WEBVTT 00:00:01.000 --> 00:00:04.000 Good morning, class. Nice to see you here, 00:00:04.000 --> 00:00:08.000 you loyal holdouts, the stalwarts who haven't gone home early for 00:00:08.000 --> 00:00:12.000 Thanksgiving. You recall that last time we were talking about the 00:00:12.000 --> 00:00:16.000 Matevoidic system, and much of the rationale for 00:00:16.000 --> 00:00:20.000 studying it stems from two reasons. First of all, it recapitulates in a 00:00:20.000 --> 00:00:25.000 formal sense what happens during embryogenesis, 00:00:25.000 --> 00:00:29.000 i.e. one has relatively undifferentiated stem cells which 00:00:29.000 --> 00:00:33.000 are able to differentiate into a number of different directions by 00:00:33.000 --> 00:00:37.000 committing themselves to either the myeloid or lymphoid compartment, 00:00:37.000 --> 00:00:41.000 and then going down yet other pathways, more detailed pathways to 00:00:41.000 --> 00:00:46.000 generate a whole variety of cell types. 00:00:46.000 --> 00:00:50.000 Secondly, we really understand the differentiation pathways of 00:00:50.000 --> 00:00:54.000 Matevoisis better than we understand any tissue in the body, 00:00:54.000 --> 00:00:59.000 in no small part because it's much easier to study the soluble cells in 00:00:59.000 --> 00:01:03.000 the blood and in the immune system than it is to study how these 00:01:03.000 --> 00:01:08.000 processes happen in normal tissues. But having said that, 00:01:08.000 --> 00:01:13.000 I want to emphasize the fact that in each of our tissues there are 00:01:13.000 --> 00:01:18.000 oligopotential stem cells. When I say oligopotential I mean 00:01:18.000 --> 00:01:24.000 they can go down several different pathways. Recall up there on that 00:01:24.000 --> 00:01:29.000 diagram we talked about pluripotential which means multiple, 00:01:29.000 --> 00:01:34.000 and today we're going to talk a bit about todipotential stem cells, 00:01:34.000 --> 00:01:39.000 which are able to disperse descendants into all the different 00:01:39.000 --> 00:01:45.000 differentiation lineages in the body. 00:01:45.000 --> 00:01:50.000 At the end of our last lecture, we were focusing on the red blood 00:01:50.000 --> 00:01:56.000 cells. And this is sometimes called erythropoiesis, 00:01:56.000 --> 00:02:02.000 which is to say the process by which red blood cells are generated. 00:02:02.000 --> 00:02:06.000 We mentioned the concept of homeostasis, and homeostasis just 00:02:06.000 --> 00:02:11.000 refers to the fact that all of these systems are in very delicate balance 00:02:11.000 --> 00:02:15.000 so that the body can respond to the physiologic needs of the organism at 00:02:15.000 --> 00:02:20.000 any one point in time. We talked about the fact that for 00:02:20.000 --> 00:02:24.000 example when there's a massive infection in the body, 00:02:24.000 --> 00:02:29.000 then the homeostatic mechanisms allow an increase in these kinds of 00:02:29.000 --> 00:02:34.000 immune cells in order to encounter the infection. 00:02:34.000 --> 00:02:38.000 And at the end of our last lecture, we were talking about this specific 00:02:38.000 --> 00:02:43.000 branch, and how in fact homeostasis is maintained there. 00:02:43.000 --> 00:02:48.000 And what we see here is a series of committed progenitors. 00:02:48.000 --> 00:02:52.000 So when I talk about committed progenitors I'm referring to cells 00:02:52.000 --> 00:02:57.000 that have already made the commitment to go down one 00:02:57.000 --> 00:03:02.000 or another pathway. They're not yet fully differentiated. 00:03:02.000 --> 00:03:06.000 As you can see here, we have first forming cells and 00:03:06.000 --> 00:03:10.000 colony forming cells. We don't need to remember all the 00:03:10.000 --> 00:03:14.000 different abbreviations except to say that these cells here are in a 00:03:14.000 --> 00:03:18.000 relative undifferentiated state. And the only end stage 00:03:18.000 --> 00:03:22.000 differentiation comes at the very end here when we get to red blood 00:03:22.000 --> 00:03:26.000 cells. We said in general that it's the case that most highly 00:03:26.000 --> 00:03:30.000 differentiated cells are post-mitotic, which is to say 00:03:30.000 --> 00:03:34.000 they're never going to reenter into the growth and division cycle of the 00:03:34.000 --> 00:03:38.000 cell that we talked about earlier in the semester. 00:03:38.000 --> 00:03:42.000 And that's obviously dictated here by the fact that this erythrocyte 00:03:42.000 --> 00:03:46.000 lacks a nucleus, i.e. during the final stage of 00:03:46.000 --> 00:03:50.000 differentiation, in addition to accumulating large 00:03:50.000 --> 00:03:54.000 amounts of hemoglobin in its cytoplasm, this cell actually pops 00:03:54.000 --> 00:03:58.000 out its nucleus, and that obviously represents an 00:03:58.000 --> 00:04:02.000 irrevocable change in that cell can never again enter into growth 00:04:02.000 --> 00:04:06.000 and division cycle. The immediate precursor of an 00:04:06.000 --> 00:04:12.000 erythrocyte is often called an erythroblast. And the term blast 00:04:12.000 --> 00:04:17.000 here refers to a cell of embryonic appearance. Blast is used often to 00:04:17.000 --> 00:04:23.000 indicate, we'll mention that again shortly, a cell which looks very 00:04:23.000 --> 00:04:28.000 primitive, and embryonic, and undifferentiated. And that ends 00:04:28.000 --> 00:04:34.000 up going into an erythrocyte, which we said is actually a synonym 00:04:34.000 --> 00:04:40.000 for a red blood cell, an RBC, a red blood cell. 00:04:40.000 --> 00:04:44.000 And we talked about the fact that this progression is actually 00:04:44.000 --> 00:04:49.000 maintained and furthered by the stimulus of the compound called 00:04:49.000 --> 00:04:53.000 erythropoietin. So, we're using some of the same 00:04:53.000 --> 00:04:58.000 words over and over again. And erythropoietin is essentially a 00:04:58.000 --> 00:05:03.000 growth factor which stimulates the end stage differentiation of the 00:05:03.000 --> 00:05:08.000 erythroblast into the erythrocyte. 00:05:08.000 --> 00:05:13.000 Epo, as erythropoietin's often abbreviated, is actually made in the 00:05:13.000 --> 00:05:19.000 kidneys. And it's made in the kidneys in response to the 00:05:19.000 --> 00:05:25.000 physiological stimulus of hypoxia. Hypoxia means inadequate 00:05:25.000 --> 00:05:31.000 oxygenation of the tissues. You might ask, well, why is red 00:05:31.000 --> 00:05:37.000 blood cell contractions controlled, as they are, in the kidney? 00:05:37.000 --> 00:05:41.000 And the fact is, we don't really know why evolution 00:05:41.000 --> 00:05:45.000 has chosen the kidney as the site of monitoring the degree of oxygenation 00:05:45.000 --> 00:05:49.000 of the blood. And in response to hypoxia, it begins to crank out 00:05:49.000 --> 00:05:53.000 erythropoietin, or Epo. You can think of 00:05:53.000 --> 00:05:57.000 erythropoietin as an extracellular liggon just like a growth factor. 00:05:57.000 --> 00:06:01.000 It has its own cognate receptor on the surface of the erythroblast, 00:06:01.000 --> 00:06:06.000 and when Epo released by the kidney hits an erythroblast in the context 00:06:06.000 --> 00:06:11.000 of the bone marrow, it actually has two effects. 00:06:11.000 --> 00:06:16.000 It happens to be the case that roughly even 95% of the erythroblast 00:06:16.000 --> 00:06:21.000 that are made routinely are forced to go into apitosis under routine 00:06:21.000 --> 00:06:26.000 conditions. So, this is an enormously wasteful 00:06:26.000 --> 00:06:31.000 system, i.e. as every moment we speak, 90 or 95% of the erythroblast 00:06:31.000 --> 00:06:37.000 that have come into existence in your bone marrow apitose. 00:06:37.000 --> 00:06:43.000 They never go into end stage differentiation. 00:06:43.000 --> 00:06:50.000 But when Epo is around, Epo provides a strong anti-apoptotic 00:06:50.000 --> 00:06:56.000 signal to the red blood saves some and maybe even all of the 00:06:56.000 --> 00:07:03.000 erythroblasts from their normal fate of undergoing apitosis. 00:07:03.000 --> 00:07:07.000 So here, if we imagine there are actually two fates, 00:07:07.000 --> 00:07:12.000 one is to become an erythrocyte, and the other is to apitose, where 00:07:12.000 --> 00:07:16.000 the aptisosis is paradoxically enough the dominant fate of the cell, 00:07:16.000 --> 00:07:21.000 the moment that an Epo comes on the scene, it blocks this alternative 00:07:21.000 --> 00:07:25.000 fate, allowing these cells to mature. Epo at the same time stimulates the 00:07:25.000 --> 00:07:30.000 erythroblast to differentiate. Now, you might as yourself the 00:07:30.000 --> 00:07:35.000 question, why is there this enormously inefficient process? 00:07:35.000 --> 00:07:38.000 An enormous effort is made to crank out large, astronomical numbers of 00:07:38.000 --> 00:07:42.000 erythroblasts, and yet most of them are wasted even 00:07:42.000 --> 00:07:46.000 before they've had a chance to undergo end stage differentiation. 00:07:46.000 --> 00:07:50.000 And the rationale here is as follows. This is a terrific system 00:07:50.000 --> 00:07:54.000 for rapidly ramping up the level of red blood cells in your circulation 00:07:54.000 --> 00:07:58.000 because here, within a matter of a day or two, one can crank up, 00:07:58.000 --> 00:08:02.000 actually in a matter of hours, you can crank up the rate of 00:08:02.000 --> 00:08:06.000 production of red blood cells by maybe even a factor of ten. 00:08:06.000 --> 00:08:10.000 Instead of having 90% of the erythroblast apitose, 00:08:10.000 --> 00:08:14.000 let's say 0% of them do so, and therefore, instead of having 10% 00:08:14.000 --> 00:08:18.000 of the erythroblasts becoming red blood cells, 100% of them will do so. 00:08:18.000 --> 00:08:22.000 And therefore, you have the virtually miraculous 00:08:22.000 --> 00:08:26.000 response that if you go from here high up in the rocky mountains at 00:08:26.000 --> 00:08:30.000 ten or 12,000 feet, within a matter of two or three days, 00:08:30.000 --> 00:08:34.000 your red blood cell concentration actually has compensated, 00:08:34.000 --> 00:08:38.000 has risen up to create the oxygen caring capacity that enables you to 00:08:38.000 --> 00:08:42.000 deal with the thin oxygen, with the low oxygen tension that's 00:08:42.000 --> 00:08:47.000 present at high altitudes. Now, having said that, 00:08:47.000 --> 00:08:52.000 the fact is that there is an Epo receptor on the surface of the 00:08:52.000 --> 00:08:58.000 erythroblast, and what we see there is the following. 00:08:58.000 --> 00:09:01.000 Let's talk about the erythroblast and just blow it up a little bit. 00:09:01.000 --> 00:09:05.000 So, here's the erythroblast. That's the undifferentiated 00:09:05.000 --> 00:09:08.000 precursor. And by the way, the erythroblast is actually still a 00:09:08.000 --> 00:09:12.000 white blood cell. Often we call a white blood cell a 00:09:12.000 --> 00:09:15.000 leukocyte. You may know that gluco means white. So, 00:09:15.000 --> 00:09:19.000 a leukocyte, it's still white. And after the erythropotent 00:09:19.000 --> 00:09:22.000 impinges on it, one of the things it starts doing is 00:09:22.000 --> 00:09:26.000 to make the hemoglobin, which turns it into a red blood cell. 00:09:26.000 --> 00:09:30.000 At this stage, it's still white. On the surface of the erythroblast 00:09:30.000 --> 00:09:35.000 are these Epo receptors. I'll just abbreviate them like this, 00:09:35.000 --> 00:09:40.000 Epo receptor, and once it binds the liggon Epo just like the growth 00:09:40.000 --> 00:09:45.000 factor receptors, we talked early in the receptors 00:09:45.000 --> 00:09:49.000 signals are sent into the erythroblast to stimulate both 00:09:49.000 --> 00:09:54.000 differentiation and to prevent the initiation of the cell suicide 00:09:54.000 --> 00:09:59.000 program that we call apitosis. Interestingly, one of the things 00:09:59.000 --> 00:10:04.000 that happens normally is the following, that when these signals 00:10:04.000 --> 00:10:09.000 come in, there is an enzyme called a phosphotase which is attracted 00:10:09.000 --> 00:10:14.000 to the receptor. The Epo receptor works like a 00:10:14.000 --> 00:10:18.000 tyrosine kinase growth factor receptor that we talked about 00:10:18.000 --> 00:10:22.000 earlier in the semester. And here, we have an enzyme, 00:10:22.000 --> 00:10:27.000 a phosphotase, which actually counteracts the function of the 00:10:27.000 --> 00:10:31.000 tyrosine kinases. So, after the Epo receptor has 00:10:31.000 --> 00:10:35.000 bound its liggon, here's the plasma membrane, 00:10:35.000 --> 00:10:40.000 it has a whole series of I'll draw Y here for tyrosine. 00:10:40.000 --> 00:10:43.000 It has a whole series of phosphates attached to it because of the 00:10:43.000 --> 00:10:47.000 actions of tyrosine kinase enzymes that are associated with its 00:10:47.000 --> 00:10:51.000 cytoplasmic domain indirect analogy to what we talked about in the case 00:10:51.000 --> 00:10:54.000 of growth factor receptors. But, one of the things that happens 00:10:54.000 --> 00:10:58.000 is that this phosphotase, which removes phosphates, then gloms 00:10:58.000 --> 00:11:02.000 onto the receptor like this. It grabs hold of some of these 00:11:02.000 --> 00:11:06.000 tyrosine kinases. And what this phosphotase does is 00:11:06.000 --> 00:11:10.000 reach around. It reaches around and it begins to prune off all of these 00:11:10.000 --> 00:11:14.000 phosphates because that's what a phosphate does. 00:11:14.000 --> 00:11:19.000 It cuts away all the phosphates, thereby directly reversing the 00:11:19.000 --> 00:11:23.000 previous actions of the tyrosine kinase that led to the formation of 00:11:23.000 --> 00:11:27.000 these phosphates, and that in turn allows downstream 00:11:27.000 --> 00:11:32.000 signaling to occur. This is obviously a functional 00:11:32.000 --> 00:11:36.000 negative feedback loop, i.e. whenever there is an agonist 00:11:36.000 --> 00:11:40.000 you want an antagonist. Whenever there's a stimulus which 00:11:40.000 --> 00:11:44.000 is induced in the body, there has to be an inhibitory signal, 00:11:44.000 --> 00:11:48.000 and this is part of the whole issue of homeostasis, 00:11:48.000 --> 00:11:52.000 the balance between forward and backward. Interestingly enough, 00:11:52.000 --> 00:11:56.000 there's a family in Finland, I believe, which has a mutant receptor. 00:11:56.000 --> 00:12:01.000 And their mutant receptor lacks this tyrosine. 00:12:01.000 --> 00:12:04.000 And what happens as a consequence is that that particular tyrosine 00:12:04.000 --> 00:12:07.000 doesn't get phosphorolated. Because that tyrosine doesn't get 00:12:07.000 --> 00:12:11.000 phosphorolated, the phosphotase cannot be attracted 00:12:11.000 --> 00:12:14.000 to the receptor because there isn't a tyrosine there. 00:12:14.000 --> 00:12:18.000 There's some other amino acid residue. I don't know what it is. 00:12:18.000 --> 00:12:21.000 It's not important, but it's not a tyrosine. And this cannot happen 00:12:21.000 --> 00:12:24.000 because they don't have this tyrosine. This phosphotase could 00:12:24.000 --> 00:12:28.000 not be attracted to the receptor to shut it down as it normally 00:12:28.000 --> 00:12:32.000 would be. So normally homeostasis is 00:12:32.000 --> 00:12:36.000 imbalanced, and several members of this family have become Olympic 00:12:36.000 --> 00:12:41.000 cross-country ski winners. They've become Olympic champions. 00:12:41.000 --> 00:12:45.000 Why? Because their Epo receptor's hyperactive. Because the Epo 00:12:45.000 --> 00:12:49.000 receptor's hyperactive, they have higher than normal levels 00:12:49.000 --> 00:12:54.000 of red blood cells in the circulation, and this clearly allows 00:12:54.000 --> 00:12:58.000 them to function better in cross country skiing, 00:12:58.000 --> 00:13:03.000 which as you know is a really physically demanding task. 00:13:03.000 --> 00:13:06.000 Again, I'm not saying this is a good thing for them necessarily. 00:13:06.000 --> 00:13:10.000 There are other things in life besides, believe it or not, 00:13:10.000 --> 00:13:14.000 winning cross country Olympic competitions because as I mentioned 00:13:14.000 --> 00:13:18.000 last time, having too many red blood cells in your circulation, 00:13:18.000 --> 00:13:22.000 there's a downside to it which is that you have a much greater 00:13:22.000 --> 00:13:26.000 tendency to have occlusions, to have blood clots in your 00:13:26.000 --> 00:13:30.000 circulation which obviously is not a very good thing to have. 00:13:30.000 --> 00:13:38.000 Oh, so is there a threshold of Epo 00:13:38.000 --> 00:13:41.000 receptor activation before phosphotase shuts it down? 00:13:41.000 --> 00:13:44.000 These things are not really well understood, are not well studied. 00:13:44.000 --> 00:13:47.000 The fact is, you might be able to say we should make a mathematical 00:13:47.000 --> 00:13:51.000 model of all of these different circuitry. But the fact is if you 00:13:51.000 --> 00:13:54.000 want to make a mathematical model, you have to know some of the 00:13:54.000 --> 00:13:57.000 constants. You have to know some of the parameters, the binding 00:13:57.000 --> 00:14:00.000 constants. And in fact, for most of the 00:14:00.000 --> 00:14:04.000 signaling interactions, no one's ever really studied them in 00:14:04.000 --> 00:14:08.000 such great detail. So, one really doesn't know how 00:14:08.000 --> 00:14:11.000 much phosphate you need here before the phosphotase becomes really 00:14:11.000 --> 00:14:15.000 active. And so, there's not a really good 00:14:15.000 --> 00:14:19.000 mathematical model of this feedback loop, even though we know without 00:14:19.000 --> 00:14:22.000 any doubt that it exists. So, I want to get into other issues 00:14:22.000 --> 00:14:26.000 that are related to the whole issue of accumulated differentiation 00:14:26.000 --> 00:14:30.000 traits as one moves down this pathway. 00:14:30.000 --> 00:14:34.000 Again, we've used this as a model for how differentiation takes place 00:14:34.000 --> 00:14:38.000 in the entire body. The faith that's been implicit in 00:14:38.000 --> 00:14:42.000 this kind of scheme for the last 20 or 30 years is that this acquisition 00:14:42.000 --> 00:14:47.000 of different kinds of phenotypes is not accompanied by genetic changes, 00:14:47.000 --> 00:14:51.000 that is, in the genomes of these cells. I.e. one can accomplish 00:14:51.000 --> 00:14:55.000 these different kinds of differentiation not by rearranging 00:14:55.000 --> 00:15:00.000 genes but just by rearranging transcriptional programs, 00:15:00.000 --> 00:15:03.000 and that the DNA sequence of these cells as they proliferate and 00:15:03.000 --> 00:15:07.000 differentiate is fully unchanged. And that's a matter of faith 00:15:07.000 --> 00:15:11.000 because you could say to me, how do you know that it's really 00:15:11.000 --> 00:15:15.000 true. The fact is that people have looked at genes in many kinds of 00:15:15.000 --> 00:15:18.000 cell types, but it's essentially impossible, or it has been at least 00:15:18.000 --> 00:15:22.000 until recently, to preclude the possibility that as 00:15:22.000 --> 00:15:26.000 cells move down these differentiation pathways, 00:15:26.000 --> 00:15:30.000 they begin to change the nucleotide sequences of different 00:15:30.000 --> 00:15:33.000 ones of their genes. In fact, I've already told you about 00:15:33.000 --> 00:15:37.000 one instance where that's clearly the case. And that is in the 00:15:37.000 --> 00:15:41.000 differentiation of the B cells of the immune system, 00:15:41.000 --> 00:15:45.000 which happen to be right up here on this chart, because as you recall 00:15:45.000 --> 00:15:48.000 from our discussion vis-à-vis immunology, the B cells actually do 00:15:48.000 --> 00:15:52.000 rearrange their genes in order to cobble together DNA sequences that 00:15:52.000 --> 00:15:56.000 together are able to enable them to make antibodies that are able to 00:15:56.000 --> 00:16:00.000 react to specific antigens. So there, there's no doubt at all 00:16:00.000 --> 00:16:04.000 that there's a somatic rearrangement of the genes, somatic meaning it's 00:16:04.000 --> 00:16:08.000 not a germ line change. It's happening in the soma outside 00:16:08.000 --> 00:16:12.000 of the germ line. There's a somatic mutation. 00:16:12.000 --> 00:16:16.000 It's not a mutation that's deleterious, but rather is directed 00:16:16.000 --> 00:16:20.000 towards a physiologically normal and desirable end point. 00:16:20.000 --> 00:16:24.000 But for example, how do you know that when you remember things in the 00:16:24.000 --> 00:16:28.000 brain, part of the memory does not derive from changing the DNA 00:16:28.000 --> 00:16:32.000 sequence and different neurons in the brain? 00:16:32.000 --> 00:16:36.000 What's the molecular basis of memory? Could it be that each time we learn 00:16:36.000 --> 00:16:41.000 some things that there are different nucleotide sequences, 00:16:41.000 --> 00:16:45.000 critical nucleotide sequences, that are changed in neurons in the 00:16:45.000 --> 00:16:50.000 brain, and that those nucleotide sequence changes represent an 00:16:50.000 --> 00:16:54.000 important basis for ensuring that memory is retained over decades of 00:16:54.000 --> 00:16:59.000 time. Or, rather than having genetic changes in the brain, 00:16:59.000 --> 00:17:03.000 might it all be epigenetic, i. . all the other changes that happen 00:17:03.000 --> 00:17:08.000 to the cell besides changing DNA sequences in the chromosomal DNA. 00:17:08.000 --> 00:17:13.000 So, here we're dealing with the dialectic between epigenetic and 00:17:13.000 --> 00:17:19.000 genetic. And, have we talked about DNA methylation 00:17:19.000 --> 00:17:24.000 here? Yes, so we talked about DNA methylation, and do you recall or 00:17:24.000 --> 00:17:30.000 having discussed the fact that when DNA gets methylated, 00:17:30.000 --> 00:17:36.000 that suppresses the transcription of a gene. 00:17:36.000 --> 00:17:39.000 But that doesn't change the nucleotide sequence, 00:17:39.000 --> 00:17:43.000 and that methylation configuration of a gene can be passed to one cell 00:17:43.000 --> 00:17:46.000 generation to the next. It's heritable, but it's not 00:17:46.000 --> 00:17:50.000 genetic in the strictest sense of the term, i.e. 00:17:50.000 --> 00:17:54.000 it doesn't involve a change in nucleotide sequence, 00:17:54.000 --> 00:17:58.000 which is what we want to limit this term to referring. 00:17:58.000 --> 00:18:02.000 So, epigenic can represent all the changes in the cell including DNA 00:18:02.000 --> 00:18:07.000 methylation, alterations in transcription, 00:18:07.000 --> 00:18:12.000 and all other downstream events that result in changes in the cell. 00:18:12.000 --> 00:18:17.000 And how can one address this? Well, there are different ways of 00:18:17.000 --> 00:18:22.000 addressing this question or addressing the possibility that in 00:18:22.000 --> 00:18:27.000 fact there are changes in the nucleotide sequence of the gene. 00:18:27.000 --> 00:18:32.000 One way to do this is the following. And that is to take cells from an 00:18:32.000 --> 00:18:37.000 early embryo, and here we see an early vertebrate embryo. 00:18:37.000 --> 00:18:42.000 This looks really more like a frog embryo or a slightly different shape, 00:18:42.000 --> 00:18:47.000 and here we see an early embryo. It's after a blastula. It's called 00:18:47.000 --> 00:18:52.000 a blastocyst. Here again we have the word blast. 00:18:52.000 --> 00:18:57.000 How about one question per lecture? We have to have some equity here. 00:18:57.000 --> 00:19:02.000 Other people can ask questions. It's good to ask questions, 00:19:02.000 --> 00:19:06.000 but how about one per lecture; that's fair, equitable. 00:19:06.000 --> 00:19:10.000 All right, so here's an early vertebrate embryo. 00:19:10.000 --> 00:19:14.000 Here we see the blastocyst. This comes after the earlier stages 00:19:14.000 --> 00:19:18.000 in the embryo, and here we see the inner cell mass. 00:19:18.000 --> 00:19:22.000 And as it turns out, the inner cell mass is going to be the precursor of 00:19:22.000 --> 00:19:26.000 many of the tissues of the ultimately arising embryo. 00:19:26.000 --> 00:19:30.000 And here, one can do an interesting experiment. One can take cells out 00:19:30.000 --> 00:19:34.000 of the inner cell mass. And one can begin to propagate them 00:19:34.000 --> 00:19:38.000 in culture. And what one ends up with is embryonic stem cells. 00:19:38.000 --> 00:19:42.000 And the intrinsic interest of embryonic stem cells is manifold. 00:19:42.000 --> 00:19:46.000 For one thing, you can take embryonic stem cells and you can 00:19:46.000 --> 00:19:51.000 genetically alter them. You can put a new gene in, 00:19:51.000 --> 00:19:55.000 in the case of a mouse, or you can take another gene out. 00:19:55.000 --> 00:19:59.000 And then what you can do is you can inject the genetically altered 00:19:59.000 --> 00:20:04.000 embryonic stem cell into the blastocyst of another embryo. 00:20:04.000 --> 00:20:08.000 So let's say we take the cells out of the inner cell mass. 00:20:08.000 --> 00:20:13.000 We develop embryonic stem cells. We can call them ES cells. That's 00:20:13.000 --> 00:20:17.000 what they're called in the trade, ES cells. We take them out. We can 00:20:17.000 --> 00:20:22.000 propagate them in culture. And then, what we can find is we'll 00:20:22.000 --> 00:20:26.000 put a genetic marker in those ES cells. Let's say we put in those 00:20:26.000 --> 00:20:31.000 embryonic stem cells the marker for the gene beta-galactosidase. 00:20:31.000 --> 00:20:35.000 And beta-galactosidase in the presence of a proper indicator, 00:20:35.000 --> 00:20:39.000 if you put a proper indicator and make a cell turn blue. 00:20:39.000 --> 00:20:43.000 So now we have an ES cell line that produces the beta-galactosidase 00:20:43.000 --> 00:20:47.000 enzyme. The beta-galactosidase enzyme beta-gal itself has no effect 00:20:47.000 --> 00:20:51.000 on the biology of the cells. It's only a marker. And now, 00:20:51.000 --> 00:20:55.000 we take those ES cells, and we inject them into another embryo, 00:20:55.000 --> 00:21:00.000 a wild type embryo that lacks this beta-gal marker. 00:21:00.000 --> 00:21:05.000 And what we can see is that we inject the ES cells into this 00:21:05.000 --> 00:21:10.000 blastocyst. The injected ES cells will now insinuate themselves, 00:21:10.000 --> 00:21:15.000 will now intrude into the massive cells in this embryo into which we 00:21:15.000 --> 00:21:20.000 injected the ES cells, and they will become part of the 00:21:20.000 --> 00:21:25.000 entire embryo genesis that follows. I.e. soon these foreign ES cells 00:21:25.000 --> 00:21:30.000 will weasel their way into this inner cell mass. 00:21:30.000 --> 00:21:34.000 And they will become established and become functionally equivalent to 00:21:34.000 --> 00:21:38.000 the inner cell mass cells that were resident there prior to this 00:21:38.000 --> 00:21:42.000 injection. And what you can do then is follow the subsequent fate of, 00:21:42.000 --> 00:21:46.000 in this case, a mouse. And what will happen often is that you can 00:21:46.000 --> 00:21:50.000 find blue cells all over the mouse sometimes in the paws, 00:21:50.000 --> 00:21:54.000 sometimes in the coat. Let's imagine that the hair would turn 00:21:54.000 --> 00:21:58.000 blue, which in fact is not the case. But let's imagine the hair would 00:21:58.000 --> 00:22:02.000 turn blue. So here's the mouse, 00:22:02.000 --> 00:22:06.000 happy because it's part of an important experiment. 00:22:06.000 --> 00:22:11.000 And what you'll sometimes see is that, well, remember that art was 00:22:11.000 --> 00:22:16.000 not my forte. Anyhow, here you might see stripes of blue 00:22:16.000 --> 00:22:20.000 cells on the skin. The hair won't turn blue actually, 00:22:20.000 --> 00:22:25.000 but the skin may if you give it the proper indicator. 00:22:25.000 --> 00:22:29.000 And what this indicates is that in this case, the cells that were 00:22:29.000 --> 00:22:34.000 injected into the blastocyst could become part of lineages which 00:22:34.000 --> 00:22:39.000 committed themselves to becoming skin cells. 00:22:39.000 --> 00:22:43.000 Or, the cells in the brain might be blue. Or, the cells in the gut 00:22:43.000 --> 00:22:47.000 might be blue. Or under certain conditions, 00:22:47.000 --> 00:22:51.000 the cells in the intestine might be blue. In telling you that, 00:22:51.000 --> 00:22:55.000 I mean to indicate that the cells that we injected into this 00:22:55.000 --> 00:23:00.000 blastocyst, which carry beta-gal were totipotent. 00:23:00.000 --> 00:23:04.000 They could create all the tissues of the mouse under the proper 00:23:04.000 --> 00:23:08.000 conditions. The proper conditions are obviously being put into this 00:23:08.000 --> 00:23:12.000 very special environment in which all kinds of differentiation 00:23:12.000 --> 00:23:16.000 inducing signals, which we don't really understand, 00:23:16.000 --> 00:23:20.000 can induce this cell to commit itself to enter into one or another 00:23:20.000 --> 00:23:24.000 differentiation lineage. And in principal, you can make a 00:23:24.000 --> 00:23:28.000 whole organism out of an ES cell. ES cell has as much plasticity, as 00:23:28.000 --> 00:23:32.000 much flexibility, as a fertilized egg. 00:23:32.000 --> 00:23:36.000 It has not yet lost the ability to make all the parts of the body. 00:23:36.000 --> 00:23:40.000 On some occasions, the ES cell will even get into the gonads of the 00:23:40.000 --> 00:23:45.000 mouse, which are down here somewhere. And if that's so, 00:23:45.000 --> 00:23:49.000 if the ES cell which you injected has been able to seed the formation 00:23:49.000 --> 00:23:54.000 of these cells down here, then what will happen is that either 00:23:54.000 --> 00:23:58.000 the sperm or the egg coming from this mouse will now transmit 00:23:58.000 --> 00:24:04.000 the blue gene. And now, in the next generation, 00:24:04.000 --> 00:24:10.000 all of the mice will inherit the blue beta-galactosidase gene in all 00:24:10.000 --> 00:24:16.000 of their cells because now this will have entered into the germ line. 00:24:16.000 --> 00:24:22.000 If these blue cells happen to colonize the testes, 00:24:22.000 --> 00:24:28.000 the ovary, or the testes, then these blue cells will become 00:24:28.000 --> 00:24:32.000 ancestors to the sperm or the egg. And now, in the next generation, 00:24:32.000 --> 00:24:36.000 mice will inherit a blue gene in all of their cells. 00:24:36.000 --> 00:24:40.000 And now this mouse is really happy because it's now part of an 00:24:40.000 --> 00:24:44.000 extremely important experiment because now all of its cells will 00:24:44.000 --> 00:24:47.000 become blue, having inherited them as part of the oocyte which led to 00:24:47.000 --> 00:24:51.000 its formation. In this kind of an animal, 00:24:51.000 --> 00:24:55.000 we call this animal a kind of a chimera. Chimera is a mythical 00:24:55.000 --> 00:24:59.000 beast which is, let's say, half human and half horse 00:24:59.000 --> 00:25:02.000 or something like that. Or a chimera means it has 00:25:02.000 --> 00:25:06.000 genetically different parts in it. That is not to say that these parts 00:25:06.000 --> 00:25:09.000 carrying the blue gene are necessarily defective, 00:25:09.000 --> 00:25:13.000 they're just genetically different, one from the other. But they can 00:25:13.000 --> 00:25:16.000 participate in embryogenesis in a fashion that's indistinguishable 00:25:16.000 --> 00:25:20.000 from the non-blue cells. They just do everything they're 00:25:20.000 --> 00:25:23.000 supposed to do, and they pretend as if they were in 00:25:23.000 --> 00:25:27.000 this embryo from the get go, from the very beginning, from the 00:25:27.000 --> 00:25:31.000 moment of fertilization. So they are totipotent. 00:25:31.000 --> 00:25:34.000 There's an alternative experiment you can do, and you can take the ES 00:25:34.000 --> 00:25:38.000 cells, and you can inject them under the skin of a mouse, 00:25:38.000 --> 00:25:41.000 let's say. So now, you're putting them in a very unfamiliar 00:25:41.000 --> 00:25:45.000 environment. And what you see then on many occasions is you can 00:25:45.000 --> 00:25:49.000 actually get a tumor. You can get what's called an 00:25:49.000 --> 00:26:00.000 embryonal carcinoma. 00:26:00.000 --> 00:26:03.000 Now you'll say, well, so what? That's not so 00:26:03.000 --> 00:26:07.000 interesting. But it's very interesting. Why? 00:26:07.000 --> 00:26:10.000 Because if you look at the genome of those embryonal carcinoma cells 00:26:10.000 --> 00:26:14.000 which we can call EC cells if you want, those cells are genetically 00:26:14.000 --> 00:26:17.000 full wild type. And yet, we're getting a tumor here. 00:26:17.000 --> 00:26:21.000 So, it means that these cells, which have been placed in a fully 00:26:21.000 --> 00:26:24.000 unfamiliar environment under the skin or in the belly of a mouse will 00:26:24.000 --> 00:26:28.000 begin to form a tumor. And in fact, they represent the 00:26:28.000 --> 00:26:31.000 only type of cell that we know about where a cell having a wild type 00:26:31.000 --> 00:26:35.000 genome can actually give you a tumor. 00:26:35.000 --> 00:26:39.000 As you sensed from our previous discussions, all other kinds of 00:26:39.000 --> 00:26:44.000 human cancer cells we know about have to have mutant genes in order 00:26:44.000 --> 00:26:48.000 for them to grow as a malignancy. These cells are fully wild type and 00:26:48.000 --> 00:26:53.000 can grow as an embryonal carcinoma. They are very primitive. These 00:26:53.000 --> 00:26:57.000 cells have quite a bit of autonomy. They're not so responsive to all 00:26:57.000 --> 00:27:02.000 the growth factors that normally are required by many cells throughout 00:27:02.000 --> 00:27:07.000 the soma of an animal throughout the tissues. 00:27:07.000 --> 00:27:10.000 So this allows us to begin to move on and ask other kinds of questions. 00:27:10.000 --> 00:27:14.000 For example, you can take these embryonal carcinoma cells. 00:27:14.000 --> 00:27:18.000 You put them in a Petri dish, and you can actually induce them to 00:27:18.000 --> 00:27:22.000 differentiate into different cell types in vitro. 00:27:22.000 --> 00:27:26.000 How can you do that? Well, we're just beginning to learn 00:27:26.000 --> 00:27:30.000 how to do that. We don't really know how to do that. 00:27:30.000 --> 00:27:34.000 But, if you give them the right cocktail of growth factors, 00:27:34.000 --> 00:27:38.000 they might begin to form muscle cells. If you give them another 00:27:38.000 --> 00:27:43.000 cocktail of growth factors, they might begin to give pancreatic 00:27:43.000 --> 00:27:47.000 eyelid cells that form insulin, or in this case cartilage cells. 00:27:47.000 --> 00:27:52.000 And presumably, the cocktail of growth factors you're providing each 00:27:52.000 --> 00:27:56.000 one of these cells with in vitro, i.e. in the Petri dish, is mimicking 00:27:56.000 --> 00:28:00.000 the growth factor environment that each of these cell types is 00:28:00.000 --> 00:28:04.000 experiencing within the embryo. In other words, 00:28:04.000 --> 00:28:08.000 cells in different parts of the embryo experience different 00:28:08.000 --> 00:28:12.000 combinations of growth factors that persuade them to commit themselves 00:28:12.000 --> 00:28:16.000 to becoming these kind of cells, these kind of cells, and these kind 00:28:16.000 --> 00:28:20.000 of cells. And therefore, one of the promises of embryonic 00:28:20.000 --> 00:28:24.000 stem cell research is the possibility of being able to 00:28:24.000 --> 00:28:28.000 regenerate different kinds of tissues in a fashion that I just 00:28:28.000 --> 00:28:32.000 showed you here. But this whole experiment in the 00:28:32.000 --> 00:28:36.000 case of human beings is ethically extremely controversial. 00:28:36.000 --> 00:28:40.000 Why? Because the experiment starts out making these ES cells here, 00:28:40.000 --> 00:28:44.000 and if we want to start out with an early embryo like this, 00:28:44.000 --> 00:28:48.000 start out with a blastocyst, in the case of a human blastocyst, 00:28:48.000 --> 00:28:52.000 this human blastocyst has the potential under the proper 00:28:52.000 --> 00:28:56.000 conditions of becoming a newborn human being. And therefore, 00:28:56.000 --> 00:29:00.000 we have this enormous ethical conflict in this country. 00:29:00.000 --> 00:29:04.000 Is this blastocyst already a human being? Can you already afford to 00:29:04.000 --> 00:29:08.000 truncate the life of this blastocyst at this stage of development, 00:29:08.000 --> 00:29:13.000 and in so doing, are you actually extinguishing human life, 00:29:13.000 --> 00:29:17.000 or is this organism, if you want to call it that, already still much too 00:29:17.000 --> 00:29:22.000 primitive to consider it to be equal to human life? 00:29:22.000 --> 00:29:26.000 And here, I would not, unlike my political views, 00:29:26.000 --> 00:29:31.000 be forward enough to venture an opinion because it's really 00:29:31.000 --> 00:29:35.000 something that no one really can argue about in any objective way. 00:29:35.000 --> 00:29:40.000 It's all a matter of opinion. Is this a human being already, 00:29:40.000 --> 00:29:44.000 or is it simply an inanimate cluster, a clump of cells? 00:29:44.000 --> 00:29:48.000 Now, in principal, how could we do this? 00:29:48.000 --> 00:29:52.000 How could we actually create this kind of tissue therapy? 00:29:52.000 --> 00:29:56.000 Because the fact is, as you get older, your tissues start falling 00:29:56.000 --> 00:30:00.000 apart. You haven't experienced that. 00:30:00.000 --> 00:30:04.000 But I have. And the fact is that even if you try to stay in shape, 00:30:04.000 --> 00:30:09.000 things just start falling apart. And the older you get, 00:30:09.000 --> 00:30:13.000 the more they fall apart. Even people who eat well, 00:30:13.000 --> 00:30:18.000 which I do, and exercise well, which I don't, even they fall apart. 00:30:18.000 --> 00:30:22.000 And so the question is, are there way of replacing and repairing 00:30:22.000 --> 00:30:27.000 tissue? And this would, in principal, represent one such 00:30:27.000 --> 00:30:31.000 strategy because it means that you could possibly inject replacement 00:30:31.000 --> 00:30:36.000 cells into an agent tissue and generate cells which could then 00:30:36.000 --> 00:30:40.000 restore and regeneration function which has somehow inevitably 00:30:40.000 --> 00:30:45.000 deteriorated over the decades. Well, that raises the question of 00:30:45.000 --> 00:30:50.000 how you can actually get a blastocyst, how you can make a 00:30:50.000 --> 00:30:56.000 blastocyst like this. To state an obvious thing which you 00:30:56.000 --> 00:31:01.000 might already have intuited, let's say you had such cells 00:31:01.000 --> 00:31:05.000 differentiated from various cell types that you want to inject into 00:31:05.000 --> 00:31:09.000 somebody's muscle or into their liver if they had diabetes and had 00:31:09.000 --> 00:31:13.000 lost their beta cells, or into their cartilage if they 00:31:13.000 --> 00:31:17.000 banged up their knee during basketball practice or something 00:31:17.000 --> 00:31:21.000 like that, or jogging, which is allegedly good for you. 00:31:21.000 --> 00:31:25.000 Who knows? How could you deal with that? Well, the fact is, 00:31:25.000 --> 00:31:29.000 let's imagine there were such a blastocyst which we'd produce in 00:31:29.000 --> 00:31:34.000 this fashion that we differentiated like this. 00:31:34.000 --> 00:31:37.000 OK, this is now the sequence of events. There's an important 00:31:37.000 --> 00:31:40.000 consideration we have to take into account, and that is if this 00:31:40.000 --> 00:31:44.000 blastocyst came from a different person than you, 00:31:44.000 --> 00:31:47.000 and we induced these cells to differentiate, 00:31:47.000 --> 00:31:51.000 and we injected those differentiation cells into your 00:31:51.000 --> 00:31:54.000 muscle, things wouldn't work. Why? Because these cells, if the 00:31:54.000 --> 00:31:57.000 blastocyst originated in a different person than yourself would be 00:31:57.000 --> 00:32:01.000 genetically different from you, and would be recognized as foreign 00:32:01.000 --> 00:32:04.000 tissue by your immune system. So even though you were getting an 00:32:04.000 --> 00:32:08.000 injection of cells which could regenerate your muscle perfectly 00:32:08.000 --> 00:32:11.000 well, those cells would never be given a chance to establish 00:32:11.000 --> 00:32:15.000 themselves and to thrive, and to reconstruct the tissue simple 00:32:15.000 --> 00:32:18.000 because the immune system would regard those cells as being 00:32:18.000 --> 00:32:22.000 foreigners and would go after them hammer and tongs trying to get rid 00:32:22.000 --> 00:32:25.000 of them in the same way it tries to get rid of all kinds of foreign 00:32:25.000 --> 00:32:29.000 invaders. I.e. the only way you could avoid it is 00:32:29.000 --> 00:32:33.000 if this blastocyst was genetically identical to you. 00:32:33.000 --> 00:32:37.000 But how can you make a blastocyst which is genetically identical to 00:32:37.000 --> 00:32:41.000 you? Well, I'm glad I asked that question. That's really the big 00:32:41.000 --> 00:32:45.000 challenge we have here because we don't want to create a situation 00:32:45.000 --> 00:32:49.000 where we have to restore somebody's tissues, but the only way we can 00:32:49.000 --> 00:32:53.000 restore them is to leave them immunosuppressed for the rest of 00:32:53.000 --> 00:32:57.000 their lives. When I say immunosuppressed I mean we have to 00:32:57.000 --> 00:33:01.000 prevent their immune system from attacking all of these cells that 00:33:01.000 --> 00:33:05.000 we've injected in them, these foreign cells, in the same way 00:33:05.000 --> 00:33:09.000 that we have to suppress the immune system of any person who has 00:33:09.000 --> 00:33:13.000 received a graft from another individual including often bone 00:33:13.000 --> 00:33:18.000 marrow transplants. In all cases, we have at least for a 00:33:18.000 --> 00:33:24.000 while to prevent their immune system from attacking and eliminating these 00:33:24.000 --> 00:33:29.000 engrafted cells. And this is where the whole strategy 00:33:29.000 --> 00:33:33.000 comes for the whole process of cloning. You may recall the case of 00:33:33.000 --> 00:33:37.000 Dolly about five years ago, and let's remember what happened 00:33:37.000 --> 00:33:41.000 here because this would a momentous experiment in mammalian biology. 00:33:41.000 --> 00:33:45.000 It asked the question, really, if you take cells from a somatic tissue, 00:33:45.000 --> 00:33:49.000 from here, or here, or here, are those cells, 00:33:49.000 --> 00:33:53.000 in principal, still totipotent, i.e. is the nucleus, is the genome 00:33:53.000 --> 00:33:57.000 of those cells totipotent, or has the genome, the chromosomal 00:33:57.000 --> 00:34:01.000 complement of cells in their cells undergone some kind of irrevocable, 00:34:01.000 --> 00:34:05.000 irreversible change, which precludes those cells from ever 00:34:05.000 --> 00:34:08.000 becoming totipotent? Well, in fact, 00:34:08.000 --> 00:34:12.000 if you take mammary epithelial cells from the breast of a human being or 00:34:12.000 --> 00:34:15.000 from the breast of a ewe and you put them into the blastocyst, 00:34:15.000 --> 00:34:18.000 nothing's going to happen. Those introduced mammary epithelial 00:34:18.000 --> 00:34:22.000 cells will not be able to establish themselves in the blastocyst. 00:34:22.000 --> 00:34:25.000 And, we will not be able to insinuate themselves amidst the 00:34:25.000 --> 00:34:29.000 inner cell mass, and they will not be able to 00:34:29.000 --> 00:34:33.000 participate in embryogenesis. So therefore, the epigenetic program 00:34:33.000 --> 00:34:38.000 in these somatic cells seems to be irrevocably set to preclude the 00:34:38.000 --> 00:34:44.000 participation of the already differentiated mammary epithelial 00:34:44.000 --> 00:34:49.000 cells in subsequent embryogenesis. Therefore, you could not do this 00:34:49.000 --> 00:34:54.000 experiment all over again of introducing cells into the inner 00:34:54.000 --> 00:35:00.000 cell mass as I just described over here, injecting them into this. 00:35:00.000 --> 00:35:04.000 But still, that doesn't answer the question. The issue is not whether 00:35:04.000 --> 00:35:08.000 the mammary epithelial cell is irrevocably committed to being a 00:35:08.000 --> 00:35:12.000 mammary epithelial cell. The issue: is its genome capable 00:35:12.000 --> 00:35:16.000 under the proper circumstances of becoming an early embryonic cell. 00:35:16.000 --> 00:35:21.000 And therefore, what was done is the following. One took mammary 00:35:21.000 --> 00:35:25.000 epithelial cells, in this case from Dolly's quote 00:35:25.000 --> 00:35:29.000 unquote "mother, one prepared nuclei from these 00:35:29.000 --> 00:35:33.000 cells, taking them out of the cytoplasm, and then one got 00:35:33.000 --> 00:35:38.000 fertilized eggs or eggs that have been induced to become. 00:35:38.000 --> 00:35:42.000 So here's an oocyte. An oocyte is an unfertilized egg. 00:35:42.000 --> 00:35:46.000 In principle, you can activate an oocyte by putting a sperm in, 00:35:46.000 --> 00:35:51.000 or in fact it's actually better if you take the oocyte and you fool it 00:35:51.000 --> 00:35:55.000 into thinking it's become fertilized by treating it with different salts, 00:35:55.000 --> 00:36:00.000 high potassium concentration, and so forth. 00:36:00.000 --> 00:36:04.000 And that will induce the egg to say I've been fertilized. 00:36:04.000 --> 00:36:09.000 I better start embryogenesis. But what you do in this case is the 00:36:09.000 --> 00:36:13.000 following. The egg has its own haploid nucleus here, 00:36:13.000 --> 00:36:18.000 and you can take a little needle. And, you suck that nucleus right 00:36:18.000 --> 00:36:23.000 out of the egg. So, you've enucleated it. 00:36:23.000 --> 00:36:27.000 That's what you've done, and now the egg is enucleate. 00:36:27.000 --> 00:36:32.000 It doesn't have a nucleus in it. But keep in mind, 00:36:32.000 --> 00:36:36.000 much of what happens during early embryogenesis is programmed not only 00:36:36.000 --> 00:36:41.000 by the genes but by all array of cytoplasmic proteins that are 00:36:41.000 --> 00:36:46.000 present throughout the egg, and which play critical roles in 00:36:46.000 --> 00:36:50.000 determining the subsequent course of embryogenesis. 00:36:50.000 --> 00:36:55.000 So now what you can do is you inject into this enucleate oocyte 00:36:55.000 --> 00:37:00.000 the nucleus of a mammary epithelial cell. 00:37:00.000 --> 00:37:05.000 The mammary epithelial cell is obviously highly differentiated. 00:37:05.000 --> 00:37:10.000 It's there to make milk. We'll call it an MEC if you want, 00:37:10.000 --> 00:37:15.000 and you put that in there, and under certain circumstances, 00:37:15.000 --> 00:37:20.000 and then you can treat this with a little bit of salt to mimic the 00:37:20.000 --> 00:37:25.000 physiological stimulus that comes after the sperm hits the egg. 00:37:25.000 --> 00:37:31.000 And now this egg will think it's been fertilized. 00:37:31.000 --> 00:37:35.000 And now it will begin to divide. But keep in mind, the genome of 00:37:35.000 --> 00:37:39.000 this quote unquote "unfertilized egg" has come not from the sperm and 00:37:39.000 --> 00:37:44.000 the preexisting nucleus of the egg. It's come because the nucleus has 00:37:44.000 --> 00:37:48.000 been injected from a mammary epithelial cell. 00:37:48.000 --> 00:37:52.000 An experience over the last 30 years had indicated that this will 00:37:52.000 --> 00:37:57.000 never work. But finally somebody in Scotland, a man named Ian Wilmouth 00:37:57.000 --> 00:38:01.000 tinkered enough with the conditions of these cells that he could 00:38:01.000 --> 00:38:05.000 actually get it to work not so often, maybe one, or two, 00:38:05.000 --> 00:38:10.000 or three times out of 100 tries. But on those conditions, 00:38:10.000 --> 00:38:14.000 this thing would begin to divide. The nucleus would begin to divide 00:38:14.000 --> 00:38:19.000 its diploid. Keep in mind that when a sperm comes into an egg, 00:38:19.000 --> 00:38:23.000 the egg is haploid. The sperm is haploid. Together they make a 00:38:23.000 --> 00:38:27.000 diploid genome. This introduced genomus diploid, 00:38:27.000 --> 00:38:32.000 and the question is, the critical question is, can the genes in this 00:38:32.000 --> 00:38:36.000 introduced nucleus totally rearrange their transcriptional program so 00:38:36.000 --> 00:38:41.000 that even though these genes might all be intact in terms of nucleotide 00:38:41.000 --> 00:38:45.000 sequence, can the entire infinitely complex array of DNA associated 00:38:45.000 --> 00:38:50.000 proteins, I.e. the proteins that constitute 00:38:50.000 --> 00:38:54.000 the chromatin which is not only the histones but also the transcription 00:38:54.000 --> 00:38:59.000 factors, the TF's, can they all jump on and jump off as 00:38:59.000 --> 00:39:03.000 they should to mimic and replicate the spectrum of transcription 00:39:03.000 --> 00:39:08.000 factors that is normally present shortly after an egg is fertilized? 00:39:08.000 --> 00:39:12.000 If they can do that, then this embryo can begin to 00:39:12.000 --> 00:39:16.000 replicate, and can ultimately develop into a complete embryo. 00:39:16.000 --> 00:39:20.000 If they can't, then embryogenesis is going to be truncated shortly 00:39:20.000 --> 00:39:24.000 thereafter maybe at the two cell stage, at the four cell stage, 00:39:24.000 --> 00:39:28.000 at the 16 cell stage, but shortly thereafter, not because of the DNA 00:39:28.000 --> 00:39:32.000 sequences being defective, but because the spectrum of 00:39:32.000 --> 00:39:36.000 transcription factors is up and down regulates certain genes is in fact 00:39:36.000 --> 00:39:40.000 not been able to re-assort themselves in response to what? 00:39:40.000 --> 00:39:44.000 Initially, in response to the signals coming from the cytoplasm 00:39:44.000 --> 00:39:48.000 because one might imagine, correctly so, that the nucleus in 00:39:48.000 --> 00:39:53.000 here is getting signals from the cytoplasm telling it, 00:39:53.000 --> 00:39:57.000 in effect, telling this nucleus, you should behave functionally as if 00:39:57.000 --> 00:40:01.000 you were the nucleus of a fertilized egg. In other words, 00:40:01.000 --> 00:40:05.000 the environment of proteins here is influencing the behavior 00:40:05.000 --> 00:40:09.000 of this nucleus. That goes backwards to our normal 00:40:09.000 --> 00:40:12.000 way of thinking because keep in mind our normal vectoral way of thinking 00:40:12.000 --> 00:40:14.000 is that the nucleus is influencing the cytoplasm. 00:40:14.000 --> 00:40:17.000 That's the direction of information flow. But here, 00:40:17.000 --> 00:40:20.000 we're having a different situation. Here, the cytoplasm is telling this 00:40:20.000 --> 00:40:23.000 injected nucleus, well, you used to be a mammary 00:40:23.000 --> 00:40:25.000 epithelial cell nucleus, but now you've got to take on a 00:40:25.000 --> 00:40:28.000 different job. And we're going to force you to do 00:40:28.000 --> 00:40:32.000 so. And to the extent that happens, 00:40:32.000 --> 00:40:36.000 then in principle, one can end up having a normal embryo. 00:40:36.000 --> 00:40:40.000 And, it happened actually on rare occasion that this worked. 00:40:40.000 --> 00:40:44.000 Here they used actual electrical stimulus rather than salt to get the 00:40:44.000 --> 00:40:48.000 nucleus to divide. This electrical stimulus, 00:40:48.000 --> 00:40:52.000 again, was to mimic the stimulus that the sperm entering the egg 00:40:52.000 --> 00:40:56.000 normally provides, thereby activating the egg and 00:40:56.000 --> 00:41:00.000 forcing the entire fertilized egg to proliferate. 00:41:00.000 --> 00:41:03.000 And so, once this starts developing, let's say, the blastocyst stage, 00:41:03.000 --> 00:41:07.000 here we have a blastocyst. You can see the inner cell mass 00:41:07.000 --> 00:41:11.000 once again here. This can be transferred into a 00:41:11.000 --> 00:41:14.000 pseudo-pregnant ewe. Pseudo-pregnant means you take a 00:41:14.000 --> 00:41:18.000 female ewe and you inject it with a series of hormones that persuade her 00:41:18.000 --> 00:41:22.000 reproductive system including prolactin, and progesterone, 00:41:22.000 --> 00:41:25.000 or estrogen, persuade her reproductive system, 00:41:25.000 --> 00:41:29.000 her uterus, that she's pregnant. You inject this early embryo into 00:41:29.000 --> 00:41:33.000 her, and this early embryo will then implant into the wall of her uterus 00:41:33.000 --> 00:41:37.000 and begin to develop. And if it all works well, 00:41:37.000 --> 00:41:41.000 you get a Dolly is born. You get a new sheep coming out of this. 00:41:41.000 --> 00:41:46.000 It doesn't work so often, one, two, three, four times after out of 00:41:46.000 --> 00:41:50.000 a hundred, and very often in the great majority of cases, 00:41:50.000 --> 00:41:55.000 there are mis-births, mis-carriages, which happen in the middle of 00:41:55.000 --> 00:41:59.000 embryogenesis. So, almost in the great majority of 00:41:59.000 --> 00:42:04.000 cases, this fails. Somehow, the reprogramming of this 00:42:04.000 --> 00:42:08.000 nucleus, which is what we're talking about, reprogramming it in terms of 00:42:08.000 --> 00:42:12.000 its transcriptional program, goes awry. And therefore, bad 00:42:12.000 --> 00:42:17.000 things happen. The fact that on a rare occasion 00:42:17.000 --> 00:42:21.000 gets and succeeds here already is extremely interesting because it 00:42:21.000 --> 00:42:25.000 proves irrevocably that the genome of a mammary epithelial cell is in 00:42:25.000 --> 00:42:30.000 principle competent to program entire embryonic development. 00:42:30.000 --> 00:42:34.000 And that means that during the development of Dolly's mother, 00:42:34.000 --> 00:42:39.000 we'll put her up here, as she developed from one cell into 1, 00:42:39.000 --> 00:42:44.000 00 or 10,000 billion cells, as that development occurred the DNA 00:42:44.000 --> 00:42:49.000 sequences that went from the fertilized egg to her didn't really 00:42:49.000 --> 00:42:53.000 change. I.e. the DNA sequences that were in one of her mammary 00:42:53.000 --> 00:42:58.000 epithelial cells were intact, and as capable in principle of 00:42:58.000 --> 00:43:03.000 launching the full-fledged development as would be 00:43:03.000 --> 00:43:08.000 a fertilized egg. And that is one of the proofs, 00:43:08.000 --> 00:43:12.000 by the way, that in fact differentiation does not involve, 00:43:12.000 --> 00:43:16.000 with some rare exceptions, alterations in DNA sequence. 00:43:16.000 --> 00:43:20.000 This, in turn, ends up being connected with the whole issue of 00:43:20.000 --> 00:43:24.000 embryonic stem cells. Let's say that I wanted to have my 00:43:24.000 --> 00:43:28.000 muscles regenerated, although they're still pretty good. 00:43:28.000 --> 00:43:33.000 So, I take a skin cell of mine, and I inject the skin cell. 00:43:33.000 --> 00:43:36.000 I take the nucleus out, and I inject it into an oocyte. 00:43:36.000 --> 00:43:40.000 And then I let the oocyte develop up to this stage. 00:43:40.000 --> 00:43:44.000 And I don't put the oocyte back into a sheep or another woman, 00:43:44.000 --> 00:43:48.000 although I could in principle. I actually take the cells out of the 00:43:48.000 --> 00:43:51.000 inner cell mass. Those are ES cells, 00:43:51.000 --> 00:43:55.000 and I begin to use them to regenerate my muscles to do this 00:43:55.000 --> 00:43:59.000 strategy. So, the cells are, in this case, 00:43:59.000 --> 00:44:03.000 not used for reproductive cloning, which is what this is here. 00:44:03.000 --> 00:44:07.000 They're used for therapeutic cloning, where instead of taking these cells 00:44:07.000 --> 00:44:11.000 and the ES cells and allowing them to form a whole embryo, 00:44:11.000 --> 00:44:15.000 they're used to form a cell line of ES cells from the blastocyst from 00:44:15.000 --> 00:44:19.000 the inner cell mass. What we talked about before, 00:44:19.000 --> 00:44:23.000 here you see the blastocyst with the inner cell mass here. 00:44:23.000 --> 00:44:27.000 You see it again. But now, rather than allowing this blastocyst 00:44:27.000 --> 00:44:31.000 to continue development, we simply extract cells from it and 00:44:31.000 --> 00:44:34.000 again create ES cells. I could create therefore in 00:44:34.000 --> 00:44:38.000 principle, ES cells, which are genetically identical to 00:44:38.000 --> 00:44:41.000 all the cells in my body, and any one of you could as well. 00:44:41.000 --> 00:44:44.000 And here, there's not only one, but there's two ethical complications. 00:44:44.000 --> 00:44:48.000 First of all, here we're starting human life with the intent of 00:44:48.000 --> 00:44:51.000 truncating it very early, and secondly, where are the oocytes 00:44:51.000 --> 00:44:54.000 going to come from? Well, you could say you can get 00:44:54.000 --> 00:44:58.000 them from some women, but producing oocytes from a human 00:44:58.000 --> 00:45:02.000 female isn't so easy. You have to inject her with all 00:45:02.000 --> 00:45:06.000 kinds of stimulatory hormones, choreogramatatrophic hormones. It's 00:45:06.000 --> 00:45:10.000 an unpleasant procedure. Usually women are paid $5, 00:45:10.000 --> 00:45:14.000 00 or $10,000 to produce some oocytes. Well, 00:45:14.000 --> 00:45:18.000 you say, that's OK, but is that OK? I don't know. 00:45:18.000 --> 00:45:22.000 Is it OK to pay a woman to donate her oocytes to make herself into an 00:45:22.000 --> 00:45:26.000 oocyte factory? I don't know. You have to judge. 00:45:26.000 --> 00:45:30.000 I think there's arguments both for and against it. 00:45:30.000 --> 00:45:34.000 Clearly, any one of us would be extraordinarily naïve if we thought 00:45:34.000 --> 00:45:39.000 that this was a procedure which had no ethical encumbrances in it. 00:45:39.000 --> 00:45:43.000 And, you have to think about them for yourself. Still, 00:45:43.000 --> 00:45:48.000 the potentials are enormous, and therefore the question exists. 00:45:48.000 --> 00:45:53.000 Will there be ways in the future of taking differentiated cells from 00:45:53.000 --> 00:45:57.000 one's tissue, and in fact using them in these ways to make ES cells 00:45:57.000 --> 00:46:02.000 without having to go through an oocyte, and without having the 00:46:02.000 --> 00:46:06.000 potential of creating human life. The alternative to this has been to 00:46:06.000 --> 00:46:10.000 do the following, to go into our normal tissues and 00:46:10.000 --> 00:46:14.000 pull out adult stem cells. What do I mean by adult stem cells? 00:46:14.000 --> 00:46:18.000 These are not stem cells that are totipotent. These are stem cells 00:46:18.000 --> 00:46:22.000 which are in my muscles and regenerating muscle mass, 00:46:22.000 --> 00:46:26.000 which happens believe it or not. These are stem cells which might be 00:46:26.000 --> 00:46:30.000 in my skin and are continually regenerating skin cells. 00:46:30.000 --> 00:46:34.000 Keep in mind that in the maintenance of all our normal tissues there are 00:46:34.000 --> 00:46:38.000 stem cells whose configuration can formally be depicted like this with 00:46:38.000 --> 00:46:42.000 the transit amplifying cells we talked about before. 00:46:42.000 --> 00:46:46.000 And maybe, if one took the stem cells out of an adult tissue right 00:46:46.000 --> 00:46:50.000 here, if we had a way of extracting them, those could be propagated in 00:46:50.000 --> 00:46:54.000 vitro, and then injected back in. Those are so-called adult stem 00:46:54.000 --> 00:46:58.000 cells. And the individuals who are against 00:46:58.000 --> 00:47:02.000 this kind of manipulation of human embryos and so forth say that adult 00:47:02.000 --> 00:47:06.000 stem cells are really the solution. You take stem cells out of a 00:47:06.000 --> 00:47:10.000 person's tissue, you expand them. Ex vivo means out 00:47:10.000 --> 00:47:14.000 of the body, in vitro, and then you use them. You inject 00:47:14.000 --> 00:47:19.000 them into somebody's tissue to regenerate their tissue. 00:47:19.000 --> 00:47:23.000 There's only one problem with that. It's ethically far less encumbered 00:47:23.000 --> 00:47:27.000 obviously, but it doesn't work that well. In fact, 00:47:27.000 --> 00:47:31.000 some people think it hardly works at all, that the exceptions are really 00:47:31.000 --> 00:47:36.000 rather far and few between. And so, this issue will long be or 00:47:36.000 --> 00:47:41.000 continue to be debated. But it obviously represents a very 00:47:41.000 --> 00:47:46.000 new and exciting area of biomedical research. And interestingly enough, 00:47:46.000 --> 00:47:52.000 it impinges as well in a fully unexpected way on cancer because 00:47:52.000 --> 00:47:57.000 this whole paradigm of stem cells, it turns out, also applies to cancer 00:47:57.000 --> 00:48:01.000 cells. If you were to have asked me two or 00:48:01.000 --> 00:48:05.000 three years ago, what did the cells in the tumor look 00:48:05.000 --> 00:48:09.000 like? I would draw a picture like this, that these are a series of 00:48:09.000 --> 00:48:12.000 exponentially growing cells so that all the cancer cells, 00:48:12.000 --> 00:48:16.000 all the neoplastic cells in the tumor mass are biologically 00:48:16.000 --> 00:48:20.000 equivalent to one another. They all have the same mutant 00:48:20.000 --> 00:48:23.000 genome, and they all are capable of multiplying exponentially. 00:48:23.000 --> 00:48:27.000 But it turns out that work in the Matavoidic system on Matevoidic 00:48:27.000 --> 00:48:31.000 tumors like leukemias, and now on breast cancers, 00:48:31.000 --> 00:48:36.000 yields a very different results, because it turns out that the way 00:48:36.000 --> 00:48:44.000 that the tumors are organized looks like this. The tumors also are 00:48:44.000 --> 00:48:52.000 organized in this hierarchical array just like normal tissue. 00:48:52.000 --> 00:49:00.000 How do we know that? Again, I'm glad I asked that question. 00:49:00.000 --> 00:49:04.000 Because if you take these cells out of the tumor and put them in another 00:49:04.000 --> 00:49:08.000 mouse, let's say, you get a new tumor. 00:49:08.000 --> 00:49:13.000 These cells are tumorogenic, I.e. they concede a new tumor. 00:49:13.000 --> 00:49:17.000 If you take these cells out of the tumor, they have the same mutant 00:49:17.000 --> 00:49:21.000 genome. They constitute the bulk, the vast mass of the cancer cells in 00:49:21.000 --> 00:49:26.000 a tumor. You put these into a mouse, and they're non-tumorogenic. 00:49:26.000 --> 00:49:30.000 And, in some kinds of tumors, the tumorogenic cells can represent 00:49:30.000 --> 00:49:35.000 only 1 or 2% of the total mass of cancer cells in the tumor. 00:49:35.000 --> 00:49:38.000 And from this, we begin to realize that you look 00:49:38.000 --> 00:49:42.000 inside tumors: the tumors deviate minimally from the organization of 00:49:42.000 --> 00:49:46.000 normal tissue. They also depend on self-renewing 00:49:46.000 --> 00:49:50.000 stem cells which can make transit amplifying cells and can give end 00:49:50.000 --> 00:49:53.000 stage cells, which although they're neoplastic, have many of the 00:49:53.000 --> 00:49:57.000 differentiated characteristics of the normal tissue from which they 00:49:57.000 --> 00:50:01.000 arose. And this has enormous implications for, 00:50:01.000 --> 00:50:05.000 for example, therapies against tumors. 00:50:05.000 --> 00:50:09.000 If you ask somebody, how do you develop and how you judge 00:50:09.000 --> 00:50:13.000 the success of an anticancer treatment? You talk to somebody 00:50:13.000 --> 00:50:17.000 like from the pharmaceutical industry. And let's say that's easy. 00:50:17.000 --> 00:50:21.000 If you have a new drug, and that drug reduces the mass of a 00:50:21.000 --> 00:50:26.000 tumor by 50%, that means that you've done something really good. 00:50:26.000 --> 00:50:30.000 But let's look what's going on here. If these cells are 99% of the tumor 00:50:30.000 --> 00:50:34.000 in terms of the mass and these cells are 1% of the tumor, 00:50:34.000 --> 00:50:38.000 let's say you've invented a new drug which wipes out all of these cells 00:50:38.000 --> 00:50:42.000 but doesn't touch these cells. The bulk of the tumor has shrunk and 00:50:42.000 --> 00:50:46.000 everybody will say, eureka, we've succeeded in curing 00:50:46.000 --> 00:50:50.000 cancer. But keep in mind that the self-renewing capacity of the tumor 00:50:50.000 --> 00:50:53.000 rests in these cells. And if these cells are allowed to 00:50:53.000 --> 00:50:57.000 survive, then they'll start proliferating again and regenerate 00:50:57.000 --> 00:51:01.000 the entire tumor mass. And you won't really know that you 00:51:01.000 --> 00:51:05.000 had any success because these cells look like all the other tumor cells 00:51:05.000 --> 00:51:10.000 under the microscope. But biologically, they're very 00:51:10.000 --> 00:51:14.000 different. And therefore, the future of cancer therapy, 00:51:14.000 --> 00:51:19.000 and it will take five or ten years to do this, has to begin to focus on 00:51:19.000 --> 00:51:23.000 getting rid of these self-renewing stem cells which create this 00:51:23.000 --> 00:51:28.000 enormous regenerative capacity on the part of tumors. 00:51:28.000 --> 00:51:32.000 See you next Monday. Have a great vacation. 00:51:32.000 --> 00:51:37.000 Eat much turkey, and get some exercise, and don't smoke.