1 00:00:01 --> 00:00:05 The following content is provided by MIT OpenCourseWare under a creative 2 00:00:05 --> 00:00:09 commons license. Additional information about our license and 3 00:00:09 --> 00:00:14 MIT OpenCourseWare in general is available at ocw.mit.edu In 4 00:00:14 --> 00:00:18 particular, we have a class quiz up there. Take a moment if you 5 00:00:18 --> 00:00:23 haven't. You've heard a lot of these terms before. One you 6 00:00:23 --> 00:00:27 haven't. I have a particularly wonderful item to make 7 00:00:27 --> 00:00:33 up for the tough exam. It is a flashing, 8 00:00:33 --> 00:00:39 isn't that so cool? It's a flashing jellyfish ball. OK, 9 00:00:39 --> 00:00:45 so let's see what we can do. Yes, I know, this is a post-test item. OK, 10 00:00:45 --> 00:00:51 settle down. Please, we have a lot to cover. You've 11 00:00:51 --> 00:00:57 heard most of these terms before. This lecture is all about these 12 00:00:57 --> 00:01:05 terms phrased in a different way. So I want to make sure we are 13 00:01:05 --> 00:01:16 together before we start. [Student speaks] Yes, ma'am? OK, so the final function of 14 00:01:16 --> 00:01:27 a cell, OK. Potency in the pink top. I saw your hand first, 15 00:01:27 --> 00:01:36 yeah? Yeah, OK, potency. This is a very big one in stem cells, 16 00:01:36 --> 00:01:43 the number of possible fates that a cell can assume, 17 00:01:43 --> 00:01:50 commitment, decision to make a cell type, differentiation, 18 00:01:50 --> 00:01:57 someone on the side of the room must have some thoughts. Yeah? 19 00:01:57 --> 00:02:05 OK, I'll give that to you, to the final transition here. 20 00:02:05 --> 00:02:09 Do you want one that's orange rather than pink? God knows I don't want 21 00:02:09 --> 00:02:13 to be genderist here, but there we go. The 22 00:02:13 --> 00:02:17 differentiation, the process by which a cell assumes 23 00:02:17 --> 00:02:21 its final fate. And what we are going to discuss today 24 00:02:21 --> 00:02:25 is the kind of gradations of commitments in differentiation, 25 00:02:25 --> 00:02:30 commitment particularly, some of the steps of commitment. 26 00:02:30 --> 00:02:35 The last one we have not discussed in class, but if anyone wants to 27 00:02:35 --> 00:02:41 have a go, I will, yeah? Lineage, the term lineage in 28 00:02:41 --> 00:02:49 biology. 29 00:02:49 --> 00:02:55 Ish, ish. Let's try another one. Good, OK, a group of cells or a 30 00:02:55 --> 00:03:01 group of cell types that descends from a common precursor, 31 00:03:01 --> 00:03:07 so, this is a funny term, lineage, because it's used a lot in 32 00:03:07 --> 00:03:13 stem cell biology. Let's think about it for a moment. The 33 00:03:13 --> 00:03:19 definition I have is the set of cell types that normally arise from a 34 00:03:19 --> 00:03:25 precursor cell. Now, of course, if you think hard, 35 00:03:25 --> 00:03:31 all cells arise from a fertilized egg from a zygote. So, 36 00:03:31 --> 00:03:38 really, all cell types are of a common lineage. 37 00:03:38 --> 00:03:43 And this gets to the question of semantics, and where you put your 38 00:03:43 --> 00:03:48 definition boxes. OK, in stem cell biology, and a lot of 39 00:03:48 --> 00:03:54 biology, the term lineage refers to a subset of cells arising from a 40 00:03:54 --> 00:03:59 common precursor that is somewhat arbitrarily defined. All right, 41 00:03:59 --> 00:04:05 so this diagram which you've seen over several lectures 42 00:04:05 --> 00:04:10 is really important. That's just what we've been talking 43 00:04:10 --> 00:04:14 about, the progression from uncommitted cells through committed 44 00:04:14 --> 00:04:18 cells through differentiated cells. And so, we are in the how-to two 45 00:04:18 --> 00:04:22 module, talking about stem cells. Stem cells are in the news all the 46 00:04:22 --> 00:04:26 time, and I want to, today, take you beyond the 47 00:04:26 --> 00:04:30 newspapers, beyond the magazines, and tell you what I think stem cells 48 00:04:30 --> 00:04:35 are all about. So, if you look at our game board of 49 00:04:35 --> 00:04:41 life, we are, I would say, halfway plus through the semester, 50 00:04:41 --> 00:04:46 and we've gone through all of these things, sticking out here into stem 51 00:04:46 --> 00:04:52 cells. So, where are we? OK, so what's a stem cell? 52 00:04:52 --> 00:04:57 Let me go back for one moment, and let's talk about stem cells. 53 00:04:57 --> 00:05:03 For those of you who don't have a handout, you really should get it. 54 00:05:03 --> 00:05:09 It's two pages up front here. So, let's do some board stuff, 55 00:05:09 --> 00:05:15 stem cell, which I will heretofore abbreviate SC is a cell type that I 56 00:05:15 --> 00:05:21 am going to refer to as somewhat committed. So that's deliberately 57 00:05:21 --> 00:05:27 vague. This is not a committed cell type, but it's somewhat committed. 58 00:05:27 --> 00:05:34 It knows more or less what it's going to become. 59 00:05:34 --> 00:05:41 It can be either unipotent or pluripotent or totipotent. And that 60 00:05:41 --> 00:05:48 stem cell, by definition, will go on and make an asymmetric 61 00:05:48 --> 00:05:55 cell division. So, it will give rise to itself, 62 00:05:55 --> 00:06:03 another, not itself, but another stem cell. 63 00:06:03 --> 00:06:09 And it will also give rise to a committed, and here's jargon you 64 00:06:09 --> 00:06:16 should know, progenitor. And so, this is an asymmetric with one S, 65 00:06:16 --> 00:06:23 asymmetric cell division. So, you'll end up with two daughters 66 00:06:23 --> 00:06:30 that are different from one another, a committed cell, and a stem cell. 67 00:06:30 --> 00:06:37 And so, the stem cell in a sense is self renewing, 68 00:06:37 --> 00:06:45 or is self renewing. And then, this is one of the big deals about 69 00:06:45 --> 00:06:53 stem cells. That committed progenitor will go on to divide, 70 00:06:53 --> 00:07:01 and it eventually gives rise to one or more differentiated cell types. 71 00:07:01 --> 00:07:07 OK, in those differentiated cells will come from a specific lineage. 72 00:07:07 --> 00:07:17 So, the stem cell is 73 00:07:17 --> 00:07:21 undifferentiated. The committed progenitor is undifferentiated, 74 00:07:21 --> 00:07:25 and eventually you'll end up with a differentiated cell. You can look 75 00:07:25 --> 00:07:29 at your first handout. Here it is; I've drawn it for you out. You need 76 00:07:29 --> 00:07:33 to know this, so I've done it in two places. There is the asymmetric 77 00:07:33 --> 00:07:37 division stem cell plus progenitor, and the progenitor goes on to give 78 00:07:37 --> 00:07:41 these differentiated cell types. There is no magic about this. This 79 00:07:41 --> 00:07:45 is the same process we've been talking about all along except for 80 00:07:45 --> 00:07:50 the fact of the self renewing thing. So, in the normal embryo, 81 00:07:50 --> 00:07:55 cells go on down the pathway of commitment towards differentiation. 82 00:07:55 --> 00:08:00 But they generally do not renew themselves. 83 00:08:00 --> 00:08:05 So, a zygote, for example, although it's totipotent is not 84 00:08:05 --> 00:08:11 considered a stem cell because it doesn't self renew. And that's true 85 00:08:11 --> 00:08:16 of most embryonic cells. They do not self renew and so they are not 86 00:08:16 --> 00:08:22 considered stem cells. So, what's the deal with stem cells, 87 00:08:22 --> 00:08:28 and why, when I Googled stem cells in Google News last night did I come 88 00:08:28 --> 00:08:34 up with several thousand entries for the previous week? 89 00:08:34 --> 00:08:38 This is the deal. One of the great goals of biologists is to repair 90 00:08:38 --> 00:08:43 organs, to repair damaged tissues. And this has been very difficult to 91 00:08:43 --> 00:08:48 do. We touched on this a few lectures ago., 92 00:08:48 --> 00:08:53 stem cells have some kind of promise a some kind of universal repair kit. 93 00:08:53 --> 00:08:57 And the idea is this, that one could isolate some kind of 94 00:08:57 --> 00:09:02 magic stem cell, or some kind of stem cell, 95 00:09:02 --> 00:09:07 and it would be autologous, which means it would be matching 96 00:09:07 --> 00:09:14 your own cells. OK, so you isolate some kind of 97 00:09:14 --> 00:09:22 autologous, self matching stem cell. That stem cell under ideal 98 00:09:22 --> 00:09:30 conditions is pluripotent, or that set of stem cells, and can 99 00:09:30 --> 00:09:38 be coaxed to form many different cell types depending 100 00:09:38 --> 00:09:45 on how you treat it. So, you can imagine adding some kind 101 00:09:45 --> 00:09:51 of factor. And if you like, you can call that an 102 00:09:51 --> 00:09:56 inducer as we have been talking about secreted factors 103 00:09:56 --> 00:10:02 in developmental biology. And that inducer would turn those stem 104 00:10:02 --> 00:10:07 cells into progenitors. And those progenitors would 105 00:10:07 --> 00:10:15 have a specific -- 106 00:10:15 --> 00:10:20 -- future fate by virtue of which factor you treated the stem cells 107 00:10:20 --> 00:10:26 with. You would then take those progenitors, inject them into 108 00:10:26 --> 00:10:37 someone whose body needed repair -- 109 00:10:37 --> 00:10:42 And the notion is that these progenitors would go on to 110 00:10:42 --> 00:10:48 differentiate and repair whatever needed repairing. 111 00:10:48 --> 00:10:59 OK, so this is the dream. And 112 00:10:59 --> 00:11:03 this is why you can find headlines like this everywhere. These are 113 00:11:03 --> 00:11:07 some of the ones I pulled out last night: Stem Cells May Help Repair 114 00:11:07 --> 00:11:11 Stroke Damage. Stem Cells May Repair Broken Bones. Note the use 115 00:11:11 --> 00:11:15 of the term may. OK, there's a lot of hype about stem 116 00:11:15 --> 00:11:19 cells and not much that's been proven. There's a lot of money 117 00:11:19 --> 00:11:23 involved in stem cells, trying to isolate stem cells to 118 00:11:23 --> 00:11:27 repair people, and there is also, 119 00:11:27 --> 00:11:31 there are also advertisements where you pay people to take your stem 120 00:11:31 --> 00:11:35 cells, and to store them. So, poured blood refers to the blood 121 00:11:35 --> 00:11:40 of the umbilical cord of a newborn, which is believed to be, it was 122 00:11:40 --> 00:11:45 known to be full of stem cells of a certain kinds. And 123 00:11:45 --> 00:11:50 you can pay someone a couple of thousand dollars to freeze that 124 00:11:50 --> 00:11:55 group of cells and keep it in case the child needs some kind of stem 125 00:11:55 --> 00:12:00 cell therapy in the future. So, what's the deal? Do stem cells 126 00:12:00 --> 00:12:07 actually exist? 127 00:12:07 --> 00:12:20 Well, yes, OK. 128 00:12:20 --> 00:12:23 Do stem cells exist or are they just hype? They do exist, 129 00:12:23 --> 00:12:27 and the idea is that sometime in embryogenesis, 130 00:12:27 --> 00:12:31 as cells are normally going on and making the different cell types, 131 00:12:31 --> 00:12:34 some cells are put aside, that the body is going to use later on for 132 00:12:34 --> 00:12:38 repairing itself. And there are some examples, 133 00:12:38 --> 00:12:42 and I will tell you a couple which are really fantastic illustrations 134 00:12:42 --> 00:12:47 of this. So, do stem cells exist? 135 00:12:47 --> 00:12:55 Yes. Where? Likely in the older embryo, and the adult, 136 00:12:55 --> 00:13:03 and the particular organs that contains stem cells is not clear. 137 00:13:03 --> 00:13:11 There are several very good examples, but it's not clear whether or not 138 00:13:11 --> 00:13:17 some, yes, all, not clear. It's not clear whether all organs 139 00:13:17 --> 00:13:22 contain stem cells. So, let's look at this a bit more, 140 00:13:22 --> 00:13:27 and let's go back to talking about how stem cells were discovered. Why 141 00:13:27 --> 00:13:36 are we having this conversation? 142 00:13:36 --> 00:13:41 And I'm going to be referring to number two on your handout. So, 143 00:13:41 --> 00:13:46 let's talk about the discovery of stem cells. The discovery of stem 144 00:13:46 --> 00:13:52 cells was an accident, and it came about when people 145 00:13:52 --> 00:13:57 started looking to see how long cells lived in a particular organ. 146 00:13:57 --> 00:14:03 And the way you do this is by using a protocol called a pulse 147 00:14:03 --> 00:14:10 chase experiment. 148 00:14:10 --> 00:14:20 And a pulse chase experiment gets to the question of turnover rate, 149 00:14:20 --> 00:14:31 or if you want, half-life of cells in an organ. 150 00:14:31 --> 00:14:35 OK, this is the way it goes. You've got this as number two on your 151 00:14:35 --> 00:14:39 handouts. You take a cell population, or you just take the 152 00:14:39 --> 00:14:44 whole organism if you like, you feed it something called 153 00:14:44 --> 00:14:48 bromodeoxyuridine. It doesn't have to be this, but this is a good one. 154 00:14:48 --> 00:14:53 Bromodeoxyuridine is a nucleotide. Deoxyuridine, so, 155 00:14:53 --> 00:14:57 you know uracil is normally an RNA, but if you make the deoxy form, 156 00:14:57 --> 00:15:02 it gets incorporated into DNA. The bromo part allows it, 157 00:15:02 --> 00:15:06 later on, to be detected by various colorimetric assays, 158 00:15:06 --> 00:15:10 and you can add BRDU to an organism for a short time. This is called a 159 00:15:10 --> 00:15:15 pulse. The BRDU is incorporated into DNA of those cells that are 160 00:15:15 --> 00:15:19 undergoing DNA synthesis, and then by various means you wash 161 00:15:19 --> 00:15:23 out the BRDU. And what you get is the labeled cell 162 00:15:23 --> 00:15:28 population that had undergone DNA replication during this pulse 163 00:15:28 --> 00:15:32 period. And then, if you follow this group of cells 164 00:15:32 --> 00:15:36 over a period of hours or days or weeks or months or years, 165 00:15:36 --> 00:15:41 you can look and see what happens to those labeled cells. 166 00:15:41 --> 00:15:45 So, in my example, I started off with for labeled cells 167 00:15:45 --> 00:15:49 over some period of time, the number of labeled cells per 168 00:15:49 --> 00:15:54 total unit number of other cells decreases by half. And that gives 169 00:15:54 --> 00:15:58 you the half-life of the population. And if you do this for many organs, 170 00:15:58 --> 00:16:02 you find that adult organs do not just sit there with a cohort of 171 00:16:02 --> 00:16:08 cells that doesn't divide. There is an enormous amount of cell 172 00:16:08 --> 00:16:14 division in adult organs. And we talked briefly about this 173 00:16:14 --> 00:16:21 previously. So, for example, red blood cells have a 174 00:16:21 --> 00:16:28 half-life of about 120 days. And that actually means that there are 175 00:16:28 --> 00:16:35 more than ten to the 7th new cells produced per day. 176 00:16:35 --> 00:16:40 The intestine is something we touched on previously. Cells in the 177 00:16:40 --> 00:16:45 intestine, some of the cells in the intestine had a half-life of three 178 00:16:45 --> 00:16:51 to five days, and you're producing about ten to the tenth new cells per 179 00:16:51 --> 00:16:56 day. Obviously, the number that you're producing per 180 00:16:56 --> 00:17:02 day depends on the total size of the population. 181 00:17:02 --> 00:17:11 Skin has a half-life of about 14 days, hair on your head, 182 00:17:11 --> 00:17:20 a half-life of about four years, and your eyebrows and eyelashes, 183 00:17:20 --> 00:17:30 half life of about 30 days. One of the mysterious half-lives are the 184 00:17:30 --> 00:17:37 neural cells, your nerve cells. It was believed for a long time that 185 00:17:37 --> 00:17:41 nerve cells never divided, and once you've got all your nerve 186 00:17:41 --> 00:17:45 cells by about age two, you never made any more. In fact, 187 00:17:45 --> 00:17:49 that doesn't seem to be true. And there are certainly populations of 188 00:17:49 --> 00:17:53 neurons that divide. But we don't really know the half-lives for those 189 00:17:53 --> 00:17:57 cells. So, this was very interesting data, 190 00:17:57 --> 00:18:01 and it said that there had to be some way that the organism was using 191 00:18:01 --> 00:18:05 to replenish these cells that were dying, and to repopulate the organs 192 00:18:05 --> 00:18:10 so that things functioned properly. And in fact, you can go further 193 00:18:10 --> 00:18:14 than this because you can not only count the labeled cells, 194 00:18:14 --> 00:18:18 you can ask what those labeled cells become. So, you can look at your 195 00:18:18 --> 00:18:22 labeled cell population and assay the fate of those labeled cells. 196 00:18:22 --> 00:18:26 And if the cells want to differentiate from an initially 197 00:18:26 --> 00:18:30 undifferentiated population, you know that these differentiated 198 00:18:30 --> 00:18:34 cells must have been derived from stem cells or from progenitors 199 00:18:34 --> 00:18:39 by definition. OK, so what organs do we know have 200 00:18:39 --> 00:18:43 got stem cells? Let's talk a bit about this. The 201 00:18:43 --> 00:18:48 test is a great example, and the spermatogonia, 202 00:18:48 --> 00:18:53 the diploid precursors of the spermatozoa are dividing cells that 203 00:18:53 --> 00:18:57 divide throughout life, and go on to give rise to themselves 204 00:18:57 --> 00:19:02 so they can replenish themselves. And they go on to give to these 205 00:19:02 --> 00:19:06 primary spermatocytes, which are the first step in the 206 00:19:06 --> 00:19:10 cascade or in the lineage of cells that are going to differentiate as a 207 00:19:10 --> 00:19:14 spermatozoa. OK, and you know you can do these. You 208 00:19:14 --> 00:19:18 can look very clearly, and mathematically look and see the 209 00:19:18 --> 00:19:22 number of cells, do this pulse chase analysis, 210 00:19:22 --> 00:19:26 and know that the spermatigonea must be stem cells. This is a very 211 00:19:26 --> 00:19:30 important stem cell lineage. It's the hematopoietic lineage. 212 00:19:30 --> 00:19:34 In the bone marrow, there is some kind of pluripotential 213 00:19:34 --> 00:19:39 hematopoietic cell that gives rise to all of myeloid progenitors, 214 00:19:39 --> 00:19:43 so all the red blood cells, and the various other cells in the blood 215 00:19:43 --> 00:19:48 stream as well as to all the immune cells. And those come from a 216 00:19:48 --> 00:19:53 single progenitor, a single pluripotent cell. This is 217 00:19:53 --> 00:19:57 called the hematopoietic lineage. And we will talk more about 218 00:19:57 --> 00:20:01 that in a moment. This is an example that I mentioned 219 00:20:01 --> 00:20:05 to you many lectures ago. Newt limbs, if they are amputated, will 220 00:20:05 --> 00:20:09 regrow. The reason that they regrow is that there seem to be a 221 00:20:09 --> 00:20:13 population of cells in the lab which are called blastema cells, 222 00:20:13 --> 00:20:16 and those are the cells which can go on and to form the entire limb 223 00:20:16 --> 00:20:20 again. And that's something obviously we can't do, 224 00:20:20 --> 00:20:24 but people are very interested in. That's been a tough system to look 225 00:20:24 --> 00:20:28 at. This might be a better system to try and understand the 226 00:20:28 --> 00:20:31 details of regeneration. So, this is a planarian. This is a 227 00:20:31 --> 00:20:35 flatworm. These are little guys. They can grow up to a couple of 228 00:20:35 --> 00:20:39 centimeters, or a few centimeters. They are very pervasive animals in 229 00:20:39 --> 00:20:43 the animal kingdom. And they have this extraordinary property, 230 00:20:43 --> 00:20:47 as many simple animals do, that you can cut them into pieces, 231 00:20:47 --> 00:20:50 and they will regenerate the whole animal. So, you can take out the 232 00:20:50 --> 00:20:54 head. And over time, it will regenerate the tail. You 233 00:20:54 --> 00:20:58 can take off the middle; it will regenerate a whole animal, 234 00:20:58 --> 00:21:02 and so on. And this bottom picture is a planarian that's stained for 235 00:21:02 --> 00:21:06 BRDU incorporation, or for another marker as well. 236 00:21:06 --> 00:21:09 And each of these dots is a cell called a neoblast. 237 00:21:09 --> 00:21:12 The neoblasts are the stem cells of planaria that can regenerate the 238 00:21:12 --> 00:21:15 whole animal. And if you look right up front here, 239 00:21:15 --> 00:21:18 it's not very distinct, but you will see a region where 240 00:21:18 --> 00:21:21 there are no neoblasts. And that is the one region of the animal 241 00:21:21 --> 00:21:25 that cannot regenerate. So, if you cut off the very tip of the 242 00:21:25 --> 00:21:28 sort of nose equivalent region. It will not regenerate a new animal 243 00:21:28 --> 00:21:32 because there are no neoblasts. And the system is being studied 244 00:21:32 --> 00:21:37 here at MIT by Professor Reddien of the Whitehead Institute, 245 00:21:37 --> 00:21:42 who is a new faculty member, and who some of you might went to Europe 246 00:21:42 --> 00:21:47 with sometime. OK, so let's talk about isolating stem 247 00:21:47 --> 00:21:52 cells, and how you do this. If one is going to use stem cells for 248 00:21:52 --> 00:21:58 repair, you've got to be able to isolate these things. 249 00:21:58 --> 00:22:05 And the challenge of isolating stem cells is that they are rare. For 250 00:22:05 --> 00:22:13 example, in the bone marrow, the hematopoietic stem cells, 251 00:22:13 --> 00:22:21 which I'm abbreviating HSC, comprise about 0.01% of the bone marrow. 252 00:22:21 --> 00:22:29 And I would say it's fair to say 253 00:22:29 --> 00:22:33 that no one has really seen a cell and said, oh, this is a stem cell. 254 00:22:33 --> 00:22:37 It's hard to pinpoint exactly what a stem cell really looks like. It's 255 00:22:37 --> 00:22:41 just a cell. But it's got particular properties, 256 00:22:41 --> 00:22:45 and you need the appropriate way to look at the cells and see these 257 00:22:45 --> 00:22:49 properties. One way that's been used to isolate stem cells is 258 00:22:49 --> 00:22:53 something called FACS, or fluorescence activated cell 259 00:22:53 --> 00:22:57 sorting. I'll talk about it in a moment. And there are two ways 260 00:22:57 --> 00:23:01 that FACS is being used to isolate stem cells. One is by getting a 261 00:23:01 --> 00:23:05 group of cells called SP cells where the SP stands for side population. 262 00:23:05 --> 00:23:10 We'll talk about that more in a moment. And the other is through 263 00:23:10 --> 00:23:15 the use of cell surface proteins that are characteristic, 264 00:23:15 --> 00:23:20 or enriched in stem cells. And this has been many decades of work 265 00:23:20 --> 00:23:25 by many people to come up with a set of criteria by which one can enrich 266 00:23:25 --> 00:23:30 for stem cells in a particular population. 267 00:23:30 --> 00:23:34 So, here's the way. Actually, before I go through that, let me go 268 00:23:34 --> 00:23:39 through the assays, and then we will go through all the 269 00:23:39 --> 00:23:44 slides together. How do you assay for stem cells? 270 00:23:44 --> 00:23:49 Well, one of the ways you know you have a stem cell is if you have 271 00:23:49 --> 00:23:54 something that can act as a stem cell. And there are two assays 272 00:23:54 --> 00:23:59 that you should be aware of. One of them is a repopulation assay usually 273 00:23:59 --> 00:24:04 done by transplant, and very often you remove some 274 00:24:04 --> 00:24:09 endogenous group of cells, and then try to replace that group 275 00:24:09 --> 00:24:15 of cells by using stem cells. So, you remove some set of 276 00:24:15 --> 00:24:27 differentiated cells. 277 00:24:27 --> 00:24:32 And then, you try to replace with transplanted stem cells. And 278 00:24:32 --> 00:24:38 the other way you can do this is in some kind of in vitro culture assay 279 00:24:38 --> 00:24:44 which I will talk about later on. All right, so with this in mind, 280 00:24:44 --> 00:24:49 let's go through some of the slides. You've all heard of bone marrow 281 00:24:49 --> 00:24:55 transplants, which people who have leukemia and other associated 282 00:24:55 --> 00:25:01 disorders undergo to repair themselves to get rid of the 283 00:25:01 --> 00:25:07 leukemia cells to get rid of the cancer. 284 00:25:07 --> 00:25:11 This is how it works in a mouse. And this is a repopulation assay. 285 00:25:11 --> 00:25:15 I'm going to use bone marrow transplants as an example. You take 286 00:25:15 --> 00:25:20 your mouse, or your person if you are undergoing bone marrow 287 00:25:20 --> 00:25:24 transplants. And you irradiate to destroy the bone 288 00:25:24 --> 00:25:28 marrow. The reason you do that is to make space for new cells to come 289 00:25:28 --> 00:25:33 in to expand and grow. If you just put your new cells into 290 00:25:33 --> 00:25:37 an animal with an intact bone marrow, they kind of disappear 291 00:25:37 --> 00:25:42 amidst the masses. So, you have to give the cells your 292 00:25:42 --> 00:25:47 assaying a chance. And then, that irradiated mouse would die. 293 00:25:47 --> 00:25:51 That irradiated person would die. But you get them, 294 00:25:51 --> 00:25:56 now, an injection of normal bone marrow. And if things go well, 295 00:25:56 --> 00:26:01 the mouse or the person lives. And there was a Nobel Prize given 296 00:26:01 --> 00:26:05 out some years ago for developing bone marrow transplantation. One of 297 00:26:05 --> 00:26:10 the gold standards in the stem cell field is asking whether or not this 298 00:26:10 --> 00:26:15 rescued mouse has regenerated stem cells because you can imagine that 299 00:26:15 --> 00:26:19 what you're doing in this case is giving cells that are the 300 00:26:19 --> 00:26:24 progenitors. They are one step down from the stem cells. Or they might 301 00:26:24 --> 00:26:29 even be partly differentiated. So, you can imagine that you are 302 00:26:29 --> 00:26:33 restoring this mouse is life by giving cells that are 303 00:26:33 --> 00:26:38 not self-renewing. Excuse me, one of the gold standards 304 00:26:38 --> 00:26:43 in the stem cell field is to take this rescued mouse, 305 00:26:43 --> 00:26:48 isolate more stem cells or more putative stem cells, 306 00:26:48 --> 00:26:53 and take those and then tried to rescue another mouse. And 307 00:26:53 --> 00:26:58 in the hematopoietic system, you can do this over and over again. 308 00:26:58 --> 00:27:03 So, how many stem cells do you need to repopulate a mouse? 309 00:27:03 --> 00:27:06 Actually, you need one, and I will tell you how this is 310 00:27:06 --> 00:27:10 done. So, the idea of the first thing when you are trying to do 311 00:27:10 --> 00:27:13 assays to figure out how many stem cells you need for rescue. You take 312 00:27:13 --> 00:27:17 your bone marrow; you stain it for some stem cell marker. You have 313 00:27:17 --> 00:27:21 this in front of you. OK, this is number nine of the first 314 00:27:21 --> 00:27:24 page of your handout. You stain somehow for a stem cell marker. 315 00:27:24 --> 00:27:28 I'll tell you in a moment how you sought the stained cells through 316 00:27:28 --> 00:27:32 this fluorescence activated cell sorter. 317 00:27:32 --> 00:27:35 And you isolate from it a pure-ish population of stem cells, 318 00:27:35 --> 00:27:39 an enriched population of stem cells. And then, 319 00:27:39 --> 00:27:43 you do a dilution assay where you inject different 320 00:27:43 --> 00:27:46 numbers of cells into a recipient irradiated mouse. Now, 321 00:27:46 --> 00:27:50 you don't actually injects one, ten, and 100 cells. You inject one 322 00:27:50 --> 00:27:54 cell, and millions of carrier cells to help those cells along, 323 00:27:54 --> 00:27:57 OK? The one cell would just disappear in your syringe. So, 324 00:27:57 --> 00:28:01 you've got to give it some companions. But 325 00:28:01 --> 00:28:05 the bottom line is you really only need one stem cell to rescue the 326 00:28:05 --> 00:28:09 life of that mouse. It can go and repopulate the entire 327 00:28:09 --> 00:28:13 hematopoietic system, all the blood cells, all the immune 328 00:28:13 --> 00:28:17 cells. What is this fluorescence activated cell sorter? 329 00:28:17 --> 00:28:22 Fantastic machine. It looks like this. Again, you have this in front 330 00:28:22 --> 00:28:26 of you, so look up here. The idea is that you take a reservoir of 331 00:28:26 --> 00:28:30 cells that you have labeled with particular antibodies. And these 332 00:28:30 --> 00:28:35 are living cells. OK, you label them in certain ways, 333 00:28:35 --> 00:28:39 and they can be labeled with fluorescent antibodies or 334 00:28:39 --> 00:28:43 fluorescent dyes. I'll tell you a dye example in a moment. And 335 00:28:43 --> 00:28:47 you put them in a reservoir, and you trip them out so that one drop of 336 00:28:47 --> 00:28:52 liquid contains one cell on average, or zero cells. And you let those 337 00:28:52 --> 00:28:56 cells drip through a laser and a fluorescence detector. The laser 338 00:28:56 --> 00:29:00 activates the cells. They fluoresce, and you set the detector to activate 339 00:29:00 --> 00:29:04 a charging collar at a specific wavelength of 340 00:29:04 --> 00:29:09 fluorescence. And when the charging collar is 341 00:29:09 --> 00:29:14 activated, it will activate some deflecting plates which will give 342 00:29:14 --> 00:29:19 charge to cells of particular colors, and move them into particular tubes 343 00:29:19 --> 00:29:24 so that they are sorted on the basis of their color. OK, 344 00:29:24 --> 00:29:29 this is done one cell at a time but it's really quick. And 345 00:29:29 --> 00:29:34 you can purify millions of cells to do these kinds of assays. 346 00:29:34 --> 00:29:37 This kind of thing is not done for human bone marrow transplants. 347 00:29:37 --> 00:29:40 There, you take a much bigger population of cells from the bone 348 00:29:40 --> 00:29:44 marrow, and you generally don't purify them very much. But 349 00:29:44 --> 00:29:47 if you want to do specific stem cell assays, and many others, 350 00:29:47 --> 00:29:51 the fax machine is really fantastic. So, what do you get out of this? 351 00:29:51 --> 00:29:54 Well, you can plot what the cells look like. So, 352 00:29:54 --> 00:29:58 here in this example, I've got cells that are labeled in 353 00:29:58 --> 00:30:01 red and green. And you can label them according to this 354 00:30:01 --> 00:30:05 plot as to whether they have no label, red label, 355 00:30:05 --> 00:30:09 green label, or both red and green. This is a real example of an 356 00:30:09 --> 00:30:14 experiment that was done here at MIT a decade ago in Richard Mulligan's 357 00:30:14 --> 00:30:19 lab. And this is what a real FACS plot looks like. OK, 358 00:30:19 --> 00:30:24 it's a mess. Every little is a cell. But for reasons known only to 359 00:30:24 --> 00:30:29 the Mulligan lab, Peggy Goodell who is now a 360 00:30:29 --> 00:30:34 professor in her own laboratory, they assayed this little region of 361 00:30:34 --> 00:30:39 cells in the bottom left-hand corner for their stem cell properties. 362 00:30:39 --> 00:30:43 They called these SP cells or side population cells, 363 00:30:43 --> 00:30:47 and they found to their surprise that these SP cells were highly 364 00:30:47 --> 00:30:51 enriched for hematopoietic stem cells 1,000 fold or more. And 365 00:30:51 --> 00:30:56 in fact, if you take the very bottom left-hand corner where there are 366 00:30:56 --> 00:31:00 almost no cells, you get a 10,000 fold enrichment. 367 00:31:00 --> 00:31:05 So, the way they sorted these was with a dye called Hest 3342. 368 00:31:05 --> 00:31:09 This is a vital dye. It stains the DNA but it doesn't tell the cells. 369 00:31:09 --> 00:31:13 And somehow, these SP exclude the dye, or efflux it, 370 00:31:13 --> 00:31:17 remove it from the cell. And it's really not clear what that has 371 00:31:17 --> 00:31:21 to do with stem cellness, but this is still one of the very 372 00:31:21 --> 00:31:25 best ways to isolate stem cells from almost every organ. These SP cells, 373 00:31:25 --> 00:31:29 these things that don't stain with these DNA dyes seem to be the ones 374 00:31:29 --> 00:31:35 that are stem cell-like. OK, so let's move on to the question 375 00:31:35 --> 00:31:41 to several questions that I want to discuss with you that fall under the 376 00:31:41 --> 00:31:47 umbrella of regulation and control of stem cell fate. And 377 00:31:47 --> 00:31:54 there's several questions I like to pose to you. Firstly, 378 00:31:54 --> 00:32:00 what makes a stem cell self renewing? What's the molecular basis for that? 379 00:32:00 --> 00:32:06 What makes a stem cell decide whether it's going to make 380 00:32:06 --> 00:32:15 progenitors or not? 381 00:32:15 --> 00:32:20 And the big one for the stem cell field, what controls the potency of 382 00:32:20 --> 00:32:25 a stem cell? Well, this is where we step back into the 383 00:32:25 --> 00:32:30 developmental biology that we've been talking about because it all 384 00:32:30 --> 00:32:36 controls at some level by gene expression. 385 00:32:36 --> 00:32:40 And in particular, there are both intrinsic and 386 00:32:40 --> 00:32:45 extrinsic factors that seem to control certainly the first two 387 00:32:45 --> 00:32:49 points on my list, the self renewing and the progenitor 388 00:32:49 --> 00:32:54 aspect, and perhaps also the potency also. 389 00:32:54 --> 00:32:58 Intrinsic factors, cell autonomous factors, 390 00:32:58 --> 00:33:05 determinants -- 391 00:33:05 --> 00:33:11 -- and extrinsic factors, non autonomous factors, secreted 392 00:33:11 --> 00:33:18 ligands, inducers, and these and extrinsic factors have 393 00:33:18 --> 00:33:25 been given a special name in the stem cell field just for argument's 394 00:33:25 --> 00:33:32 sake. They are called the niche where the niche contains all the 395 00:33:32 --> 00:33:39 cells that influence stem cell activity, cells that influence 396 00:33:39 --> 00:33:45 stem cell activity. OK, this is just a term. You should 397 00:33:45 --> 00:33:49 know it because if you read it you will know it. So, 398 00:33:49 --> 00:33:53 here's how it works. The surrounding cells in the niche are 399 00:33:53 --> 00:33:57 cells that seem to maintain stem cells usually in acquiescent state. 400 00:33:57 --> 00:34:01 So, it's believed that in most organs, stem cells are 401 00:34:01 --> 00:34:05 sitting there quietly. They're not dividing very much, 402 00:34:05 --> 00:34:10 but they can be stimulated to divide, and this is on the second page of 403 00:34:10 --> 00:34:15 your handout. They can be stimulated to divide by some kind of 404 00:34:15 --> 00:34:19 environmental input, and this changes the surrounding 405 00:34:19 --> 00:34:24 cells. And the environmental input could also be a secreted ligand for 406 00:34:24 --> 00:34:29 example. And the surrounding cells then induce 407 00:34:29 --> 00:34:34 the stem cells to be activated. And they go on to make progenitors, 408 00:34:34 --> 00:34:38 and of course also to self renew. This is a fancy way of saying that 409 00:34:38 --> 00:34:42 cell fate is controlled by induction. OK, 410 00:34:42 --> 00:34:46 so you should be nodding. The should not be anything new for you 411 00:34:46 --> 00:34:50 at this point. It's phrased a little differently, 412 00:34:50 --> 00:34:54 but that's all. This is a very interesting example of control of 413 00:34:54 --> 00:34:58 cell fate by surrounding cells. This is from my colleague, 414 00:34:58 --> 00:35:02 Professor Fuchs at Rockefeller who studies the hair follicle, 415 00:35:02 --> 00:35:06 and who has shown that this region called the bulge is the 416 00:35:06 --> 00:35:10 source of stem cells. So, this brown thing is the hair 417 00:35:10 --> 00:35:14 that sits in a shaft of cells that got a bunch of interesting cells 418 00:35:14 --> 00:35:18 around it. In particular, there's a rather inconspicuous group 419 00:35:18 --> 00:35:22 of cells on one side called the bulge. And some years ago, 420 00:35:22 --> 00:35:26 there is another group of cells. I'm also going to refer to two at 421 00:35:26 --> 00:35:30 the bottom, which is called the dermal propeller. 422 00:35:30 --> 00:35:33 But some years ago, Professor Fuchs took the cells of 423 00:35:33 --> 00:35:37 the bulge and she transplanted them into a mouse that didn't have any 424 00:35:37 --> 00:35:41 hair. It's called a nude mouse. It has lots of problems including no 425 00:35:41 --> 00:35:45 hair. But when she did that, here's the control and here's a 426 00:35:45 --> 00:35:48 mouse into which these stem cells have been transplanted. And 427 00:35:48 --> 00:35:52 you can see all of a sudden this poor little nude mouse has got tufts 428 00:35:52 --> 00:35:56 of hair. OK, and in fact, these hairs actually 429 00:35:56 --> 00:36:00 glow-in-the-dark. They've been labeled with green. It's 430 00:36:00 --> 00:36:03 really cool, OK? The cells that were transplanted, 431 00:36:03 --> 00:36:07 this is something you know, as well. They were lineage labeled, 432 00:36:07 --> 00:36:11 and so, you could prove that these hairs came from the transplanted 433 00:36:11 --> 00:36:15 cells. All right, so let's look. So, 434 00:36:15 --> 00:36:18 during the hair cycle, so you're hairs grow cyclically. 435 00:36:18 --> 00:36:22 And there are a whole bunch of processes including growth, 436 00:36:22 --> 00:36:26 regression, induction of growth, and new growth. And during that 437 00:36:26 --> 00:36:30 period of time, the bulge and the dermal papillae 438 00:36:30 --> 00:36:34 are in relatively different positions. 439 00:36:34 --> 00:36:38 So, during a process of growth, they are far away from each other, 440 00:36:38 --> 00:36:43 and then as the hair and growth regresses, they come closer until 441 00:36:43 --> 00:36:47 they're actually touching each other during the process of induction of 442 00:36:47 --> 00:36:52 new growth. And it's at that time that new growth in 443 00:36:52 --> 00:36:56 the hair is stimulated. And it's clear that the dermal papillae 444 00:36:56 --> 00:37:01 is signaling to the bulge cells. And here's what it's using to 445 00:37:01 --> 00:37:05 signal. It's using something called the wind pathway, 446 00:37:05 --> 00:37:09 and in particular, a molecule called beta-catenin. If 447 00:37:09 --> 00:37:13 you think back several lectures, we talked about beta-catenin is one 448 00:37:13 --> 00:37:17 of the things that told the embryo to make its back rather than its 449 00:37:17 --> 00:37:21 belly. So, here's a different use of the same molecule in stimulating 450 00:37:21 --> 00:37:25 hairs to grow. And so this is a particularly cool example of cells 451 00:37:25 --> 00:37:29 coming together at particular points to stimulate stem cells to go on and 452 00:37:29 --> 00:37:36 to make progenitors. All right, let's move on to 453 00:37:36 --> 00:37:46 something important called embryonic stem cells And all right -- 454 00:37:46 --> 00:37:56 -- also called ES sells. So, 455 00:37:56 --> 00:38:00 although I told you that most adult organs are likely to have stem cells, 456 00:38:00 --> 00:38:04 and this has been very clearly shown for many, there are many organs 457 00:38:04 --> 00:38:09 where it's not clear whether they have stem cells, 458 00:38:09 --> 00:38:13 or it's very difficult to isolate them. Stem cells are rare in all 459 00:38:13 --> 00:38:17 organs, and things like neural stem cells in the nervous system seemed 460 00:38:17 --> 00:38:22 to be a exceedingly rare and difficult to isolate. So, 461 00:38:22 --> 00:38:26 the push has been to try to find a source of stem cells that would be 462 00:38:26 --> 00:38:31 more plentiful and more useful for repairing lots of different organs. 463 00:38:31 --> 00:38:36 And that's where the embryo comes in this particular kind of stem cell 464 00:38:36 --> 00:38:42 called an embryonic stem cell. And the idea is if one takes an embryo 465 00:38:42 --> 00:38:47 or part of it, puts it into culture, 466 00:38:47 --> 00:38:53 after many steps you get out cells that are called ES cells. And 467 00:38:53 --> 00:38:58 these ES cells are pluripotent. They are not totipotent. But they 468 00:38:58 --> 00:39:04 are very, if I can be forgiven, they are very pluripotent bouquet, 469 00:39:04 --> 00:39:10 and they are pluripotent, and you can control their cell fate. 470 00:39:10 --> 00:39:13 So, let me going to the slides, and we will talk more about this 471 00:39:13 --> 00:39:17 through the slides. So, in mammalian development, 472 00:39:17 --> 00:39:20 and we're going to talk about mammals here specifically, 473 00:39:20 --> 00:39:24 mammalian development, the blastula forms. And at a certain point in 474 00:39:24 --> 00:39:28 development, a group of cells called the inner cell mass segregates from 475 00:39:28 --> 00:39:32 the rest of the cells which form a shell around the embryo. 476 00:39:32 --> 00:39:36 We mentioned this previously. This yellow stuff is fluid, 477 00:39:36 --> 00:39:40 and this little group of cells called the ICM, 478 00:39:40 --> 00:39:44 or inner cell mass, is the thing that's going to give 479 00:39:44 --> 00:39:48 rise to the embryo proper. The cells surrounding are going to give 480 00:39:48 --> 00:39:53 rise to the placenta and the other extra embryonic components. So, 481 00:39:53 --> 00:39:57 the idea in trying to get these ES cells is to take the inner cell mass 482 00:39:57 --> 00:40:01 of an early embryo, take it out of the embryo, 483 00:40:01 --> 00:40:05 and put it in a Petri dish that's got nutrients and various factors to 484 00:40:05 --> 00:40:09 disperse the cells such that you've got single cells dispersed in the 485 00:40:09 --> 00:40:13 plate, and give them nutrients. And over time, 486 00:40:13 --> 00:40:17 those cells will grow, and they will form clumps, 487 00:40:17 --> 00:40:21 each of which is derived from a single embryonic cell. OK, 488 00:40:21 --> 00:40:25 now, normal embryonic cells do not do this. OK, they will normally go 489 00:40:25 --> 00:40:29 on and differentiate, and stop dividing, but there's 490 00:40:29 --> 00:40:33 something that happens during this culture process that is abnormal. 491 00:40:33 --> 00:40:37 And it turns the cells into groups of cells that can self renew. OK, 492 00:40:37 --> 00:40:42 so you've turned these cells into self renewing cells. And 493 00:40:42 --> 00:40:46 each of these groups of cells or colonies that you get may be able to 494 00:40:46 --> 00:40:51 grow into a stem cell line. And I'll talk about cell lines in a 495 00:40:51 --> 00:40:55 moment. I'll talk about cell lines now. So, you might not be familiar 496 00:40:55 --> 00:41:00 with the term cell line. What is the cell line? 497 00:41:00 --> 00:41:04 A cell line is a cell population, a homogeneous cell population that 498 00:41:04 --> 00:41:08 could grow continuously in culture. So, it's self renewing. OK, 499 00:41:08 --> 00:41:12 so all cell lines are self renewing. A stem cell line is a cell line 500 00:41:12 --> 00:41:17 that has the capacity to go on and differentiate into specific normal 501 00:41:17 --> 00:41:21 cell types. So, there are many cell lines that will 502 00:41:21 --> 00:41:25 grow continuously in culture, but they will never go on and 503 00:41:25 --> 00:41:30 differentiate as anything. They are very abnormal cells. 504 00:41:30 --> 00:41:34 They are useful for many studies, but they are not stem cells. The 505 00:41:34 --> 00:41:38 stem cells not only can grow continuously, but they can go on and 506 00:41:38 --> 00:41:42 differentiate. But you are dealing with an abnormal cells here. This 507 00:41:42 --> 00:41:47 is not a normal embryonic cell. OK, how do you test the potency of these 508 00:41:47 --> 00:41:51 ES cells done in the following way? The ES cells from a mouse, a black 509 00:41:51 --> 00:41:55 mouse, are taken, and they are injected into an early 510 00:41:55 --> 00:42:00 embryo of an embryo derived from white parents. 511 00:42:00 --> 00:42:04 And if you do that, the ES cells incorporate into the 512 00:42:04 --> 00:42:08 embryo. You then take the embryos, and put them into a surrogate 513 00:42:08 --> 00:42:12 mother. And when the mice are born, when the babies are born, 514 00:42:12 --> 00:42:16 you can see that they often are not just pure white. They often got 515 00:42:16 --> 00:42:20 black stripes, and you can look at the various 516 00:42:20 --> 00:42:24 organs and show that these ES cells have incorporated into various 517 00:42:24 --> 00:42:28 organs. So, these ES cells are highly potent. And 518 00:42:28 --> 00:42:32 the idea is that you can take these ES cells and add to them 519 00:42:32 --> 00:42:37 various factors. So, you can add a particular red 520 00:42:37 --> 00:42:41 factor that will turn an ES line into heart muscle cells or pancreas 521 00:42:41 --> 00:42:46 cells or cartilage cells. And you can use those cells in your stem 522 00:42:46 --> 00:42:51 cell repair assays. OK, and this is true. You can take ES 523 00:42:51 --> 00:42:55 cells, and you can do exactly this to them. And then, 524 00:42:55 --> 00:43:00 they will become these different kinds of differentiated derivatives. 525 00:43:00 --> 00:43:05 So, this is really cool. And the push has been to try to get 526 00:43:05 --> 00:43:09 this to work not for mice but for humans. And this is where the huge 527 00:43:09 --> 00:43:13 controversy in the stem cell field comes from. So, 528 00:43:13 --> 00:43:17 the controversy comes from the fact that you need embryos to get these 529 00:43:17 --> 00:43:22 stem cells. You get the embryos from in vitro fertilization that we 530 00:43:22 --> 00:43:26 touched on a few lectures ago. Eggs are isolated by ovarian stimulation. 531 00:43:26 --> 00:43:30 They are fertilized in vitro, and they are allowed to grow in 532 00:43:30 --> 00:43:35 vitro for a week or so. And at that point, 533 00:43:35 --> 00:43:39 there are harvested or killed to make the ES line. And 534 00:43:39 --> 00:43:44 this is the very controversial point, whether or not it is ethically OK to 535 00:43:44 --> 00:43:48 harvest these embryos, and turn them in to stem cell lines 536 00:43:48 --> 00:43:53 or not. Presently, there is no federal funding that is 537 00:43:53 --> 00:43:58 allowed to be used to make human ES cell lines. There are some that 538 00:43:58 --> 00:44:02 exist, and President Bush has told scientists that they need to use the 539 00:44:02 --> 00:44:07 ones that exist. However, they are not very good cell 540 00:44:07 --> 00:44:12 lines, and so scientists have used private funding to make new human ES 541 00:44:12 --> 00:44:17 lines that hopefully will be more useful. I don't think there's any 542 00:44:17 --> 00:44:22 right or wrong answer whether this is OK or not. My opinion is that 543 00:44:22 --> 00:44:27 this is an OK thing to do, with all due respect to the embryo. 544 00:44:27 --> 00:44:32 I think one can save people's lives, well, and the embryo at this point 545 00:44:32 --> 00:44:37 is not a differentiated entity. But it is an embryo. And so, 546 00:44:37 --> 00:44:41 this is an ethical issue. There's opinion here, and it's good for you 547 00:44:41 --> 00:44:46 to think about what your opinion about this type of research is. OK, 548 00:44:46 --> 00:44:50 so let's move on. And I'm going to, in the last minute, 549 00:44:50 --> 00:44:55 just touch on something called stem cell plasticity. 550 00:44:55 --> 00:45:09 One of the things that has come out 551 00:45:09 --> 00:45:13 of the human ES work or all the ES work, and that has come out of the 552 00:45:13 --> 00:45:18 quest not to use embryos for stem cells is to ask whether or not one 553 00:45:18 --> 00:45:22 can turn one stem cell line into another. So, it's easy, 554 00:45:22 --> 00:45:27 or relatively easy, to get hematopoietic stem cells. And 555 00:45:27 --> 00:45:32 wouldn't it be wonderful if you take those hematopoietic stem cells and 556 00:45:32 --> 00:45:36 turn them into brain stem cells, and fix people who have Parkinson's 557 00:45:36 --> 00:45:41 or Alzheimer's? OK, wouldn't it be wonderful to fix 558 00:45:41 --> 00:45:45 people who have muscular dystrophy by turning hematopoietic stem cells 559 00:45:45 --> 00:45:49 that you can get lots of into muscle cells? So, the idea is maybe you 560 00:45:49 --> 00:45:53 could turn something from one lineage, a hematopoietic stem cell 561 00:45:53 --> 00:45:57 into a stem cell from another lineage and get it to 562 00:45:57 --> 00:46:01 do something else? And the hypothesis, 563 00:46:01 --> 00:46:05 then, is that if you do appropriate experiments, you may be able to 564 00:46:05 --> 00:46:09 figure out where the stem cells from one lineage can contribute to 565 00:46:09 --> 00:46:13 another. This is the second to last slide on your handout. So, 566 00:46:13 --> 00:46:17 this is the way the experiments were done. It's the last thing I'm going 567 00:46:17 --> 00:46:21 to tell you. Bear with me. You can take a mouse that's been dyed green, 568 00:46:21 --> 00:46:25 and its hematopoietic stem cells are expressing GFP, 569 00:46:25 --> 00:46:29 which is a green fluorescent protein. You use that mouse as a 570 00:46:29 --> 00:46:33 daughter for bone marrow transplants. 571 00:46:33 --> 00:46:37 You put in the great cells. Not only do you rescue the bone marrow, 572 00:46:37 --> 00:46:41 you also ask whether other organs have got green cells in them, 573 00:46:41 --> 00:46:45 indicating that the hematopoietic cells can contribute to other 574 00:46:45 --> 00:46:49 lineages. And when you do that, 575 00:46:49 --> 00:46:53 initially people got very excited because you saw results like this. 576 00:46:53 --> 00:46:57 And this is data from my colleague, Dr. Camargo over at the Whitehead 577 00:46:57 --> 00:47:01 Institute. When this experiment was done, he could show that the liver 578 00:47:01 --> 00:47:05 of such an animal had lots of green cells, suggesting that the 579 00:47:05 --> 00:47:10 hematopoietic stem cells could also be liver stem cells. 580 00:47:10 --> 00:47:14 But when he looked more closely, this is a complete artifact. And 581 00:47:14 --> 00:47:19 what had happened was that the hematopoietic stem cells had 582 00:47:19 --> 00:47:23 actually fused with the liver cells, making the liver cells green. And 583 00:47:23 --> 00:47:28 in fact, presently, there is no data to suggest that you 584 00:47:28 --> 47:31 can interconvert stem cell lineages. And I'll stop there. Thank you.