WEBVTT 00:00:16.960 --> 00:00:19.660 ADAM MARTIN: So to start out, I just 00:00:19.660 --> 00:00:24.770 wanted to mention we talk a lot about dogma in the lab. 00:00:24.770 --> 00:00:27.240 So we talk about dogma, right? 00:00:27.240 --> 00:00:30.100 There's the central dogma, which is DNA. 00:00:33.820 --> 00:00:36.910 Information flows from DNA to RNA to protein. 00:00:39.880 --> 00:00:44.630 I'm going to describe another dogma, if you will, 00:00:44.630 --> 00:00:50.740 which is that life starts as sort of a fertilized egg. 00:00:50.740 --> 00:00:52.180 So you get a fertilized egg. 00:00:58.420 --> 00:01:02.470 And this fertilized egg, which you see in the video up here, 00:01:02.470 --> 00:01:04.060 undergoes development. 00:01:08.880 --> 00:01:11.680 And as part of that development there 00:01:11.680 --> 00:01:14.860 is what is known as differentiation, 00:01:14.860 --> 00:01:19.940 where cells acquire more specialized cell types. 00:01:19.940 --> 00:01:23.250 So there's development and differentiation. 00:01:26.390 --> 00:01:29.830 And this results in adult cell types that 00:01:29.830 --> 00:01:31.450 are known as differentiated. 00:01:35.400 --> 00:01:40.870 So it results in differentiated/adult cell types 00:01:40.870 --> 00:01:43.570 with specialized functions. 00:01:43.570 --> 00:01:47.260 And you see just a few of them up on the slide above. 00:01:47.260 --> 00:01:51.670 There are, in fact, thousands of different sort of cell types 00:01:51.670 --> 00:01:54.340 that humans have. 00:01:54.340 --> 00:02:01.450 But the dogma, at least up until fairly recently-- 00:02:01.450 --> 00:02:08.620 in the past couple of decades or so or at least before 1960, 00:02:08.620 --> 00:02:10.030 1950s-- 00:02:10.030 --> 00:02:12.070 is that this is you unidirectional, 00:02:12.070 --> 00:02:15.760 that basically the development goes in one direction, where 00:02:15.760 --> 00:02:19.720 you go from the fertilized egg to the differentiated adult 00:02:19.720 --> 00:02:22.370 cell types. 00:02:22.370 --> 00:02:25.570 And so this state down here-- 00:02:25.570 --> 00:02:29.180 the differentiated adult cell types are fairly stable, right? 00:02:29.180 --> 00:02:31.040 I mean, if you look at your neighbor 00:02:31.040 --> 00:02:32.930 you see they don't have muscle growing 00:02:32.930 --> 00:02:34.820 on the outside of their bodies. 00:02:34.820 --> 00:02:38.060 Their skin cells stay skin cells. 00:02:38.060 --> 00:02:42.440 And that is a relatively stable state. 00:02:42.440 --> 00:02:43.670 OK. 00:02:43.670 --> 00:02:48.650 Now, last week you learned about a contradiction 00:02:48.650 --> 00:02:58.190 to the central dogma, which is the behavior of retroviruses, 00:02:58.190 --> 00:03:01.880 which have reverse transcriptase, which 00:03:01.880 --> 00:03:07.640 can sort of go backwards up this pathway, where you can get DNA 00:03:07.640 --> 00:03:09.720 from RNA. 00:03:09.720 --> 00:03:10.220 OK. 00:03:10.220 --> 00:03:12.770 And today, we're going to talk about how 00:03:12.770 --> 00:03:19.460 you can sort of overcome this you unidirectional path here. 00:03:19.460 --> 00:03:22.880 And this is going to be what we will call reprogramming. 00:03:29.240 --> 00:03:33.860 But first, I want to talk about this differentiation process 00:03:33.860 --> 00:03:36.380 because it's important for us to understand this 00:03:36.380 --> 00:03:40.140 before we get into the reprogramming event. 00:03:40.140 --> 00:03:44.840 And so the fertilized egg is capable of producing all 00:03:44.840 --> 00:03:46.430 of these different cell types. 00:03:46.430 --> 00:03:47.390 OK. 00:03:47.390 --> 00:03:53.300 So the fertilized egg will be known as totipotent, 00:03:53.300 --> 00:04:00.185 meaning it has the potential to form all cell types. 00:04:04.470 --> 00:04:08.450 And then over development, this potency 00:04:08.450 --> 00:04:12.350 goes down such that when cells differentiate 00:04:12.350 --> 00:04:15.080 into their final adult form there 00:04:15.080 --> 00:04:19.160 is a much more limited repertoire of cell types 00:04:19.160 --> 00:04:22.100 that the cell can go into. 00:04:22.100 --> 00:04:23.870 And one way of thinking about this 00:04:23.870 --> 00:04:27.830 is that if you think of this marble run here, 00:04:27.830 --> 00:04:31.740 the totipotent state is right at the top here, right? 00:04:31.740 --> 00:04:33.710 If I put a marble in the top here, 00:04:33.710 --> 00:04:37.580 it has the potential of going into any of these three 00:04:37.580 --> 00:04:39.290 sort of different fates. 00:04:39.290 --> 00:04:41.470 OK. 00:04:41.470 --> 00:04:45.060 So this is the totipotent state up at the top. 00:04:45.060 --> 00:04:46.570 And you'll see these marbles will 00:04:46.570 --> 00:04:50.960 be able to go into any of these three different states here. 00:04:50.960 --> 00:04:51.460 OK. 00:04:51.460 --> 00:04:54.820 But you see how that marble went down on this path. 00:04:54.820 --> 00:04:57.250 Some marbles go down in this path. 00:04:57.250 --> 00:05:01.720 And so you can think of cells going through development 00:05:01.720 --> 00:05:06.130 is sort of getting funneled into these distinct paths. 00:05:06.130 --> 00:05:09.050 And in this case, it's kind of random. 00:05:09.050 --> 00:05:11.680 But in development, it relies on signaling 00:05:11.680 --> 00:05:14.470 between cells and interaction between cells 00:05:14.470 --> 00:05:15.985 in the multicellular organism. 00:05:20.350 --> 00:05:22.840 So as an example, I want to tell you 00:05:22.840 --> 00:05:26.390 about early mammalian development 00:05:26.390 --> 00:05:29.680 and the differentiation of cell types 00:05:29.680 --> 00:05:31.255 in the early mammalian embryo. 00:05:38.060 --> 00:05:38.560 OK. 00:05:38.560 --> 00:05:43.312 So we start with an oocyte. 00:05:43.312 --> 00:05:45.910 That's the female gamete. 00:05:45.910 --> 00:05:47.500 And the male gamete, the sperm. 00:05:51.550 --> 00:05:53.920 So these can come together to form the zygote. 00:05:57.740 --> 00:06:01.810 If I draw a little circle in the middle, it's a nucleus. 00:06:01.810 --> 00:06:05.440 And this zygote is totipotent. 00:06:12.220 --> 00:06:15.970 And the embryo undergoes cleavage divisions, 00:06:15.970 --> 00:06:16.990 which I'll show here. 00:06:16.990 --> 00:06:21.040 You see how that zygote divided into two cells. 00:06:21.040 --> 00:06:22.870 Now it's going to divide into four. 00:06:22.870 --> 00:06:26.185 And it'll keep dividing till it's 16 probably. 00:06:28.990 --> 00:06:32.980 So you get cleavage divisions that 00:06:32.980 --> 00:06:36.332 generate more than one cell. 00:06:36.332 --> 00:06:37.540 You start with a single cell. 00:06:37.540 --> 00:06:40.450 The cleavage divisions give you more than one cell. 00:06:40.450 --> 00:06:43.300 And what you see up here now is a stage 00:06:43.300 --> 00:06:48.950 known as the blastocyst or the blastula. 00:06:48.950 --> 00:06:52.460 You can see there's a hollow sort of inner fluid-filled area 00:06:52.460 --> 00:06:53.240 here. 00:06:53.240 --> 00:06:55.070 And you see there are cells around 00:06:55.070 --> 00:06:57.530 that fluid-filled cavity. 00:06:57.530 --> 00:07:00.200 And there's a thickening on one side of the embryo. 00:07:00.200 --> 00:07:02.060 OK. 00:07:02.060 --> 00:07:03.710 So I'm going to draw that out here. 00:07:07.500 --> 00:07:08.000 Sorry. 00:07:08.000 --> 00:07:09.650 Mine is a little flat up here. 00:07:09.650 --> 00:07:12.910 It should be perfectly spherical. 00:07:12.910 --> 00:07:18.260 And there is this thickening on one side of the blastula, which 00:07:18.260 --> 00:07:22.610 is a bunch of cells that are kind of interior 00:07:22.610 --> 00:07:25.370 on the inside of the embryo. 00:07:25.370 --> 00:07:29.375 These cells on the interior are known as the inner cell mass. 00:07:35.240 --> 00:07:39.230 And these cells are now restricted 00:07:39.230 --> 00:07:41.150 in what they can become. 00:07:41.150 --> 00:07:43.815 These cells will become the embryo proper. 00:07:46.520 --> 00:07:48.240 So they'll become the cell types that 00:07:48.240 --> 00:07:52.520 will be a part of the fetus. 00:07:52.520 --> 00:07:56.315 These cells on the outside are known as the trophoblast cells. 00:08:00.620 --> 00:08:05.700 And these cells will form part of the placenta-- 00:08:05.700 --> 00:08:08.340 the embryonic portion of the placenta. 00:08:08.340 --> 00:08:12.690 So they form part of the placenta. 00:08:12.690 --> 00:08:14.780 And they're important for this embryo 00:08:14.780 --> 00:08:17.690 being able to implant into the uterine wall. 00:08:21.410 --> 00:08:25.550 So if we think about-- this is the first example 00:08:25.550 --> 00:08:31.610 of differentiation in the early mammalian embryo 00:08:31.610 --> 00:08:34.340 because you can see, based on what I said here, 00:08:34.340 --> 00:08:36.600 these cells are becoming restricted 00:08:36.600 --> 00:08:39.559 in what they can become. 00:08:39.559 --> 00:08:41.510 So this is kind of like the first branch 00:08:41.510 --> 00:08:44.520 point in differentiation. 00:08:44.520 --> 00:08:48.590 So we get some more marbles going down here. 00:08:51.780 --> 00:08:52.280 All right. 00:08:52.280 --> 00:08:55.250 So this would be the sort of stage right 00:08:55.250 --> 00:08:58.580 before the blastula, before differentiation. 00:08:58.580 --> 00:09:00.050 And you can see these marbles-- 00:09:00.050 --> 00:09:04.010 when I let them, they will either go this way 00:09:04.010 --> 00:09:06.770 and become one fate, or they'll go the other way 00:09:06.770 --> 00:09:15.220 and be sort of directed down another potential fate. 00:09:15.220 --> 00:09:17.970 There we go. 00:09:17.970 --> 00:09:21.100 So this is the first sort of branch point, if you will, 00:09:21.100 --> 00:09:25.190 in fate determination for the mammalian embryo. 00:09:25.190 --> 00:09:30.380 And so this branch point here is different from this 00:09:30.380 --> 00:09:33.530 because once the marble goes one way or the other, 00:09:33.530 --> 00:09:37.320 it's restricted in what fate it can become. 00:09:37.320 --> 00:09:37.820 OK. 00:09:37.820 --> 00:09:44.750 So these cells here are not totipotent 00:09:44.750 --> 00:09:48.950 because they can't form the trophoblast. 00:09:48.950 --> 00:09:52.580 They can't form the part of the placenta needed by the embryo. 00:09:52.580 --> 00:09:55.160 But they do form the embryo proper. 00:09:55.160 --> 00:09:58.130 So they are still capable of many fates. 00:09:58.130 --> 00:10:03.650 And so these cells are known as pluripotent, which 00:10:03.650 --> 00:10:08.850 means capable of many things 00:10:08.850 --> 00:10:09.350 OK. 00:10:09.350 --> 00:10:11.390 And so a type of cell type, which 00:10:11.390 --> 00:10:19.610 I'm sure you've heard about, is called Embryonic Stem cells, 00:10:19.610 --> 00:10:20.840 or ES cells. 00:10:23.660 --> 00:10:27.890 These ES cells are derived from the inner cell mass. 00:10:27.890 --> 00:10:29.210 So these would be-- 00:10:29.210 --> 00:10:31.670 if they were taken out of the blastula-- 00:10:31.670 --> 00:10:35.160 sorry-- this is called the blastula-- 00:10:35.160 --> 00:10:39.390 if they are taken from the blastula and cultured in vitro, 00:10:39.390 --> 00:10:41.690 these would be sort of embryonic stem cells 00:10:41.690 --> 00:10:46.470 that can be propagated. 00:10:46.470 --> 00:10:54.050 And they form the embryo proper, so these are pluripotent stem 00:10:54.050 --> 00:11:00.680 cells that can basically-- they are still capable of forming 00:11:00.680 --> 00:11:04.310 sort of any of the cell types in the embryo-- 00:11:04.310 --> 00:11:14.450 capable of forming embryonic cell types. 00:11:28.630 --> 00:11:31.090 Now let's look at the next slide. 00:11:31.090 --> 00:11:32.860 So this branch point-- 00:11:32.860 --> 00:11:36.280 this first branch point sort of in development 00:11:36.280 --> 00:11:40.360 is associated with changes in gene expression. 00:11:40.360 --> 00:11:43.900 So there are changes in gene expression. 00:11:46.740 --> 00:11:50.050 And you're seeing one up on the slide above. 00:11:50.050 --> 00:11:55.420 The slide above is a fixed blastula. 00:11:55.420 --> 00:11:57.820 And all of the nuclei in the blastula 00:11:57.820 --> 00:11:59.380 are stained with green. 00:11:59.380 --> 00:12:02.460 But there is one gene that's stained in red. 00:12:02.460 --> 00:12:04.240 It's called Oct4. 00:12:04.240 --> 00:12:06.520 And this is a transcription factor that 00:12:06.520 --> 00:12:09.130 marks sort of pluripotency. 00:12:09.130 --> 00:12:12.790 And you can see how it's expressed specifically 00:12:12.790 --> 00:12:15.610 in these cells of the inner cell mass, which 00:12:15.610 --> 00:12:21.160 are what the embryonic stem cells are derived from. 00:12:21.160 --> 00:12:25.340 So there are clearly changes in gene expression. 00:12:25.340 --> 00:12:28.840 And one question you might have is whether or not 00:12:28.840 --> 00:12:34.210 these cells that are going to form the embryo proper, 00:12:34.210 --> 00:12:37.150 whether they have lost information such that they're 00:12:37.150 --> 00:12:42.080 unable to form the part of the placenta. 00:12:42.080 --> 00:12:46.870 And you can also ask this for an adult cell. 00:12:46.870 --> 00:12:52.450 Has an adult cell in your body-- has it lost gene content such 00:12:52.450 --> 00:12:56.590 that it's unable to make an entire organism? 00:12:56.590 --> 00:12:58.210 I'm going to tell you the answer. 00:12:58.210 --> 00:13:00.250 I want you to think about what experiment 00:13:00.250 --> 00:13:03.390 would allow you to sort of determine the answer. 00:13:03.390 --> 00:13:08.140 But I'm going to tell you the answer is that there is not 00:13:08.140 --> 00:13:13.030 a loss of gene content. 00:13:13.030 --> 00:13:16.270 But this differentiation process is 00:13:16.270 --> 00:13:18.700 due to changes in gene expression 00:13:18.700 --> 00:13:21.190 for the vast majority of our cell types. 00:13:23.830 --> 00:13:26.780 So let's say you wanted to determine whether or not 00:13:26.780 --> 00:13:31.900 a differentiated cell had lost some sort of capability 00:13:31.900 --> 00:13:35.590 to regenerate an entire organism. 00:13:35.590 --> 00:13:38.410 What might be some type of experiment you could do? 00:13:43.240 --> 00:13:45.477 Brett. 00:13:45.477 --> 00:13:47.435 AUDIENCE: Creating conditions that will perhaps 00:13:47.435 --> 00:13:52.660 be similar to what embryonic stem cells or perhaps 00:13:52.660 --> 00:13:56.730 [INAUDIBLE] factors that allow for you to change 00:13:56.730 --> 00:13:58.794 the expression back to more than one impact 00:13:58.794 --> 00:14:01.272 to see if that would actually change it. 00:14:01.272 --> 00:14:02.230 ADAM MARTIN: All right. 00:14:02.230 --> 00:14:02.730 Great. 00:14:02.730 --> 00:14:05.530 So Brett had two really good points, I think. 00:14:05.530 --> 00:14:09.670 The first is to try to sort of reproduce conditions 00:14:09.670 --> 00:14:11.320 of pluripotency. 00:14:11.320 --> 00:14:14.710 Or you can even try to reproduce conditions of totipotency, 00:14:14.710 --> 00:14:15.670 right? 00:14:15.670 --> 00:14:18.580 In which case you'd want to sort of take 00:14:18.580 --> 00:14:21.310 the genetic material of the somatic cell 00:14:21.310 --> 00:14:23.680 and sort of put it back in this situation where 00:14:23.680 --> 00:14:29.110 it's present sort of in the cytoplasm of the zygote. 00:14:29.110 --> 00:14:31.660 And the other point that Brett made 00:14:31.660 --> 00:14:37.240 is to try to maybe express something that would induce 00:14:37.240 --> 00:14:40.180 this type of pluripotent state. 00:14:40.180 --> 00:14:43.300 If we knew exactly what the genes were 00:14:43.300 --> 00:14:46.450 that create this pluripotent state, 00:14:46.450 --> 00:14:49.750 maybe we could just express those genes 00:14:49.750 --> 00:14:53.590 and regenerate sort of a cell that 00:14:53.590 --> 00:14:59.290 moves from way down here all the way up to the top again. 00:14:59.290 --> 00:15:01.750 And, actually, what Brett suggested 00:15:01.750 --> 00:15:05.770 were the two experiments that won these two folks the Nobel 00:15:05.770 --> 00:15:08.980 Prize in 2012. 00:15:08.980 --> 00:15:13.600 So in 2012, the Nobel Prize was awarded jointly 00:15:13.600 --> 00:15:18.400 to Sir John Gurdon, right here, who incidentally 00:15:18.400 --> 00:15:24.220 has the best hair of all Nobel laureates, and also 00:15:24.220 --> 00:15:27.550 Shinya Yamanaka. 00:15:27.550 --> 00:15:30.940 And so these two folks were awarded the Nobel Prize 00:15:30.940 --> 00:15:36.220 for being able to show that mature differentiated cells can 00:15:36.220 --> 00:15:41.620 be reprogrammed to become pluripotent again, 00:15:41.620 --> 00:15:45.340 which basically gives us the conclusion that there's not 00:15:45.340 --> 00:15:50.950 a loss of gene content during the differentiation process. 00:15:50.950 --> 00:15:53.170 And the work from Yamanaka showed us 00:15:53.170 --> 00:15:57.100 several genes whose expression is critical to induce 00:15:57.100 --> 00:16:01.000 this type of reprogramming. 00:16:01.000 --> 00:16:05.560 And their work spanned from frogs-- 00:16:05.560 --> 00:16:10.030 so John Gurdon worked on development of frogs, 00:16:10.030 --> 00:16:13.810 specifically Xenopus laevis, which you've seen before. 00:16:13.810 --> 00:16:21.290 Shinya Yamanaka's work involved mice and also human cell lines. 00:16:21.290 --> 00:16:23.930 So I'm going to tell you about their experiments 00:16:23.930 --> 00:16:26.810 and how this-- sort of demonstrated this conclusion 00:16:26.810 --> 00:16:29.067 here. 00:16:29.067 --> 00:16:30.650 And the first thing I want to show you 00:16:30.650 --> 00:16:34.430 is an experiment, which is very simple conceptually. 00:16:34.430 --> 00:16:36.980 Technically it's very complicated, which 00:16:36.980 --> 00:16:42.680 is if you were able to take a nucleus from an adult cell 00:16:42.680 --> 00:16:45.020 that's differentiated, could you get 00:16:45.020 --> 00:16:49.100 it to change by introducing it back into the egg 00:16:49.100 --> 00:16:50.780 cell or a zygote cell? 00:16:55.490 --> 00:17:01.070 So this experiment involves having an oocyte, 00:17:01.070 --> 00:17:06.470 but in this case an oocyte where the nucleus has been removed. 00:17:06.470 --> 00:17:08.780 So you take an enucleated oocyte. 00:17:13.790 --> 00:17:16.849 And want to know if the cytoplasm of this oocyte 00:17:16.849 --> 00:17:19.220 is somehow special that would allow 00:17:19.220 --> 00:17:23.819 it to reprogram the nucleus of a differentiated cell. 00:17:23.819 --> 00:17:26.869 And so you could suck up the nucleus 00:17:26.869 --> 00:17:30.290 of a differentiated cell. 00:17:30.290 --> 00:17:35.560 So a somatic cell is another way to say differentiated. 00:17:35.560 --> 00:17:37.205 So it's a somatic cell nucleus. 00:17:41.860 --> 00:17:43.970 And the experiment conceptually then 00:17:43.970 --> 00:17:46.970 is to just take this somatic cell nucleus that you've 00:17:46.970 --> 00:17:51.890 sucked up and inject it into an oocyte without a nucleus 00:17:51.890 --> 00:17:54.320 and see if the cytoplasm of this oocyte 00:17:54.320 --> 00:17:58.970 is somehow able to change the properties of the nucleus 00:17:58.970 --> 00:18:03.000 such that it now is in an undifferentiated state. 00:18:03.000 --> 00:18:11.990 So you generate oocyte now with the somatic cell nucleus. 00:18:15.980 --> 00:18:19.220 And the question is whether the somatic cell nucleus 00:18:19.220 --> 00:18:22.880 is capable of generating the diversity of cell types 00:18:22.880 --> 00:18:26.550 that are normally present in an organism. 00:18:26.550 --> 00:18:30.170 So you could then let this go and develop. 00:18:30.170 --> 00:18:33.920 You could let it develop into a blastula, which 00:18:33.920 --> 00:18:34.730 I'm drawing here. 00:18:40.490 --> 00:18:43.540 So you could generate a blastula. 00:18:43.540 --> 00:18:47.990 And this will be a way to get embryonic stem cells derived 00:18:47.990 --> 00:18:51.870 from sort of this type of nucleus. 00:18:51.870 --> 00:18:58.190 But you could also let it grow into an entire organism. 00:18:58.190 --> 00:19:00.260 But this organism would be genetically 00:19:00.260 --> 00:19:05.570 identical to whatever organism donated this nucleus. 00:19:05.570 --> 00:19:08.210 So because you're duplicating an organism, 00:19:08.210 --> 00:19:11.190 that is known as reproductive cloning. 00:19:11.190 --> 00:19:20.930 So this is reproductive cloning, if you 00:19:20.930 --> 00:19:22.235 go all the way to the organism. 00:19:26.090 --> 00:19:31.763 I'll show you just a little video on the nuclear transfer. 00:19:31.763 --> 00:19:32.430 [VIDEO PLAYBACK] 00:19:32.430 --> 00:19:36.140 - You see now that this drilling pipe head is going to suck-- 00:19:36.140 --> 00:19:38.250 drill a little hole into the membrane. 00:19:38.250 --> 00:19:41.823 You can maybe see a little bit of the hole right 00:19:41.823 --> 00:19:42.740 here at the next part. 00:19:42.740 --> 00:19:43.323 [END PLAYBACK] 00:19:43.323 --> 00:19:46.077 ADAM MARTIN: He's talking about this pipette here. 00:19:46.077 --> 00:19:48.410 The embryo is being held with another pipette over here. 00:19:48.410 --> 00:19:49.077 [VIDEO PLAYBACK] 00:19:49.077 --> 00:19:51.090 - You can see a bit of the hole. 00:19:51.090 --> 00:19:54.030 And now the pipette's going to go in and remove the nucleus. 00:19:54.030 --> 00:19:55.910 And if you look carefully in the pipette, 00:19:55.910 --> 00:19:57.950 you'll see a line in the nucleus, which are 00:19:57.950 --> 00:20:00.530 all the chromosomes lined up. 00:20:00.530 --> 00:20:03.140 So the nucleus is going to be squirted out now because we 00:20:03.140 --> 00:20:04.470 don't need it anymore. 00:20:04.470 --> 00:20:06.140 And then we have an enucleated egg. 00:20:09.010 --> 00:20:11.690 Now, the next step is to take a set of eggs like that-- 00:20:11.690 --> 00:20:13.130 and I'll show you two-- 00:20:13.130 --> 00:20:15.980 and then transfer into them a nucleus 00:20:15.980 --> 00:20:19.200 from another kind of cell, a fully-differentiated somatic 00:20:19.200 --> 00:20:19.700 cell. 00:20:19.700 --> 00:20:23.370 So here, the enucleated egg is set on the side. 00:20:23.370 --> 00:20:26.460 And it's held by this holding pipette on the left. 00:20:26.460 --> 00:20:28.870 There's drilling the little hole in the membrane. 00:20:28.870 --> 00:20:30.260 Here we go in. 00:20:30.260 --> 00:20:32.080 Here comes the nucleus from the right. 00:20:32.080 --> 00:20:33.380 [END PLAYBACK] 00:20:33.380 --> 00:20:34.310 ADAM MARTIN: That's from a somatic zone. 00:20:34.310 --> 00:20:34.977 [VIDEO PLAYBACK] 00:20:34.977 --> 00:20:37.550 - And these pipettes are operated 00:20:37.550 --> 00:20:39.373 with a piezoelectric device. 00:20:39.373 --> 00:20:40.790 So you can't see it here, but it's 00:20:40.790 --> 00:20:43.610 like a little jackhammer going very quickly, 00:20:43.610 --> 00:20:47.000 (TRILLING SOUND) like Woody the Woodpecker, getting in there. 00:20:47.000 --> 00:20:47.583 [END PLAYBACK] 00:20:47.583 --> 00:20:49.083 ADAM MARTIN: I don't know if I would 00:20:49.083 --> 00:20:51.120 have used Woody the Woodpecker as an analogy, 00:20:51.120 --> 00:20:53.710 but that's basically the idea. 00:20:53.710 --> 00:20:57.110 So you can take a nucleus from a somatic cell 00:20:57.110 --> 00:21:01.340 and transplant it, if you will, into an oocyte and then 00:21:01.340 --> 00:21:06.380 determine whether you can get either a blastula 00:21:06.380 --> 00:21:13.430 or an entire organism from that differentiated cell's nucleus. 00:21:13.430 --> 00:21:17.030 So this is one result from Sir John Gurdon. 00:21:17.030 --> 00:21:21.710 And what the experiment was, in this case, 00:21:21.710 --> 00:21:27.900 was to take oocytes, or eggs, from this wild-type laevis frog 00:21:27.900 --> 00:21:35.040 and to transplant nuclei from donor frogs that are albino. 00:21:35.040 --> 00:21:37.610 So you can see this is an elegant experiment in that you 00:21:37.610 --> 00:21:40.160 can sort of track the origin of the nucleus, 00:21:40.160 --> 00:21:44.120 because it's genetically marked with this albino sort 00:21:44.120 --> 00:21:46.460 of phenotype. 00:21:46.460 --> 00:21:50.740 So you're getting a nuclei from-- 00:21:50.740 --> 00:21:55.060 you're getting nuclei from albino tadpoles. 00:21:55.060 --> 00:21:59.060 So these are differentiated cell nuclei. 00:21:59.060 --> 00:22:03.110 And they're transplanted into wild-type donor eggs. 00:22:03.110 --> 00:22:05.540 So normally, the wild-type frog would 00:22:05.540 --> 00:22:09.380 reproduce frogs that look like it-- that are non-albino. 00:22:09.380 --> 00:22:13.160 But in this experiment, Gurdon and his lab 00:22:13.160 --> 00:22:17.120 were able to get frogs that were albino. 00:22:17.120 --> 00:22:25.310 So these would be sort of clones of whatever albino tadpoles 00:22:25.310 --> 00:22:28.010 they got the nuclei from. 00:22:28.010 --> 00:22:32.720 So you see, in this case, it's the origin of the nucleus that 00:22:32.720 --> 00:22:36.920 is determining sort of the phenotype of these frogs. 00:22:36.920 --> 00:22:40.100 So that allowed them to show that the nucleus is getting 00:22:40.100 --> 00:22:43.610 reprogrammed from this albino tadpole 00:22:43.610 --> 00:22:47.180 and is able to still generate all of the cell types 00:22:47.180 --> 00:22:50.400 present in a normal organism. 00:22:50.400 --> 00:22:50.900 Yes. 00:22:50.900 --> 00:22:51.400 Brett. 00:22:51.400 --> 00:22:53.795 AUDIENCE: So this is an unfertilized egg 00:22:53.795 --> 00:22:55.280 that they were taking from the-- 00:22:55.280 --> 00:22:56.330 ADAM MARTIN: Yes. 00:22:56.330 --> 00:22:58.742 AUDIENCE: And so they're extracting all this DNA, 00:22:58.742 --> 00:23:01.732 then putting in a full set of DNA from the albinos. 00:23:01.732 --> 00:23:02.440 ADAM MARTIN: Yep. 00:23:02.440 --> 00:23:04.148 AUDIENCE: It would go on to differentiate 00:23:04.148 --> 00:23:05.090 a full set of DNA? 00:23:05.090 --> 00:23:05.840 ADAM MARTIN: Yeah. 00:23:05.840 --> 00:23:06.830 AUDIENCE: OK. 00:23:06.830 --> 00:23:07.580 ADAM MARTIN: Yeah. 00:23:07.580 --> 00:23:09.410 So, yeah, they're-- and actually, 00:23:09.410 --> 00:23:13.610 it was taking unfertilized eggs, which is the biggest trick. 00:23:13.610 --> 00:23:16.290 I think people had tried to do this in frogs before, 00:23:16.290 --> 00:23:19.850 and it failed because they were using fertilized eggs. 00:23:19.850 --> 00:23:24.650 And there's something about sort of the oocyte development that 00:23:24.650 --> 00:23:28.950 makes it better at reprogramming the nucleus. 00:23:28.950 --> 00:23:29.450 OK. 00:23:29.450 --> 00:23:31.730 So that's with frogs. 00:23:31.730 --> 00:23:35.150 So that experiment was actually done in the late 1950s, 00:23:35.150 --> 00:23:38.120 published in the early 1960s. 00:23:38.120 --> 00:23:43.520 And so it took another 40 years or so for the first mammal 00:23:43.520 --> 00:23:44.660 to be cloned. 00:23:44.660 --> 00:23:47.810 And you guys probably weren't even born yet. 00:23:47.810 --> 00:23:49.460 But for those of us who are older, 00:23:49.460 --> 00:23:51.080 we remember this because there was 00:23:51.080 --> 00:23:54.710 a big brouhaha over Dolly the sheep, which 00:23:54.710 --> 00:23:57.530 was the first cloned mammal. 00:23:57.530 --> 00:23:58.925 I believe that's Dolly there. 00:24:01.490 --> 00:24:04.850 So this is-- 00:24:04.850 --> 00:24:06.590 I'm not sure which one is Dolly. 00:24:06.590 --> 00:24:10.550 They're both the same type of sheep. 00:24:10.550 --> 00:24:14.270 So this was done by Ian Wilmut and his group. 00:24:14.270 --> 00:24:16.550 One thing I want to point out about this 00:24:16.550 --> 00:24:19.460 is it's incredibly inefficient. 00:24:19.460 --> 00:24:22.220 If you look over here, this Dolly 00:24:22.220 --> 00:24:27.700 resulted from over 400 oocytes having 00:24:27.700 --> 00:24:31.600 this sort of nuclear transplant take place. 00:24:31.600 --> 00:24:34.610 So it's not a very efficient process. 00:24:34.610 --> 00:24:39.100 And so that lack of efficiency is due to the fact 00:24:39.100 --> 00:24:44.320 that the nucleus is resisting getting reprogrammed. 00:24:44.320 --> 00:24:51.900 And this is a graph from one of John Gurdon's sort of-- 00:24:51.900 --> 00:24:55.600 he wrote a review article on sort of reprogramming 00:24:55.600 --> 00:24:57.550 after he won the Nobel Prize. 00:24:57.550 --> 00:24:58.870 This is from that. 00:24:58.870 --> 00:25:02.230 And what's plotted is sort of the frequency 00:25:02.230 --> 00:25:05.080 in which transfers results in sort 00:25:05.080 --> 00:25:08.950 of a differentiated organism. 00:25:08.950 --> 00:25:12.370 And what's on the x-axis is the stage 00:25:12.370 --> 00:25:15.970 of the cell that's used to transplant. 00:25:15.970 --> 00:25:20.230 And so what you see is that over the course of differentiation 00:25:20.230 --> 00:25:22.450 it gets harder and harder for the nucleus 00:25:22.450 --> 00:25:27.790 to get reprogrammed to create sort of all the cell types that 00:25:27.790 --> 00:25:32.670 are normally present in an adult animal. 00:25:32.670 --> 00:25:35.790 So nuclei do become more restricted 00:25:35.790 --> 00:25:39.360 in their ability to be reprogrammed over development. 00:25:39.360 --> 00:25:43.200 But the fact that any of them are able to be reprogrammed 00:25:43.200 --> 00:25:45.060 suggests that when they are-- 00:25:45.060 --> 00:25:47.030 that during differentiation there 00:25:47.030 --> 00:25:49.080 is not a loss of gene content. 00:25:52.550 --> 00:25:55.560 And John Gurdon has done a lot to characterize 00:25:55.560 --> 00:25:57.430 sort of the changes in the nucleus that 00:25:57.430 --> 00:26:01.150 happen during differentiation which sort of resist 00:26:01.150 --> 00:26:06.220 this reprogramming by the oocyte cytoplasm. 00:26:09.680 --> 00:26:10.180 All right. 00:26:10.180 --> 00:26:12.910 So mechanistically, what's happening? 00:26:12.910 --> 00:26:20.560 Well, one big-- one of the people 00:26:20.560 --> 00:26:24.970 who really showed what's going on there is Shinya Yamanaka. 00:26:24.970 --> 00:26:35.750 And what his work shows is that you can take just a few genes-- 00:26:35.750 --> 00:26:37.810 it turns out it is four-- 00:26:37.810 --> 00:26:41.380 and you can induce reprogramming by just 00:26:41.380 --> 00:26:47.380 expressing these four genes in an adult differentiated cell. 00:26:47.380 --> 00:26:56.640 So in this case you have a differentiated cell. 00:27:03.530 --> 00:27:06.600 And what he initially used we're fibroblasts, which are 00:27:06.600 --> 00:27:09.360 a type of differentiated cell. 00:27:09.360 --> 00:27:11.940 And what Yamanaka showed is that you 00:27:11.940 --> 00:27:15.900 can express four transcription factors, one 00:27:15.900 --> 00:27:18.660 of them being this factor here, Oct4, 00:27:18.660 --> 00:27:21.380 which is expressed in the inner cell mass 00:27:21.380 --> 00:27:23.655 and was shown to be involved in sort 00:27:23.655 --> 00:27:29.700 of maintaining pluripotency of these inner cell mass cells. 00:27:29.700 --> 00:27:34.060 So you could take Oct4 plus another transcription factor, 00:27:34.060 --> 00:27:37.290 which is important for pluripotency, plus two 00:27:37.290 --> 00:27:45.008 others and c-Myc. 00:27:45.008 --> 00:27:45.800 These are the four. 00:27:45.800 --> 00:27:48.320 So these are all transcription factors. 00:27:48.320 --> 00:27:50.930 And what they found is if you express these four 00:27:50.930 --> 00:27:55.550 transcription factors in a differentiated cell, 00:27:55.550 --> 00:28:01.280 you could get it to revert to a more pluripotent state. 00:28:01.280 --> 00:28:12.230 So this results in what is known as Induced Pluripotent Stem 00:28:12.230 --> 00:28:19.010 cells, or IPS cells, for short. 00:28:24.210 --> 00:28:28.380 And then like embryonic stem cells, 00:28:28.380 --> 00:28:33.930 here these cells can give rise to different cell types. 00:28:33.930 --> 00:28:39.030 And so one of the goals of this field of developmental biology 00:28:39.030 --> 00:28:45.720 and cell reprogramming is to use this technology 00:28:45.720 --> 00:28:49.630 to replace cells that are lost in patients. 00:28:49.630 --> 00:28:52.170 So this is known as regenerative medicine. 00:29:01.680 --> 00:29:06.840 And the idea of the theory of regenerative medicine 00:29:06.840 --> 00:29:15.420 is to take ES cells or induced pluripotent stem 00:29:15.420 --> 00:29:24.840 cells from patients and to be able to culture them in vitro-- 00:29:24.840 --> 00:29:26.205 so culture in vitro-- 00:29:32.140 --> 00:29:36.570 and then to be able to direct these cultured cells 00:29:36.570 --> 00:29:40.410 into different cell fates in order 00:29:40.410 --> 00:29:46.020 to use these cells possibly to transplant them back 00:29:46.020 --> 00:29:51.390 into an individual where maybe these cell types are dying. 00:29:51.390 --> 00:29:58.860 So you could then differentiate these cells using 00:29:58.860 --> 00:30:02.490 certain biochemical signals that you can add to the media 00:30:02.490 --> 00:30:05.370 in order to create different cell types, 00:30:05.370 --> 00:30:13.200 like neurons, muscle, maybe skin. 00:30:13.200 --> 00:30:20.010 And one example of this was shown by Shinya Yamanaka, where 00:30:20.010 --> 00:30:27.710 what they did was to take human fibroblast cells, 00:30:27.710 --> 00:30:29.900 put them back in the pluripotent state 00:30:29.900 --> 00:30:32.840 by expressing those four factors, 00:30:32.840 --> 00:30:35.270 and then get these cells to differentiate 00:30:35.270 --> 00:30:40.470 into cardiac tissue, so cardiac cells. 00:30:40.470 --> 00:30:41.480 Here's a movie. 00:30:41.480 --> 00:30:46.160 So these are from adult fibroblast cells. 00:30:46.160 --> 00:30:48.590 But now you can see they're beating sort 00:30:48.590 --> 00:30:51.380 of like a cardiac tissue would. 00:30:51.380 --> 00:30:56.940 So these were made into IPS stem cells, cultured in vitro, 00:30:56.940 --> 00:31:00.310 and then directed into a cardiac muscle fate. 00:31:03.500 --> 00:31:07.650 So the goal would then to be use these 00:31:07.650 --> 00:31:21.620 and to transplant them back into a patient, such 00:31:21.620 --> 00:31:23.390 that if a patient had, let's say, 00:31:23.390 --> 00:31:26.600 a neurodegenerative disease and was lacking 00:31:26.600 --> 00:31:28.700 a certain type of neuron, you could then 00:31:28.700 --> 00:31:33.170 take cells, start them on the path to neuronal development, 00:31:33.170 --> 00:31:36.200 and then transplant them back into that patient. 00:31:36.200 --> 00:31:42.260 And if these cells are derived from the DNA of that patient, 00:31:42.260 --> 00:31:46.130 then there won't be transplant rejection. 00:31:46.130 --> 00:31:49.460 Because what regulates transplant rejection 00:31:49.460 --> 00:31:54.530 is the major histocompatiblity complex locus. 00:31:54.530 --> 00:31:56.450 And it's polymorphic. 00:31:56.450 --> 00:31:59.810 But if you're taking a nucleus from the patient 00:31:59.810 --> 00:32:03.110 and then causing the cells to differentiate in vitro, 00:32:03.110 --> 00:32:05.120 you're taking the exact same-- 00:32:05.120 --> 00:32:07.340 you're taking clonal cells and putting them back 00:32:07.340 --> 00:32:09.380 in the same patient, such that they 00:32:09.380 --> 00:32:11.765 won't be rejected, ideally. 00:32:15.870 --> 00:32:16.370 All right. 00:32:16.370 --> 00:32:20.390 Now I want to try something with the remainder of our time. 00:32:20.390 --> 00:32:22.070 I want a group-- 00:32:22.070 --> 00:32:22.760 all right. 00:32:22.760 --> 00:32:24.320 Everyone on this side of the room 00:32:24.320 --> 00:32:27.530 over, I want you over here. 00:32:27.530 --> 00:32:29.880 And everyone on this side of the room, 00:32:29.880 --> 00:32:32.140 I want you on this side over here. 00:32:32.140 --> 00:32:33.200 OK. 00:32:33.200 --> 00:32:36.125 Maybe you guys can go over here so that we're more balanced. 00:32:39.360 --> 00:32:40.860 We're going to have a little debate. 00:32:51.340 --> 00:32:52.240 You can sit down. 00:32:52.240 --> 00:32:52.760 It's OK. 00:32:59.000 --> 00:33:01.150 Be in a position to talk to each other. 00:33:01.150 --> 00:33:04.354 I want you guys talking to each other. 00:33:04.354 --> 00:33:07.060 That was the goal of putting you close together. 00:33:07.060 --> 00:33:07.560 OK. 00:33:10.770 --> 00:33:12.480 So in the past couple weeks, there's 00:33:12.480 --> 00:33:16.200 been a little bit of a stir because there's 00:33:16.200 --> 00:33:19.800 a researcher in China who has claimed to have made 00:33:19.800 --> 00:33:22.470 the first gene-edited baby. 00:33:22.470 --> 00:33:24.570 Perhaps you have heard of this. 00:33:24.570 --> 00:33:27.630 You probably have because it's been all over the news. 00:33:27.630 --> 00:33:29.580 So I want you guys-- 00:33:29.580 --> 00:33:30.970 let's see. 00:33:30.970 --> 00:33:34.360 I want us to debate whether we should-- 00:33:34.360 --> 00:33:36.990 what are the advantages or disadvantages 00:33:36.990 --> 00:33:40.750 of gene editing or human cloning. 00:33:40.750 --> 00:33:42.420 You could talk about both. 00:33:42.420 --> 00:33:45.960 And then I want you guys to be able to present them to me. 00:33:45.960 --> 00:33:48.690 I'll write them down, and we'll have a discussion about it. 00:33:56.799 --> 00:34:00.220 Guys, continue this discussion sort of outside class. 00:34:00.220 --> 00:34:01.720 I think it's really interesting. 00:34:01.720 --> 00:34:03.670 And it's going to be something that you're 00:34:03.670 --> 00:34:07.710 going to hear a lot about in the coming years, I'm sure.