, are rapidly damages. 226 00:16:35,000 --> 00:16:39,000 And, therefore, the cells out here have a lifetime 227 00:16:39,000 --> 00:16:44,000 of only three or four days and are then induced to jump off the end of 228 00:16:44,000 --> 00:16:49,000 a gangplank and commit apoptosis. So, the cells at the tip of the 229 00:16:49,000 --> 00:16:54,000 villis are continually jumping off and dying. And what's happening is 230 00:16:54,000 --> 00:16:59,000 that down here in the bottom of the crypts we have stem cells. 231 00:16:59,000 --> 00:17:06,000 The stem cells are continually 232 00:17:06,000 --> 00:17:09,000 producing progeny that have committed themselves to 233 00:17:09,000 --> 00:17:13,000 differentiate. And the progeny, 234 00:17:13,000 --> 00:17:16,000 as you might guess from what I've just said, are continually migrating 235 00:17:16,000 --> 00:17:19,000 up the sides of the villis up to the end here. And this whole migration 236 00:17:19,000 --> 00:17:23,000 takes four or five days, and by the time they get to the tip 237 00:17:23,000 --> 00:17:26,000 and have stuck their heads into the contents of the lumen of the 238 00:17:26,000 --> 00:17:30,000 intestine for that period of three or four days. 239 00:17:30,000 --> 00:17:33,000 Finally, they're eliminated and they jump off into the abyss. 240 00:17:33,000 --> 00:17:37,000 So, there's a continuing action going on here. 241 00:17:37,000 --> 00:17:40,000 The stem cells are continually dividing. And what advantages does 242 00:17:40,000 --> 00:17:44,000 this have for us? Well, it means that cells that are 243 00:17:44,000 --> 00:17:47,000 damaged are not allowed to hang around for a very long period of 244 00:17:47,000 --> 00:17:51,000 time, i.e., cells up here in the top that are exposed to, 245 00:17:51,000 --> 00:17:55,000 for example, potential mutagenic influences are rapidly eliminated. 246 00:17:55,000 --> 00:17:58,000 Why is that good? Because the mutagenic influences up here could 247 00:17:58,000 --> 00:18:02,000 well create a mutant cell that, in principle, is able to become 248 00:18:02,000 --> 00:18:05,000 cancerous. And the body says, 249 00:18:05,000 --> 00:18:09,000 well, I don't mind if that happens because these cells up here are 250 00:18:09,000 --> 00:18:12,000 going to be eliminated anyhow. They're going to be pushed off the 251 00:18:12,000 --> 00:18:16,000 end of the diving board or the gangplank into the abyss, 252 00:18:16,000 --> 00:18:19,000 so they are continually undergoing apoptosis, not as a pathological 253 00:18:19,000 --> 00:18:23,000 process. As a normal process. They're continually being pushed 254 00:18:23,000 --> 00:18:27,000 out here. And what that means is that the 255 00:18:27,000 --> 00:18:31,000 cells down here, in the bottom of the crypt, 256 00:18:31,000 --> 00:18:36,000 are actually physically protected from the contents of the lumen of 257 00:18:36,000 --> 00:18:41,000 the small intestine because some of the cells in this crypt are 258 00:18:41,000 --> 00:18:45,000 continually secreting a kind of mucus in this area right here. 259 00:18:45,000 --> 00:18:50,000 It's called a mucin. And this mucin here creates a physical 260 00:18:50,000 --> 00:18:55,000 barrier, so the cells that are in the bottom of the crypt are never 261 00:18:55,000 --> 00:18:59,000 directly exposed to the contents of what's flowing by in the 262 00:18:59,000 --> 00:19:03,000 small intestine. And that is extremely important 263 00:19:03,000 --> 00:19:07,000 because, in fact, it means that these cells down here 264 00:19:07,000 --> 00:19:11,000 are shielded from the mutagenic influences of what might be present 265 00:19:11,000 --> 00:19:15,000 in the lumen of the small intestine. In theory, one might be able to 266 00:19:15,000 --> 00:19:19,000 evolve cells that don't mind being up here in the lumen of the small 267 00:19:19,000 --> 00:19:23,000 intestine. But, in fact, that's never been possible. 268 00:19:23,000 --> 00:19:27,000 That is to say evolution has just said, well, we can't really evolve 269 00:19:27,000 --> 00:19:31,000 cells that are resistant to the corrosive influences of what happens 270 00:19:31,000 --> 00:19:34,000 in the small intestine. And, therefore, 271 00:19:34,000 --> 00:19:38,000 we're just going to use these cells for a very short period of time and 272 00:19:38,000 --> 00:19:41,000 then get rid of them. What that also means is the 273 00:19:41,000 --> 00:19:44,000 following. The stem cells down here stay within that crypt. 274 00:19:44,000 --> 00:19:48,000 They don't migrate out. They stay there in that shielded 275 00:19:48,000 --> 00:19:51,000 site. And, in fact, if you think all this through, 276 00:19:51,000 --> 00:19:54,000 it's very important to protect these cells from becoming mutated because 277 00:19:54,000 --> 00:19:58,000 if they do become mutated they could become the precursors 278 00:19:58,000 --> 00:20:02,000 of cancer cells. If these cells become mutated out 279 00:20:02,000 --> 00:20:06,000 here, it doesn't matter because they're going to die anyhow. 280 00:20:06,000 --> 00:20:11,000 And so, we now have the following kind of dynamic. 281 00:20:11,000 --> 00:20:15,000 Here's the stem cell. I'll draw it again. I'll just 282 00:20:15,000 --> 00:20:20,000 abbreviate it stem cell. And one of the ways by which we 283 00:20:20,000 --> 00:20:24,000 want to preserve the genetic integrity of the stem cell is to 284 00:20:24,000 --> 00:20:29,000 insure that the stem cell divides as infrequently as possible. Why? 285 00:20:29,000 --> 00:20:32,000 Because the whole process of cell division is itself a fallible 286 00:20:32,000 --> 00:20:36,000 process. Every time a cell grows and divides, as we learned from the 287 00:20:36,000 --> 00:20:40,000 cell cycle, there's the possibility of different kinds of genetic 288 00:20:40,000 --> 00:20:44,000 disasters happening in which case we might end up with a mutant stem cell. 289 00:20:44,000 --> 00:20:47,000 And that mutant stem cell could in turn, I argue, 290 00:20:47,000 --> 00:20:51,000 become involved in creating a tumor. So, we have the following kind of 291 00:20:51,000 --> 00:20:55,000 arrangement. Here is the stem cell. It has one daughter that has 292 00:20:55,000 --> 00:20:59,000 committed herself to differentiate and the other that remains 293 00:20:59,000 --> 00:21:03,000 a stem cell. Well, you're saying this enormous 294 00:21:03,000 --> 00:21:08,000 amount of activity must involve a frenetic amount of cell division on 295 00:21:08,000 --> 00:21:13,000 the part of the stem cell. But that's not really the way it 296 00:21:13,000 --> 00:21:19,000 happens because this daughter cell undergoes a series of exponential 297 00:21:19,000 --> 00:21:24,000 divisions, I can't fit them all on the blackboard here, 298 00:21:24,000 --> 00:21:29,000 and might even yield a hundred descendents which then become the 299 00:21:29,000 --> 00:21:34,000 ultimate differentiated cell. This daughter cell becomes one stem 300 00:21:34,000 --> 00:21:38,000 cell. This daughter cell undergoes these exponential expansions in a 301 00:21:38,000 --> 00:21:42,000 process of creating a population of cells that are called transit 302 00:21:42,000 --> 00:21:53,000 amplifying cells. 303 00:21:53,000 --> 00:21:56,000 And at the bottom of this hierarchy, I can't draw all hundred, there may 304 00:21:56,000 --> 00:21:59,000 be a hundred of these cells, ten to the second, and these cells 305 00:21:59,000 --> 00:22:06,000 then differentiate. 306 00:22:06,000 --> 00:22:10,000 At the bottom, the hundred cells at the bottom, 307 00:22:10,000 --> 00:22:14,000 they go into the last stage of differentiation. 308 00:22:14,000 --> 00:22:18,000 They become the specialized cells that line the tips of the villis. 309 00:22:18,000 --> 00:22:22,000 Now, why is there this arrangement? Well, look at what the advantage of 310 00:22:22,000 --> 00:22:26,000 it is. The stem cell has just divided once, but this cell has 311 00:22:26,000 --> 00:22:30,000 generated a hundred progeny. And that means that the stem cell 312 00:22:30,000 --> 00:22:34,000 doesn't have to divide that often. The stem cell can divide once. 313 00:22:34,000 --> 00:22:38,000 Every time the villis needs a hundred new cells it needs to divide 314 00:22:38,000 --> 00:22:42,000 only once. And, therefore, the stem cell actually is 315 00:22:42,000 --> 00:22:46,000 one of the most slowly dividing cells in the entire gut because it 316 00:22:46,000 --> 00:22:50,000 only needs to divide episodically. Each time it divides it generates 317 00:22:50,000 --> 00:22:54,000 this enormous array of progeny. One other aspect of differentiation, 318 00:22:54,000 --> 00:22:58,000 and when I talk about differentiation here, 319 00:22:58,000 --> 00:23:02,000 I mean the acquisition by these cells of all of the traits they need 320 00:23:02,000 --> 00:23:06,000 to line the colon. When I talk about differentiation in 321 00:23:06,000 --> 00:23:12,000 the skin, I talk about the ability, the acquisition of the cells of 322 00:23:12,000 --> 00:23:17,000 becoming fully competent, fully functional skin cells. 323 00:23:17,000 --> 00:23:23,000 The same thing with neurons in the brain. And one additional important 324 00:23:23,000 --> 00:23:29,000 concept I'd like to introduce is that when cells differentiate they 325 00:23:29,000 --> 00:23:34,000 often become post-mitotic. Post-mitotic means that these cells 326 00:23:34,000 --> 00:23:38,000 give up the option of ever dividing again. In other words, 327 00:23:38,000 --> 00:23:42,000 as they acquire more and more specialized traits they say to 328 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 329 00:23:46,000 --> 00:23:50,000 in the intestine, now I'm a muscle cell, 330 00:23:50,000 --> 00:23:54,000 I'm not going to divide ever again. And this is generally true. The 331 00:23:54,000 --> 00:23:58,000 most differentiated cells in the body in general end up losing the 332 00:23:58,000 --> 00:24:02,000 ability to divide. They become post-mitotic. 333 00:24:02,000 --> 00:24:06,000 They've exited irreversibly from the cell cycle. 334 00:24:06,000 --> 00:24:11,000 They can't go back in. As you recall, we talked about 335 00:24:11,000 --> 00:24:16,000 cells going from G0 back into the active G1 phase of the cell cycle. 336 00:24:16,000 --> 00:24:20,000 That's a reversible exit from the cell cycle. Post-mitotic cells are 337 00:24:20,000 --> 00:24:25,000 irreversibly committed never to divide again. And, 338 00:24:25,000 --> 00:24:30,000 again, that holds true for almost all cells of the body. 339 00:24:30,000 --> 00:24:34,000 One exception to that is, interesting enough, in the liver. 340 00:24:34,000 --> 00:24:38,000 Because what you can do with a mouse, or even with a human, 341 00:24:38,000 --> 00:24:43,000 is you can cut away a couple lobes of the liver, major league surgery. 342 00:24:43,000 --> 00:24:47,000 And when you do that, what happens is that all the remaining cells of 343 00:24:47,000 --> 00:24:52,000 the liver, and these remaining cells in the liver are called hepatocytes, 344 00:24:52,000 --> 00:24:56,000 these hepatocytes, which until that time had been highly specialized 345 00:24:56,000 --> 00:25:00,000 differentiated cells in the liver, many of them divide, they double 346 00:25:00,000 --> 00:25:04,000 again. And in short order one ends up with 347 00:25:04,000 --> 00:25:07,000 a liver which is exactly the same size as one had before. 348 00:25:07,000 --> 00:25:10,000 And that's actually quite remarkable because there are very 349 00:25:10,000 --> 00:25:12,000 few organs in the adult human being where that will happen. 350 00:25:12,000 --> 00:25:15,000 There is, by the way, an interesting puzzle here, 351 00:25:15,000 --> 00:25:18,000 and that is the following. Let's say you cut away half the 352 00:25:18,000 --> 00:25:21,000 liver and many of the hepatocytes, which were already highly 353 00:25:21,000 --> 00:25:24,000 differentiated, began to divide again, 354 00:25:24,000 --> 00:25:27,000 so they were not serious post-mitotic cells, 355 00:25:27,000 --> 00:25:30,000 they could reenter into proliferative phase. 356 00:25:30,000 --> 00:25:34,000 How do these cells know when to stop dividing so that they end up 357 00:25:34,000 --> 00:25:38,000 regenerating a liver of exactly the right size? People have been 358 00:25:38,000 --> 00:25:43,000 looking at that for 30 or 40 years. Nobody has any idea why. Why 359 00:25:43,000 --> 00:25:47,000 doesn't the liver when all these cells divide become one and a half 360 00:25:47,000 --> 00:25:51,000 times the size of its former diameter or half the size? 361 00:25:51,000 --> 00:25:56,000 Nobody really understands that. In any case, I just want to 362 00:25:56,000 --> 00:26:00,000 indicate that there is this dynamic between differentiation and 363 00:26:00,000 --> 00:26:05,000 proliferative capacity, one in opposition to the other. 364 00:26:05,000 --> 00:26:10,000 Well, how much hematopoiesis is taking place here in our bone marrow, 365 00:26:10,000 --> 00:26:16,000 for example, where a lot of this takes place? So, 366 00:26:16,000 --> 00:26:22,000 here are just some interesting numbers. There are roughly five 367 00:26:22,000 --> 00:26:28,000 times ten to the twelfth red blood cells -- 368 00:26:28,000 --> 00:26:34,000 -- per liter of blood. 369 00:26:34,000 --> 00:26:40,000 And red blood cells, you may recall, are called erythrocytes. 370 00:26:40,000 --> 00:26:45,000 Remember, it's never good to use a short Anglo-Saxon term if you can 371 00:26:45,000 --> 00:26:51,000 use a long complicated Greek one. And each of these red blood cells 372 00:26:51,000 --> 00:26:56,000 has roughly a lifetime of 120 days. That is to say after it's made it 373 00:26:56,000 --> 00:27:02,000 sits around in the blood for roughly 120 days. 374 00:27:02,000 --> 00:27:06,000 It gets warn out. It gets gobbled up by the cells in 375 00:27:06,000 --> 00:27:11,000 the spleen. Much of the contents are recycled. The pigment in our 376 00:27:11,000 --> 00:27:16,000 stool comes from the recycling of the hemoglobin, 377 00:27:16,000 --> 00:27:20,000 the iron and the hemoglobin, and we're constantly renewing that. 378 00:27:20,000 --> 00:27:25,000 Well, the average human being, let's say, has roughly ten liters of 379 00:27:25,000 --> 00:27:32,000 blood per person. 380 00:27:32,000 --> 00:27:36,000 And if you put all those numbers together, I once calculated that the 381 00:27:36,000 --> 00:27:41,000 number of red blood cells that is being generated in the body is 382 00:27:41,000 --> 00:27:45,000 roughly ten to the tenth per hour. And you can go through the 383 00:27:45,000 --> 00:27:50,000 calculations if you want, it is fine with me, but take my word 384 00:27:50,000 --> 00:27:54,000 for it's roughly what's going on there. Ten to the tenth per hour. 385 00:27:54,000 --> 00:27:59,000 Each time I utter a word there's probably, I don't know, 386 00:27:59,000 --> 00:28:04,000 ten to the seventh new red blood cells being made in my bone marrow. 387 00:28:04,000 --> 00:28:07,000 So, this is not minor league proliferation. 388 00:28:07,000 --> 00:28:11,000 Now we begin to understand why there are ten to the sixteenth cell 389 00:28:11,000 --> 00:28:15,000 divisions in the lifetime of a human being. In fact, 390 00:28:15,000 --> 00:28:19,000 a great majority of them, to be fair, are occurring in the 391 00:28:19,000 --> 00:28:22,000 bone marrow and in the intestine. And if you have an individual who's 392 00:28:22,000 --> 00:28:26,000 being exposed to certain kinds of chemotherapy, chemotherapy, 393 00:28:26,000 --> 00:28:30,000 as you may know, is very toxic for dividing cells, 394 00:28:30,000 --> 00:28:34,000 and the side effect toxicity of anticancer chemotherapy is largely 395 00:28:34,000 --> 00:28:38,000 felt, first of all, in the bone marrow where individuals 396 00:28:38,000 --> 00:28:42,000 tend to become anemic. Anemic means they have lower than 397 00:28:42,000 --> 00:28:46,000 normal numbers of red blood cells and also they lose a lot of the 398 00:28:46,000 --> 00:28:51,000 lining of the intestine which creates all kinds of also 399 00:28:51,000 --> 00:28:55,000 unpleasantness as well. So, we're not talking about 400 00:28:55,000 --> 00:28:59,000 something that happens on rare occasion in the life 401 00:28:59,000 --> 00:29:04,000 an individual. This is a staggering amount of 402 00:29:04,000 --> 00:29:08,000 mitosis that's happening every day of our lives. Let's go back for a 403 00:29:08,000 --> 00:29:12,000 moment to this diagram here and realize that when I'm talking about 404 00:29:12,000 --> 00:29:16,000 erythrocytes, I'm only talking about one of the branches of this 405 00:29:16,000 --> 00:29:20,000 multi-branch pathway. And here we see some other 406 00:29:20,000 --> 00:29:24,000 interesting aspects of what's going on here, and I'll give you some 407 00:29:24,000 --> 00:29:28,000 proofs very shortly that this actually is what it says it is. 408 00:29:28,000 --> 00:29:32,000 It actually is organized this way. The pluripotent stem cell is capable 409 00:29:32,000 --> 00:29:36,000 of self-renewal, and it can spew off daughters which 410 00:29:36,000 --> 00:29:40,000 actually can go in two different directions. Its daughter may decide 411 00:29:40,000 --> 00:29:44,000 that it might become the precursor of the lymphoid cells in the blood 412 00:29:44,000 --> 00:29:48,000 or it might commit itself to becoming a myeloid precursor. 413 00:29:48,000 --> 00:29:52,000 So, that's already the beginning of a bifurcation. 414 00:29:52,000 --> 00:29:56,000 These cells are not yet differentiated. 415 00:29:56,000 --> 00:30:00,000 They've just made the commitment that each of them can, 416 00:30:00,000 --> 00:30:05,000 in principle, become the ancestor of highly differentiated cells. 417 00:30:05,000 --> 00:30:08,000 And these cells, we can imagine, are transient 418 00:30:08,000 --> 00:30:12,000 amplifying cells in the sense that even though they're committed to 419 00:30:12,000 --> 00:30:16,000 create progeny of one sort or another they themselves are not yet 420 00:30:16,000 --> 00:30:19,000 fully differentiated. Keep in mind in the context of the 421 00:30:19,000 --> 00:30:23,000 crypt these transient amplifying cells are on the way to becoming 422 00:30:23,000 --> 00:30:27,000 fully differentiated, but only at the bottom of this 423 00:30:27,000 --> 00:30:31,000 exponential expansion of cells do we have cells that are fully entered 424 00:30:31,000 --> 00:30:35,000 into a highly differentiated state. Here we see that these two cells are 425 00:30:35,000 --> 00:30:39,000 also stem cells in the sense that the can self-renew. 426 00:30:39,000 --> 00:30:44,000 They have a limited self-renewal capacity, but they can self-renew. 427 00:30:44,000 --> 00:30:48,000 And then they begin to create progeny which themselves can 428 00:30:48,000 --> 00:30:53,000 undertake several distinct alternative differentiation paths. 429 00:30:53,000 --> 00:30:57,000 So, the lymphoid cells can become the progenitors of the T lymphocytes 430 00:30:57,000 --> 00:31:02,000 and the B lymphocytes. And, in fact, if you recall our 431 00:31:02,000 --> 00:31:06,000 discussion of immunology, there's actually several different 432 00:31:06,000 --> 00:31:10,000 kinds of T lymphocytes and B lymphocytes. So, 433 00:31:10,000 --> 00:31:14,000 this pathway has further radiations further down. Here, 434 00:31:14,000 --> 00:31:18,000 alternatively, these are the myeloid cells. And myeloid refers to the 435 00:31:18,000 --> 00:31:22,000 bone marrow. And the myeloid cells can become these kinds of cells up 436 00:31:22,000 --> 00:31:26,000 here, eosinophils and basophils and neutrophils and monocytes, 437 00:31:26,000 --> 00:31:30,000 and this class of cells is largely involved in gobbling up infectious 438 00:31:30,000 --> 00:31:34,000 agents and as agents which are able to defend us largely against 439 00:31:34,000 --> 00:31:38,000 bacterial infections. Here's the macrophage. 440 00:31:38,000 --> 00:31:42,000 We talked about the macrophage. Remember the macrophage was this 441 00:31:42,000 --> 00:31:45,000 glutton, this pig which wandered around our tissues and gobbled up 442 00:31:45,000 --> 00:31:49,000 whatever kind of material it could find and presented it to the immune 443 00:31:49,000 --> 00:31:52,000 system. And here are several other of the lineages. 444 00:31:52,000 --> 00:31:56,000 Here is a megakaryocytic and this is an erythrocyte. 445 00:31:56,000 --> 00:32:00,000 What's a megakariocyte? Well, it has a very large nucleus. 446 00:32:00,000 --> 00:32:04,000 That's what the term implies. And what happens to the 447 00:32:04,000 --> 00:32:09,000 megakariocyte is it buds off little chunks of cytoplasm lacking nuclei. 448 00:32:09,000 --> 00:32:13,000 And these little chunks of cytoplasm become the blood platelets. 449 00:32:13,000 --> 00:32:18,000 Blood platelets lack nuclei. They're enucleate because they're 450 00:32:18,000 --> 00:32:22,000 just little bags of material which are sent out into the circulation. 451 00:32:22,000 --> 00:32:27,000 And, as we said also earlier in the semester, once the platelets are in 452 00:32:27,000 --> 00:32:31,000 the circulation they are there ready to help should there be any kind of 453 00:32:31,000 --> 00:32:36,000 wounding, any kind of hemorrhaging occurring. 454 00:32:36,000 --> 00:32:39,000 And the platelets can release upon being activated in a site of wound 455 00:32:39,000 --> 00:32:43,000 coagulation factors and growth factors for the regeneration and 456 00:32:43,000 --> 00:32:47,000 reconstruction of wound sites. And, finally, here are our friends 457 00:32:47,000 --> 00:32:51,000 the erythrocytes. So, here we have a whole sequence 458 00:32:51,000 --> 00:32:55,000 of different kinds of differentiation commitments which 459 00:32:55,000 --> 00:32:59,000 are going on at an enormous rate. How do we know that there actually 460 00:32:59,000 --> 00:33:03,000 is a pluripotent stem cell? What evidence can I provide you that 461 00:33:03,000 --> 00:33:07,000 this actually exists or it's just a figment of my normally florid 462 00:33:07,000 --> 00:33:12,000 imagination? And the most direct demonstration of that is, 463 00:33:12,000 --> 00:33:16,000 in fact, the use of bone marrow transplantation. 464 00:33:16,000 --> 00:33:21,000 So, when we talk about a bone marrow transplantation, 465 00:33:21,000 --> 00:33:26,000 or BMT as it's called in the trade, -- 466 00:33:26,000 --> 00:33:33,000 -- one can do a relatively simple 467 00:33:33,000 --> 00:33:37,000 experiment. You can take a mouse or even a human and you can irradiate 468 00:33:37,000 --> 00:33:41,000 it rather heavily. And if you irradiate it under the 469 00:33:41,000 --> 00:33:44,000 right conditions you'll actually be able to kill off all the cells in 470 00:33:44,000 --> 00:33:48,000 the bone marrow without killing off the mouse or the human being. 471 00:33:48,000 --> 00:33:52,000 In fact, there are drugs you can also use in human beings to 472 00:33:52,000 --> 00:33:55,000 eliminate virtually all the cells in the bone marrow. 473 00:33:55,000 --> 00:33:59,000 And then what you can do is you can take bone marrow from another 474 00:33:59,000 --> 00:34:03,000 organism, from another mouse or another human, 475 00:34:03,000 --> 00:34:07,000 and you inject it into the blood of the irradiated mouse or human. 476 00:34:07,000 --> 00:34:10,000 And the bone marrow cells, many of them will home to the bone 477 00:34:10,000 --> 00:34:14,000 marrow. In other words, you're injecting the bone marrow 478 00:34:14,000 --> 00:34:17,000 cells in the general circulation, but within a couple hours they'll 479 00:34:17,000 --> 00:34:21,000 all end up in the bone marrow, in the space in the middle of the 480 00:34:21,000 --> 00:34:25,000 bone because there are many kinds of cells which have this homing 481 00:34:25,000 --> 00:34:29,000 capacity. They go to the right place in the body. 482 00:34:29,000 --> 00:34:33,000 So, they can home. The injected cells can home to bone 483 00:34:33,000 --> 00:34:37,000 marrow. And then, if things are going well, 484 00:34:37,000 --> 00:34:41,000 these injected bone marrow cells will begin to proliferate and they 485 00:34:41,000 --> 00:34:45,000 will ultimately regenerate this entire cascade of differentiation 486 00:34:45,000 --> 00:34:49,000 decisions, as is indicated here. And, therefore, that individual or 487 00:34:49,000 --> 00:34:53,000 that mouse will actually be rescued. Because in the absence of such a 488 00:34:53,000 --> 00:34:58,000 rescue an individual will rapidly die. 489 00:34:58,000 --> 00:35:01,000 You can't live for very long in the absence of an active bone marrow 490 00:35:01,000 --> 00:35:05,000 because these cells here are rapidly depleted. They turn over with some 491 00:35:05,000 --> 00:35:08,000 speed. The red blood cells hang around for 120 days we said and, 492 00:35:08,000 --> 00:35:12,000 therefore, you don't need to make them immediately because there's a 493 00:35:12,000 --> 00:35:15,000 whole bunch around that have a rather slow turnover. 494 00:35:15,000 --> 00:35:19,000 But the platelets only have a lifetime of several days before 495 00:35:19,000 --> 00:35:22,000 they're lost, they're turned over. And if you don't have platelets 496 00:35:22,000 --> 00:35:26,000 you're in very bad shape because you start hemorrhaging all over the body 497 00:35:26,000 --> 00:35:29,000 because, remember, the platelets are there to stop up 498 00:35:29,000 --> 00:35:33,000 all the holes in the dike to prevent bleeding. 499 00:35:33,000 --> 00:35:37,000 These cells here are very important, the eosinophils, basophils, 500 00:35:37,000 --> 00:35:41,000 neutrophils, and even macrophages in preventing bacterial infections. 501 00:35:41,000 --> 00:35:45,000 And in the absence of having these on site one can readily succumb to 502 00:35:45,000 --> 00:35:49,000 overwhelming infections. Keep in mind that the reason why 503 00:35:49,000 --> 00:35:53,000 we're not constantly dying from bacterial infections is not because 504 00:35:53,000 --> 00:35:57,000 each of us takes an antibiotic pill every day, it's because these cells 505 00:35:57,000 --> 00:36:01,000 are on watch to kill any bacteria that happen to be in the wrong place 506 00:36:01,000 --> 00:36:05,000 in the body outside of the lumen of the gut. 507 00:36:05,000 --> 00:36:09,000 And consequently the question is always can one rescue a mouse or a 508 00:36:09,000 --> 00:36:14,000 human rapidly enough? Can one replace its bone marrow 509 00:36:14,000 --> 00:36:19,000 rapidly enough so that this disaster from losing all ones bone marrow 510 00:36:19,000 --> 00:36:23,000 doesn't overtake one and the organism dies before the bone marrow 511 00:36:23,000 --> 00:36:28,000 has had a chance to become reconstituted, regenerated, 512 00:36:28,000 --> 00:36:32,000 reconstructed. Still how do we know from all this 513 00:36:32,000 --> 00:36:36,000 that, in fact, there is a pluripotent stem cell? 514 00:36:36,000 --> 00:36:39,000 If you listened to everything I said correctly you could say, 515 00:36:39,000 --> 00:36:43,000 well, there isn't such a thing as a pluripotent stem cell. 516 00:36:43,000 --> 00:36:46,000 There are these other kinds of stem cells, this one and this one, 517 00:36:46,000 --> 00:36:50,000 or these might all be stem cells. And when I'm injecting the bone 518 00:36:50,000 --> 00:36:54,000 marrow of a donor animal into the recipient, I'm injecting a whole 519 00:36:54,000 --> 00:36:57,000 mixture of different kinds of stem cells here each of which then goes 520 00:36:57,000 --> 00:37:01,000 on and populates a specialized compartment in the bone marrow 521 00:37:01,000 --> 00:37:05,000 or in the blood. So how do we know there's one 522 00:37:05,000 --> 00:37:09,000 pluripotent stem cell? One way to prove this is the 523 00:37:09,000 --> 00:37:14,000 following. Let's say we take the bone marrow from the donor, 524 00:37:14,000 --> 00:37:18,000 that is the bone marrow that we're going to inject into the irradiated 525 00:37:18,000 --> 00:37:23,000 recipient, and we irradiate that bone marrow very lightly, 526 00:37:23,000 --> 00:37:28,000 not to kill the bone marrow cells but to introduce random chromosomal 527 00:37:28,000 --> 00:37:32,000 breaks, a very small number of random chromosomal breaks in the 528 00:37:32,000 --> 00:37:37,000 donor bone marrow. So, the purpose now of irradiation 529 00:37:37,000 --> 00:37:41,000 is quite different from what I said before. Before we wanted to give a 530 00:37:41,000 --> 00:37:45,000 heavy dose of radiation to wipe out the recipient bone marrow. 531 00:37:45,000 --> 00:37:49,000 Now we're going to just give a wee bit of radiation to the donor bone 532 00:37:49,000 --> 00:37:53,000 marrow. What's the purpose of that? The purpose of that small amount of 533 00:37:53,000 --> 00:37:58,000 radiation is to create chromosomal abnormalities. 534 00:37:58,000 --> 00:38:03,000 So, for example, if here are two homologous 535 00:38:03,000 --> 00:38:09,000 chromosomes in the donor cells. Since the radiation, the very weak 536 00:38:09,000 --> 00:38:14,000 dose of radiation is acting randomly it will create all kinds of 537 00:38:14,000 --> 00:38:20,000 abnormalities including, for example, a very specific 538 00:38:20,000 --> 00:38:25,000 chromosomal translocation so that what might happen after this is that 539 00:38:25,000 --> 00:38:31,000 a whole chunk of this chromosome is translocated over to this chromosome 540 00:38:31,000 --> 00:38:37,000 here. This is called a chromosomal translocation. 541 00:38:37,000 --> 00:38:44,000 And now here's the donor, 542 00:38:44,000 --> 00:38:48,000 these are donor bone marrow cells. And keep in mind that every donor 543 00:38:48,000 --> 00:38:52,000 bone marrow cell that gets a little bit of this radiation will get its 544 00:38:52,000 --> 00:38:56,000 own very specific randomly occurring translocation just because radiation 545 00:38:56,000 --> 00:39:00,000 is able to break chromosomes and then they will rejoin in 546 00:39:00,000 --> 00:39:04,000 unpredictable ways. What that means is that if we take 547 00:39:04,000 --> 00:39:08,000 the donor bone marrow and irradiate it very lightly so that we don't 548 00:39:08,000 --> 00:39:12,000 kill the cells but we do induce these translocations, 549 00:39:12,000 --> 00:39:17,000 one donor cell will have this translocation and another donor cell 550 00:39:17,000 --> 00:39:21,000 over here will have a totally different translocation from a 551 00:39:21,000 --> 00:39:25,000 different chromosome also induced randomly by these stochastic 552 00:39:25,000 --> 00:39:30,000 processes. So the karyotype which is the whole 553 00:39:30,000 --> 00:39:34,000 array of chromosomes of a cell, which can be viewed at the metaphase 554 00:39:34,000 --> 00:39:39,000 of mitosis when all the chromosomes condense, the karyotype of each of 555 00:39:39,000 --> 00:39:43,000 these donor bone marrow cells will be messed up slightly. 556 00:39:43,000 --> 00:39:48,000 And it will have recognizable abnormalities, 557 00:39:48,000 --> 00:39:53,000 but they're all different, one after the other. And after 558 00:39:53,000 --> 00:39:57,000 we've done this, after we've marked millions of bone 559 00:39:57,000 --> 00:40:02,000 marrow cells in the donor with these random low-dose radiations, 560 00:40:02,000 --> 00:40:06,000 we can then inject a small number of bone marrow cells into 561 00:40:06,000 --> 00:40:11,000 the recipient. And what we can sometimes find on 562 00:40:11,000 --> 00:40:15,000 occasion is if we look at the recipient after that recipient has 563 00:40:15,000 --> 00:40:19,000 been rescued, i. ., after the donor bone marrow has 564 00:40:19,000 --> 00:40:23,000 established itself within the recipient, is that the donor bone 565 00:40:23,000 --> 00:40:27,000 marrow is established in the recipient and populates all of these 566 00:40:27,000 --> 00:40:31,000 different lineages. And if we're able to look at the 567 00:40:31,000 --> 00:40:35,000 karyotype of these different kinds of cells in the recipient organism, 568 00:40:35,000 --> 00:40:39,000 we can find that in some mice all of these cells have the same very 569 00:40:39,000 --> 00:40:43,000 peculiar translocation. They have either this one or this 570 00:40:43,000 --> 00:40:46,000 one or they have yet a third translocation, 571 00:40:46,000 --> 00:40:50,000 any one of a whole series of randomly occurring mutations, 572 00:40:50,000 --> 00:40:54,000 a very peculiar idiosyncratic unusual translocation induced by the 573 00:40:54,000 --> 00:40:58,000 low-dose radiation. They all have it. 574 00:40:58,000 --> 00:41:02,000 The T cells and the B cells and the monocytes and the basophiles, 575 00:41:02,000 --> 00:41:06,000 they all have exactly the same translocation. 576 00:41:06,000 --> 00:41:10,000 Obviously, we can't do that experiment with the platelets. 577 00:41:10,000 --> 00:41:14,000 Why? Because they don't have nuclei. And we can't do that with 578 00:41:14,000 --> 00:41:18,000 the erythrocytes either, the red blood cells. Why can't we 579 00:41:18,000 --> 00:41:22,000 do that? Because in mammals, when the red blood cells are formed, 580 00:41:22,000 --> 00:41:26,000 the nuclei are spit out. Our red blood cells don't have nuclei in 581 00:41:26,000 --> 00:41:30,000 them anymore. They've become enucleate or, to put it another way, 582 00:41:30,000 --> 00:41:34,000 they have been enucleated. That is to say they've been deprived 583 00:41:34,000 --> 00:41:38,000 of their nuclei. Why? Because they're post-mitotic. 584 00:41:38,000 --> 00:41:43,000 Obviously, a cell which lacks nuclei is by definition post-mitotic. 585 00:41:43,000 --> 00:41:47,000 And our cells don't really need, red blood cells don't need nuclei. 586 00:41:47,000 --> 00:41:52,000 We know that ancestral organisms, for instance chickens, 587 00:41:52,000 --> 00:41:56,000 their red blood cells are nucleated, but our red blood cells are not 588 00:41:56,000 --> 00:42:02,000 because they're just not necessary. 589 00:42:02,000 --> 00:42:06,000 How is translocation different from crossing over? 590 00:42:06,000 --> 00:42:11,000 Crossing over occurs between two homologous chromosomes. 591 00:42:11,000 --> 00:42:15,000 So if we have a chromosome here, here's chromosome 13 and here's 592 00:42:15,000 --> 00:42:20,000 another chromosome 13, they're both chromosomes 13. 593 00:42:20,000 --> 00:42:25,000 This one came from Ma. This one came from Pa. All right? 594 00:42:25,000 --> 00:42:30,000 Each of us has a pair of homologous chromosomes. 595 00:42:30,000 --> 00:42:33,000 Here's the maternal one. Here's the paternal one. 596 00:42:33,000 --> 00:42:37,000 When we talk about crossing over we're talking about a process of 597 00:42:37,000 --> 00:42:47,000 homologous recombination. 598 00:42:47,000 --> 00:42:50,000 And when that happens we have a situation like this. 599 00:42:50,000 --> 00:42:53,000 This chunk is exchanged with this chunk over here. 600 00:42:53,000 --> 00:42:56,000 It's an equal exchange, absolutely equal down to the 601 00:42:56,000 --> 00:43:00,000 nucleotide, so that after this flipping has occurred we have two 602 00:43:00,000 --> 00:43:04,000 fully intact chromosomes. It's just that both of these 603 00:43:04,000 --> 00:43:08,000 chromosomes are fully normal. It's just that there's been a 604 00:43:08,000 --> 00:43:12,000 switching, an exchange between the two homologous chromosomes, 605 00:43:12,000 --> 00:43:16,000 the two paired chromosomes 13. When we talk about translocation 606 00:43:16,000 --> 00:43:20,000 there a chunk of chromosome 15 can go onto chromosome 7 or a chunk of 607 00:43:20,000 --> 00:43:24,000 chromosome 2 can go on chromosome 8. It's totally random, it's 608 00:43:24,000 --> 00:43:28,000 non-homologous, and it creates aberrant chromosomes. 609 00:43:28,000 --> 00:43:32,000 Neither of these recombined chromosomes is abnormal. 610 00:43:32,000 --> 00:43:36,000 It's just changed it allelic configuration. 611 00:43:36,000 --> 00:43:40,000 So, it's an important distinction. And the fact is you can pick out 612 00:43:40,000 --> 00:43:45,000 these translocated chromosomes because one even has specific dyes 613 00:43:45,000 --> 00:43:49,000 that can be able to tell you which chromosome this came from and which 614 00:43:49,000 --> 00:43:53,000 chromosome this came from. So, there is a profound difference. 615 00:43:53,000 --> 00:43:57,000 Translocations are, invariably, pathologic. When I say pathologic, 616 00:43:57,000 --> 00:44:02,000 I mean they're really sick. They're not the proper course of 617 00:44:02,000 --> 00:44:06,000 things that happens in a healthy cell. The fact, 618 00:44:06,000 --> 00:44:11,000 the very fact that we're able to generate an entire array of cells in 619 00:44:11,000 --> 00:44:15,000 the blood indicated, by necessity, that if all these 620 00:44:15,000 --> 00:44:20,000 cells have the same chromosomal translocation that they descend from 621 00:44:20,000 --> 00:44:24,000 a donor cell that originally was lightly irradiated and happened to 622 00:44:24,000 --> 00:44:28,000 receive that translocation. If we never get this array of common 623 00:44:28,000 --> 00:44:32,000 translocations in all the cells in a bone marrow recipient then we can't 624 00:44:32,000 --> 00:44:36,000 prove this, but the fact is this has been proven time and again over the 625 00:44:36,000 --> 00:44:40,000 years. And this indicates to us that this cell which is genetically 626 00:44:40,000 --> 00:44:44,000 slightly altered can, therefore, generate all these other 627 00:44:44,000 --> 00:44:48,000 cells in the body. Again, keep in mind that the 628 00:44:48,000 --> 00:44:52,000 irradiation of the donor marrow is simply to create these chromosomal 629 00:44:52,000 --> 00:44:56,000 markings. They're not necessarily good for the organism, 630 00:44:56,000 --> 00:45:00,000 but they don't compromise the viability of the cell. 631 00:45:00,000 --> 00:45:04,000 They just reshuffle the chromosomal structure. Now, 632 00:45:04,000 --> 00:45:09,000 in principle the levels of each one of these kinds of end-stage cells 633 00:45:09,000 --> 00:45:13,000 need to be carefully regulated. And, by the way, let me just note 634 00:45:13,000 --> 00:45:18,000 here, you see the T cells have the arrow going back on themselves, 635 00:45:18,000 --> 00:45:23,000 as do the B cells. That indicates that they have not become 636 00:45:23,000 --> 00:45:27,000 post-mitotic. Remember we talked about these embraces between helper 637 00:45:27,000 --> 00:45:32,000 T cells and B cells where they're walking down the allies and they get 638 00:45:32,000 --> 00:45:36,000 excited and they start multiplying? The fact that the T cells and the B 639 00:45:36,000 --> 00:45:40,000 cells are able to proliferate in response to certain antigenic 640 00:45:40,000 --> 00:45:43,000 stimuli implies that they're not post-mitotic. They still have the 641 00:45:43,000 --> 00:45:47,000 ability to proliferate, and that retained ability to 642 00:45:47,000 --> 00:45:50,000 proliferate like that of hepatocytes is indicated by these arrows that 643 00:45:50,000 --> 00:45:54,000 are looping back on themselves. Conversely, these cells are all 644 00:45:54,000 --> 00:45:57,000 essentially, as I've said before, post-mitotic. So how do we insure 645 00:45:57,000 --> 00:46:01,000 that's there proper concentrations of all of these different 646 00:46:01,000 --> 00:46:05,000 cells in the blood? And the fact is the concentrations 647 00:46:05,000 --> 00:46:10,000 of many of these cells in the blood are maintained to concentrations of 648 00:46:10,000 --> 00:46:14,000 plus or minus 10%. And this is, itself, 649 00:46:14,000 --> 00:46:19,000 a stunning testimonial to the successes of human physiology. 650 00:46:19,000 --> 00:46:24,000 We're talking here about a process which is sometimes called 651 00:46:24,000 --> 00:46:28,000 homeostasis. Homeostasis means that somehow there is a balance, 652 00:46:28,000 --> 00:46:33,000 an equilibrium that is achieved, and that there aren't profound 653 00:46:33,000 --> 00:46:38,000 fluctuations so that we always have roughly the equal level of red blood 654 00:46:38,000 --> 00:46:43,000 cells, a proper level of lymphocytes in our blood. 655 00:46:43,000 --> 00:46:47,000 And I want to get into the homeostasis which results in the 656 00:46:47,000 --> 00:46:52,000 formation of red blood cells, the RBCs, the erythrocytes in the 657 00:46:52,000 --> 00:46:56,000 blood. In fact, it happens to be the case that the 658 00:46:56,000 --> 00:47:01,000 red blood cells are one of the cell types that could actually vary quite 659 00:47:01,000 --> 00:47:06,000 profoundly in response to environment. 660 00:47:06,000 --> 00:47:09,000 To the extent that lymphocytes change, they go up and down, 661 00:47:09,000 --> 00:47:12,000 that might be due in response to an infection. So, 662 00:47:12,000 --> 00:47:15,000 if we have a serious bacterial infection we might have increased in 663 00:47:15,000 --> 00:47:19,000 the lymphocytes in the blood that have been mobilized in order to 664 00:47:19,000 --> 00:47:22,000 attack the infecting bacteria. But what about the RBCs? What 665 00:47:22,000 --> 00:47:25,000 about the red blood cells? What causes them to change? 666 00:47:25,000 --> 00:47:29,000 Well, if you move from here to Denver, Colorado or you go up skiing 667 00:47:29,000 --> 00:47:32,000 in the Rockies, you're going up to ten or twelve 668 00:47:32,000 --> 00:47:36,000 thousand feet. And within a matter of three or four 669 00:47:36,000 --> 00:47:40,000 days the concentration of your red blood cells increases very 670 00:47:40,000 --> 00:47:45,000 substantially. Why? Because obviously the oxygen 671 00:47:45,000 --> 00:47:50,000 tension at high altitude is down. And in order that your peripheral 672 00:47:50,000 --> 00:47:54,000 tissues become adequately oxygenated, the oxygen carrying capacity of the 673 00:47:54,000 --> 00:47:59,000 blood must be increased. And the way that is increased is in 674 00:47:59,000 --> 00:48:04,000 part to increase the concentration of red blood cells. 675 00:48:04,000 --> 00:48:08,000 So how does that happen? How is it possible that we can 676 00:48:08,000 --> 00:48:13,000 rapidly modulate the concentration of red blood cells? 677 00:48:13,000 --> 00:48:17,000 And the way we can do that is in part through a hormone called 678 00:48:17,000 --> 00:48:22,000 erythropoietin, EPO. We're going to talk about 679 00:48:22,000 --> 00:48:27,000 erythropoietin at the beginning of the lecture next time, 680 00:48:27,000 --> 00:48:31,000 but the homeostasis which maintains the appropriate number of red blood 681 00:48:31,000 --> 00:48:36,000 cells in our circulation is dictated in no small part by the levels of 682 00:48:36,000 --> 00:48:41,000 EPO that are in their blood. To anticipate some of the things 683 00:48:41,000 --> 00:48:45,000 we're going to say next time, when you are in a low-oxygen 684 00:48:45,000 --> 00:48:49,000 environment the levels of erythropoietin shoot up. 685 00:48:49,000 --> 00:48:53,000 And when they shoot up they insure that there is shortly thereafter a 686 00:48:53,000 --> 00:48:57,000 rapid increase in the level of circulating red blood cells which in 687 00:48:57,000 --> 00:49:01,000 turn enables the oxygen coming into your lungs to be transported more 688 00:49:01,000 --> 00:49:06,000 efficiently, more effectively into the peripheral tissues. 689 00:49:06,000 --> 00:49:10,000 You've heard about athletes perhaps who are able to dope themselves with 690 00:49:10,000 --> 00:49:14,000 erythropoietin. This is a rather devious strategy 691 00:49:14,000 --> 00:49:18,000 because it means that if they do so, they inject themselves with a little 692 00:49:18,000 --> 00:49:22,000 erythropoietin, the oxygen carrying capacity of 693 00:49:22,000 --> 00:49:26,000 their blood is temporarily increased and as a consequence they might be 694 00:49:26,000 --> 00:49:29,000 able to run further or jump higher. This, by the way, 695 00:49:29,000 --> 00:49:33,000 has its dangers. Because if you're injecting erythropoietin not in 696 00:49:33,000 --> 00:49:37,000 response to certain physiologic signals but just because you want to 697 00:49:37,000 --> 00:49:41,000 win a marathon or something, you're violating the normal 698 00:49:41,000 --> 00:49:45,000 physiologic mechanisms in the body which very carefully control the 699 00:49:45,000 --> 00:49:49,000 levels of erythropoietin. And if you inject too much 700 00:49:49,000 --> 00:49:53,000 erythropoietin you get in a very bad situation because the bone marrow 701 00:49:53,000 --> 00:49:57,000 makes more and more red blood cells. And then what happens? You start 702 00:49:57,000 --> 00:50:01,000 clotting up everywhere all over the body, and this isn't good. 703 00:50:01,000 --> 00:50:05,000 In fact, you can die. So this is not a warning against 704 00:50:05,000 --> 00:50:10,000 erythropoietin in the way that I warned you against cigarettes. 705 00:50:10,000 --> 00:50:14,000 This is just to tell you these kinds of drugs, 706 00:50:14,000 --> 00:50:19,000 or these kinds of growth factors, which they are, are maintained at 707 00:50:19,000 --> 00:50:23,000 very precise levels as we'll discuss in more detail next time. 708 00:50:23,000 --> 00:50:28,000 See you on Wednesday.