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