1 00:00:16,960 --> 00:00:19,660 ADAM MARTIN: So to start out, I just 2 00:00:19,660 --> 00:00:24,770 wanted to mention we talk a lot about dogma in the lab. 3 00:00:24,770 --> 00:00:27,240 So we talk about dogma, right? 4 00:00:27,240 --> 00:00:30,100 There's the central dogma, which is DNA. 5 00:00:33,820 --> 00:00:36,910 Information flows from DNA to RNA to protein. 6 00:00:39,880 --> 00:00:44,630 I'm going to describe another dogma, if you will, 7 00:00:44,630 --> 00:00:50,740 which is that life starts as sort of a fertilized egg. 8 00:00:50,740 --> 00:00:52,180 So you get a fertilized egg. 9 00:00:58,420 --> 00:01:02,470 And this fertilized egg, which you see in the video up here, 10 00:01:02,470 --> 00:01:04,060 undergoes development. 11 00:01:08,880 --> 00:01:11,680 And as part of that development there 12 00:01:11,680 --> 00:01:14,860 is what is known as differentiation, 13 00:01:14,860 --> 00:01:19,940 where cells acquire more specialized cell types. 14 00:01:19,940 --> 00:01:23,250 So there's development and differentiation. 15 00:01:26,390 --> 00:01:29,830 And this results in adult cell types that 16 00:01:29,830 --> 00:01:31,450 are known as differentiated. 17 00:01:35,400 --> 00:01:40,870 So it results in differentiated/adult cell types 18 00:01:40,870 --> 00:01:43,570 with specialized functions. 19 00:01:43,570 --> 00:01:47,260 And you see just a few of them up on the slide above. 20 00:01:47,260 --> 00:01:51,670 There are, in fact, thousands of different sort of cell types 21 00:01:51,670 --> 00:01:54,340 that humans have. 22 00:01:54,340 --> 00:02:01,450 But the dogma, at least up until fairly recently-- 23 00:02:01,450 --> 00:02:08,620 in the past couple of decades or so or at least before 1960, 24 00:02:08,620 --> 00:02:10,030 1950s-- 25 00:02:10,030 --> 00:02:12,070 is that this is you unidirectional, 26 00:02:12,070 --> 00:02:15,760 that basically the development goes in one direction, where 27 00:02:15,760 --> 00:02:19,720 you go from the fertilized egg to the differentiated adult 28 00:02:19,720 --> 00:02:22,370 cell types. 29 00:02:22,370 --> 00:02:25,570 And so this state down here-- 30 00:02:25,570 --> 00:02:29,180 the differentiated adult cell types are fairly stable, right? 31 00:02:29,180 --> 00:02:31,040 I mean, if you look at your neighbor 32 00:02:31,040 --> 00:02:32,930 you see they don't have muscle growing 33 00:02:32,930 --> 00:02:34,820 on the outside of their bodies. 34 00:02:34,820 --> 00:02:38,060 Their skin cells stay skin cells. 35 00:02:38,060 --> 00:02:42,440 And that is a relatively stable state. 36 00:02:42,440 --> 00:02:43,670 OK. 37 00:02:43,670 --> 00:02:48,650 Now, last week you learned about a contradiction 38 00:02:48,650 --> 00:02:58,190 to the central dogma, which is the behavior of retroviruses, 39 00:02:58,190 --> 00:03:01,880 which have reverse transcriptase, which 40 00:03:01,880 --> 00:03:07,640 can sort of go backwards up this pathway, where you can get DNA 41 00:03:07,640 --> 00:03:09,720 from RNA. 42 00:03:09,720 --> 00:03:10,220 OK. 43 00:03:10,220 --> 00:03:12,770 And today, we're going to talk about how 44 00:03:12,770 --> 00:03:19,460 you can sort of overcome this you unidirectional path here. 45 00:03:19,460 --> 00:03:22,880 And this is going to be what we will call reprogramming. 46 00:03:29,240 --> 00:03:33,860 But first, I want to talk about this differentiation process 47 00:03:33,860 --> 00:03:36,380 because it's important for us to understand this 48 00:03:36,380 --> 00:03:40,140 before we get into the reprogramming event. 49 00:03:40,140 --> 00:03:44,840 And so the fertilized egg is capable of producing all 50 00:03:44,840 --> 00:03:46,430 of these different cell types. 51 00:03:46,430 --> 00:03:47,390 OK. 52 00:03:47,390 --> 00:03:53,300 So the fertilized egg will be known as totipotent, 53 00:03:53,300 --> 00:04:00,185 meaning it has the potential to form all cell types. 54 00:04:04,470 --> 00:04:08,450 And then over development, this potency 55 00:04:08,450 --> 00:04:12,350 goes down such that when cells differentiate 56 00:04:12,350 --> 00:04:15,080 into their final adult form there 57 00:04:15,080 --> 00:04:19,160 is a much more limited repertoire of cell types 58 00:04:19,160 --> 00:04:22,100 that the cell can go into. 59 00:04:22,100 --> 00:04:23,870 And one way of thinking about this 60 00:04:23,870 --> 00:04:27,830 is that if you think of this marble run here, 61 00:04:27,830 --> 00:04:31,740 the totipotent state is right at the top here, right? 62 00:04:31,740 --> 00:04:33,710 If I put a marble in the top here, 63 00:04:33,710 --> 00:04:37,580 it has the potential of going into any of these three 64 00:04:37,580 --> 00:04:39,290 sort of different fates. 65 00:04:39,290 --> 00:04:41,470 OK. 66 00:04:41,470 --> 00:04:45,060 So this is the totipotent state up at the top. 67 00:04:45,060 --> 00:04:46,570 And you'll see these marbles will 68 00:04:46,570 --> 00:04:50,960 be able to go into any of these three different states here. 69 00:04:50,960 --> 00:04:51,460 OK. 70 00:04:51,460 --> 00:04:54,820 But you see how that marble went down on this path. 71 00:04:54,820 --> 00:04:57,250 Some marbles go down in this path. 72 00:04:57,250 --> 00:05:01,720 And so you can think of cells going through development 73 00:05:01,720 --> 00:05:06,130 is sort of getting funneled into these distinct paths. 74 00:05:06,130 --> 00:05:09,050 And in this case, it's kind of random. 75 00:05:09,050 --> 00:05:11,680 But in development, it relies on signaling 76 00:05:11,680 --> 00:05:14,470 between cells and interaction between cells 77 00:05:14,470 --> 00:05:15,985 in the multicellular organism. 78 00:05:20,350 --> 00:05:22,840 So as an example, I want to tell you 79 00:05:22,840 --> 00:05:26,390 about early mammalian development 80 00:05:26,390 --> 00:05:29,680 and the differentiation of cell types 81 00:05:29,680 --> 00:05:31,255 in the early mammalian embryo. 82 00:05:38,060 --> 00:05:38,560 OK. 83 00:05:38,560 --> 00:05:43,312 So we start with an oocyte. 84 00:05:43,312 --> 00:05:45,910 That's the female gamete. 85 00:05:45,910 --> 00:05:47,500 And the male gamete, the sperm. 86 00:05:51,550 --> 00:05:53,920 So these can come together to form the zygote. 87 00:05:57,740 --> 00:06:01,810 If I draw a little circle in the middle, it's a nucleus. 88 00:06:01,810 --> 00:06:05,440 And this zygote is totipotent. 89 00:06:12,220 --> 00:06:15,970 And the embryo undergoes cleavage divisions, 90 00:06:15,970 --> 00:06:16,990 which I'll show here. 91 00:06:16,990 --> 00:06:21,040 You see how that zygote divided into two cells. 92 00:06:21,040 --> 00:06:22,870 Now it's going to divide into four. 93 00:06:22,870 --> 00:06:26,185 And it'll keep dividing till it's 16 probably. 94 00:06:28,990 --> 00:06:32,980 So you get cleavage divisions that 95 00:06:32,980 --> 00:06:36,332 generate more than one cell. 96 00:06:36,332 --> 00:06:37,540 You start with a single cell. 97 00:06:37,540 --> 00:06:40,450 The cleavage divisions give you more than one cell. 98 00:06:40,450 --> 00:06:43,300 And what you see up here now is a stage 99 00:06:43,300 --> 00:06:48,950 known as the blastocyst or the blastula. 100 00:06:48,950 --> 00:06:52,460 You can see there's a hollow sort of inner fluid-filled area 101 00:06:52,460 --> 00:06:53,240 here. 102 00:06:53,240 --> 00:06:55,070 And you see there are cells around 103 00:06:55,070 --> 00:06:57,530 that fluid-filled cavity. 104 00:06:57,530 --> 00:07:00,200 And there's a thickening on one side of the embryo. 105 00:07:00,200 --> 00:07:02,060 OK. 106 00:07:02,060 --> 00:07:03,710 So I'm going to draw that out here. 107 00:07:07,500 --> 00:07:08,000 Sorry. 108 00:07:08,000 --> 00:07:09,650 Mine is a little flat up here. 109 00:07:09,650 --> 00:07:12,910 It should be perfectly spherical. 110 00:07:12,910 --> 00:07:18,260 And there is this thickening on one side of the blastula, which 111 00:07:18,260 --> 00:07:22,610 is a bunch of cells that are kind of interior 112 00:07:22,610 --> 00:07:25,370 on the inside of the embryo. 113 00:07:25,370 --> 00:07:29,375 These cells on the interior are known as the inner cell mass. 114 00:07:35,240 --> 00:07:39,230 And these cells are now restricted 115 00:07:39,230 --> 00:07:41,150 in what they can become. 116 00:07:41,150 --> 00:07:43,815 These cells will become the embryo proper. 117 00:07:46,520 --> 00:07:48,240 So they'll become the cell types that 118 00:07:48,240 --> 00:07:52,520 will be a part of the fetus. 119 00:07:52,520 --> 00:07:56,315 These cells on the outside are known as the trophoblast cells. 120 00:08:00,620 --> 00:08:05,700 And these cells will form part of the placenta-- 121 00:08:05,700 --> 00:08:08,340 the embryonic portion of the placenta. 122 00:08:08,340 --> 00:08:12,690 So they form part of the placenta. 123 00:08:12,690 --> 00:08:14,780 And they're important for this embryo 124 00:08:14,780 --> 00:08:17,690 being able to implant into the uterine wall. 125 00:08:21,410 --> 00:08:25,550 So if we think about-- this is the first example 126 00:08:25,550 --> 00:08:31,610 of differentiation in the early mammalian embryo 127 00:08:31,610 --> 00:08:34,340 because you can see, based on what I said here, 128 00:08:34,340 --> 00:08:36,600 these cells are becoming restricted 129 00:08:36,600 --> 00:08:39,559 in what they can become. 130 00:08:39,559 --> 00:08:41,510 So this is kind of like the first branch 131 00:08:41,510 --> 00:08:44,520 point in differentiation. 132 00:08:44,520 --> 00:08:48,590 So we get some more marbles going down here. 133 00:08:51,780 --> 00:08:52,280 All right. 134 00:08:52,280 --> 00:08:55,250 So this would be the sort of stage right 135 00:08:55,250 --> 00:08:58,580 before the blastula, before differentiation. 136 00:08:58,580 --> 00:09:00,050 And you can see these marbles-- 137 00:09:00,050 --> 00:09:04,010 when I let them, they will either go this way 138 00:09:04,010 --> 00:09:06,770 and become one fate, or they'll go the other way 139 00:09:06,770 --> 00:09:15,220 and be sort of directed down another potential fate. 140 00:09:15,220 --> 00:09:17,970 There we go. 141 00:09:17,970 --> 00:09:21,100 So this is the first sort of branch point, if you will, 142 00:09:21,100 --> 00:09:25,190 in fate determination for the mammalian embryo. 143 00:09:25,190 --> 00:09:30,380 And so this branch point here is different from this 144 00:09:30,380 --> 00:09:33,530 because once the marble goes one way or the other, 145 00:09:33,530 --> 00:09:37,320 it's restricted in what fate it can become. 146 00:09:37,320 --> 00:09:37,820 OK. 147 00:09:37,820 --> 00:09:44,750 So these cells here are not totipotent 148 00:09:44,750 --> 00:09:48,950 because they can't form the trophoblast. 149 00:09:48,950 --> 00:09:52,580 They can't form the part of the placenta needed by the embryo. 150 00:09:52,580 --> 00:09:55,160 But they do form the embryo proper. 151 00:09:55,160 --> 00:09:58,130 So they are still capable of many fates. 152 00:09:58,130 --> 00:10:03,650 And so these cells are known as pluripotent, which 153 00:10:03,650 --> 00:10:08,850 means capable of many things 154 00:10:08,850 --> 00:10:09,350 OK. 155 00:10:09,350 --> 00:10:11,390 And so a type of cell type, which 156 00:10:11,390 --> 00:10:19,610 I'm sure you've heard about, is called Embryonic Stem cells, 157 00:10:19,610 --> 00:10:20,840 or ES cells. 158 00:10:23,660 --> 00:10:27,890 These ES cells are derived from the inner cell mass. 159 00:10:27,890 --> 00:10:29,210 So these would be-- 160 00:10:29,210 --> 00:10:31,670 if they were taken out of the blastula-- 161 00:10:31,670 --> 00:10:35,160 sorry-- this is called the blastula-- 162 00:10:35,160 --> 00:10:39,390 if they are taken from the blastula and cultured in vitro, 163 00:10:39,390 --> 00:10:41,690 these would be sort of embryonic stem cells 164 00:10:41,690 --> 00:10:46,470 that can be propagated. 165 00:10:46,470 --> 00:10:54,050 And they form the embryo proper, so these are pluripotent stem 166 00:10:54,050 --> 00:11:00,680 cells that can basically-- they are still capable of forming 167 00:11:00,680 --> 00:11:04,310 sort of any of the cell types in the embryo-- 168 00:11:04,310 --> 00:11:14,450 capable of forming embryonic cell types. 169 00:11:28,630 --> 00:11:31,090 Now let's look at the next slide. 170 00:11:31,090 --> 00:11:32,860 So this branch point-- 171 00:11:32,860 --> 00:11:36,280 this first branch point sort of in development 172 00:11:36,280 --> 00:11:40,360 is associated with changes in gene expression. 173 00:11:40,360 --> 00:11:43,900 So there are changes in gene expression. 174 00:11:46,740 --> 00:11:50,050 And you're seeing one up on the slide above. 175 00:11:50,050 --> 00:11:55,420 The slide above is a fixed blastula. 176 00:11:55,420 --> 00:11:57,820 And all of the nuclei in the blastula 177 00:11:57,820 --> 00:11:59,380 are stained with green. 178 00:11:59,380 --> 00:12:02,460 But there is one gene that's stained in red. 179 00:12:02,460 --> 00:12:04,240 It's called Oct4. 180 00:12:04,240 --> 00:12:06,520 And this is a transcription factor that 181 00:12:06,520 --> 00:12:09,130 marks sort of pluripotency. 182 00:12:09,130 --> 00:12:12,790 And you can see how it's expressed specifically 183 00:12:12,790 --> 00:12:15,610 in these cells of the inner cell mass, which 184 00:12:15,610 --> 00:12:21,160 are what the embryonic stem cells are derived from. 185 00:12:21,160 --> 00:12:25,340 So there are clearly changes in gene expression. 186 00:12:25,340 --> 00:12:28,840 And one question you might have is whether or not 187 00:12:28,840 --> 00:12:34,210 these cells that are going to form the embryo proper, 188 00:12:34,210 --> 00:12:37,150 whether they have lost information such that they're 189 00:12:37,150 --> 00:12:42,080 unable to form the part of the placenta. 190 00:12:42,080 --> 00:12:46,870 And you can also ask this for an adult cell. 191 00:12:46,870 --> 00:12:52,450 Has an adult cell in your body-- has it lost gene content such 192 00:12:52,450 --> 00:12:56,590 that it's unable to make an entire organism? 193 00:12:56,590 --> 00:12:58,210 I'm going to tell you the answer. 194 00:12:58,210 --> 00:13:00,250 I want you to think about what experiment 195 00:13:00,250 --> 00:13:03,390 would allow you to sort of determine the answer. 196 00:13:03,390 --> 00:13:08,140 But I'm going to tell you the answer is that there is not 197 00:13:08,140 --> 00:13:13,030 a loss of gene content. 198 00:13:13,030 --> 00:13:16,270 But this differentiation process is 199 00:13:16,270 --> 00:13:18,700 due to changes in gene expression 200 00:13:18,700 --> 00:13:21,190 for the vast majority of our cell types. 201 00:13:23,830 --> 00:13:26,780 So let's say you wanted to determine whether or not 202 00:13:26,780 --> 00:13:31,900 a differentiated cell had lost some sort of capability 203 00:13:31,900 --> 00:13:35,590 to regenerate an entire organism. 204 00:13:35,590 --> 00:13:38,410 What might be some type of experiment you could do? 205 00:13:43,240 --> 00:13:45,477 Brett. 206 00:13:45,477 --> 00:13:47,435 AUDIENCE: Creating conditions that will perhaps 207 00:13:47,435 --> 00:13:52,660 be similar to what embryonic stem cells or perhaps 208 00:13:52,660 --> 00:13:56,730 [INAUDIBLE] factors that allow for you to change 209 00:13:56,730 --> 00:13:58,794 the expression back to more than one impact 210 00:13:58,794 --> 00:14:01,272 to see if that would actually change it. 211 00:14:01,272 --> 00:14:02,230 ADAM MARTIN: All right. 212 00:14:02,230 --> 00:14:02,730 Great. 213 00:14:02,730 --> 00:14:05,530 So Brett had two really good points, I think. 214 00:14:05,530 --> 00:14:09,670 The first is to try to sort of reproduce conditions 215 00:14:09,670 --> 00:14:11,320 of pluripotency. 216 00:14:11,320 --> 00:14:14,710 Or you can even try to reproduce conditions of totipotency, 217 00:14:14,710 --> 00:14:15,670 right? 218 00:14:15,670 --> 00:14:18,580 In which case you'd want to sort of take 219 00:14:18,580 --> 00:14:21,310 the genetic material of the somatic cell 220 00:14:21,310 --> 00:14:23,680 and sort of put it back in this situation where 221 00:14:23,680 --> 00:14:29,110 it's present sort of in the cytoplasm of the zygote. 222 00:14:29,110 --> 00:14:31,660 And the other point that Brett made 223 00:14:31,660 --> 00:14:37,240 is to try to maybe express something that would induce 224 00:14:37,240 --> 00:14:40,180 this type of pluripotent state. 225 00:14:40,180 --> 00:14:43,300 If we knew exactly what the genes were 226 00:14:43,300 --> 00:14:46,450 that create this pluripotent state, 227 00:14:46,450 --> 00:14:49,750 maybe we could just express those genes 228 00:14:49,750 --> 00:14:53,590 and regenerate sort of a cell that 229 00:14:53,590 --> 00:14:59,290 moves from way down here all the way up to the top again. 230 00:14:59,290 --> 00:15:01,750 And, actually, what Brett suggested 231 00:15:01,750 --> 00:15:05,770 were the two experiments that won these two folks the Nobel 232 00:15:05,770 --> 00:15:08,980 Prize in 2012. 233 00:15:08,980 --> 00:15:13,600 So in 2012, the Nobel Prize was awarded jointly 234 00:15:13,600 --> 00:15:18,400 to Sir John Gurdon, right here, who incidentally 235 00:15:18,400 --> 00:15:24,220 has the best hair of all Nobel laureates, and also 236 00:15:24,220 --> 00:15:27,550 Shinya Yamanaka. 237 00:15:27,550 --> 00:15:30,940 And so these two folks were awarded the Nobel Prize 238 00:15:30,940 --> 00:15:36,220 for being able to show that mature differentiated cells can 239 00:15:36,220 --> 00:15:41,620 be reprogrammed to become pluripotent again, 240 00:15:41,620 --> 00:15:45,340 which basically gives us the conclusion that there's not 241 00:15:45,340 --> 00:15:50,950 a loss of gene content during the differentiation process. 242 00:15:50,950 --> 00:15:53,170 And the work from Yamanaka showed us 243 00:15:53,170 --> 00:15:57,100 several genes whose expression is critical to induce 244 00:15:57,100 --> 00:16:01,000 this type of reprogramming. 245 00:16:01,000 --> 00:16:05,560 And their work spanned from frogs-- 246 00:16:05,560 --> 00:16:10,030 so John Gurdon worked on development of frogs, 247 00:16:10,030 --> 00:16:13,810 specifically Xenopus laevis, which you've seen before. 248 00:16:13,810 --> 00:16:21,290 Shinya Yamanaka's work involved mice and also human cell lines. 249 00:16:21,290 --> 00:16:23,930 So I'm going to tell you about their experiments 250 00:16:23,930 --> 00:16:26,810 and how this-- sort of demonstrated this conclusion 251 00:16:26,810 --> 00:16:29,067 here. 252 00:16:29,067 --> 00:16:30,650 And the first thing I want to show you 253 00:16:30,650 --> 00:16:34,430 is an experiment, which is very simple conceptually. 254 00:16:34,430 --> 00:16:36,980 Technically it's very complicated, which 255 00:16:36,980 --> 00:16:42,680 is if you were able to take a nucleus from an adult cell 256 00:16:42,680 --> 00:16:45,020 that's differentiated, could you get 257 00:16:45,020 --> 00:16:49,100 it to change by introducing it back into the egg 258 00:16:49,100 --> 00:16:50,780 cell or a zygote cell? 259 00:16:55,490 --> 00:17:01,070 So this experiment involves having an oocyte, 260 00:17:01,070 --> 00:17:06,470 but in this case an oocyte where the nucleus has been removed. 261 00:17:06,470 --> 00:17:08,780 So you take an enucleated oocyte. 262 00:17:13,790 --> 00:17:16,849 And want to know if the cytoplasm of this oocyte 263 00:17:16,849 --> 00:17:19,220 is somehow special that would allow 264 00:17:19,220 --> 00:17:23,819 it to reprogram the nucleus of a differentiated cell. 265 00:17:23,819 --> 00:17:26,869 And so you could suck up the nucleus 266 00:17:26,869 --> 00:17:30,290 of a differentiated cell. 267 00:17:30,290 --> 00:17:35,560 So a somatic cell is another way to say differentiated. 268 00:17:35,560 --> 00:17:37,205 So it's a somatic cell nucleus. 269 00:17:41,860 --> 00:17:43,970 And the experiment conceptually then 270 00:17:43,970 --> 00:17:46,970 is to just take this somatic cell nucleus that you've 271 00:17:46,970 --> 00:17:51,890 sucked up and inject it into an oocyte without a nucleus 272 00:17:51,890 --> 00:17:54,320 and see if the cytoplasm of this oocyte 273 00:17:54,320 --> 00:17:58,970 is somehow able to change the properties of the nucleus 274 00:17:58,970 --> 00:18:03,000 such that it now is in an undifferentiated state. 275 00:18:03,000 --> 00:18:11,990 So you generate oocyte now with the somatic cell nucleus. 276 00:18:15,980 --> 00:18:19,220 And the question is whether the somatic cell nucleus 277 00:18:19,220 --> 00:18:22,880 is capable of generating the diversity of cell types 278 00:18:22,880 --> 00:18:26,550 that are normally present in an organism. 279 00:18:26,550 --> 00:18:30,170 So you could then let this go and develop. 280 00:18:30,170 --> 00:18:33,920 You could let it develop into a blastula, which 281 00:18:33,920 --> 00:18:34,730 I'm drawing here. 282 00:18:40,490 --> 00:18:43,540 So you could generate a blastula. 283 00:18:43,540 --> 00:18:47,990 And this will be a way to get embryonic stem cells derived 284 00:18:47,990 --> 00:18:51,870 from sort of this type of nucleus. 285 00:18:51,870 --> 00:18:58,190 But you could also let it grow into an entire organism. 286 00:18:58,190 --> 00:19:00,260 But this organism would be genetically 287 00:19:00,260 --> 00:19:05,570 identical to whatever organism donated this nucleus. 288 00:19:05,570 --> 00:19:08,210 So because you're duplicating an organism, 289 00:19:08,210 --> 00:19:11,190 that is known as reproductive cloning. 290 00:19:11,190 --> 00:19:20,930 So this is reproductive cloning, if you 291 00:19:20,930 --> 00:19:22,235 go all the way to the organism. 292 00:19:26,090 --> 00:19:31,763 I'll show you just a little video on the nuclear transfer. 293 00:19:31,763 --> 00:19:32,430 [VIDEO PLAYBACK] 294 00:19:32,430 --> 00:19:36,140 - You see now that this drilling pipe head is going to suck-- 295 00:19:36,140 --> 00:19:38,250 drill a little hole into the membrane. 296 00:19:38,250 --> 00:19:41,823 You can maybe see a little bit of the hole right 297 00:19:41,823 --> 00:19:42,740 here at the next part. 298 00:19:42,740 --> 00:19:43,323 [END PLAYBACK] 299 00:19:43,323 --> 00:19:46,077 ADAM MARTIN: He's talking about this pipette here. 300 00:19:46,077 --> 00:19:48,410 The embryo is being held with another pipette over here. 301 00:19:48,410 --> 00:19:49,077 [VIDEO PLAYBACK] 302 00:19:49,077 --> 00:19:51,090 - You can see a bit of the hole. 303 00:19:51,090 --> 00:19:54,030 And now the pipette's going to go in and remove the nucleus. 304 00:19:54,030 --> 00:19:55,910 And if you look carefully in the pipette, 305 00:19:55,910 --> 00:19:57,950 you'll see a line in the nucleus, which are 306 00:19:57,950 --> 00:20:00,530 all the chromosomes lined up. 307 00:20:00,530 --> 00:20:03,140 So the nucleus is going to be squirted out now because we 308 00:20:03,140 --> 00:20:04,470 don't need it anymore. 309 00:20:04,470 --> 00:20:06,140 And then we have an enucleated egg. 310 00:20:09,010 --> 00:20:11,690 Now, the next step is to take a set of eggs like that-- 311 00:20:11,690 --> 00:20:13,130 and I'll show you two-- 312 00:20:13,130 --> 00:20:15,980 and then transfer into them a nucleus 313 00:20:15,980 --> 00:20:19,200 from another kind of cell, a fully-differentiated somatic 314 00:20:19,200 --> 00:20:19,700 cell. 315 00:20:19,700 --> 00:20:23,370 So here, the enucleated egg is set on the side. 316 00:20:23,370 --> 00:20:26,460 And it's held by this holding pipette on the left. 317 00:20:26,460 --> 00:20:28,870 There's drilling the little hole in the membrane. 318 00:20:28,870 --> 00:20:30,260 Here we go in. 319 00:20:30,260 --> 00:20:32,080 Here comes the nucleus from the right. 320 00:20:32,080 --> 00:20:33,380 [END PLAYBACK] 321 00:20:33,380 --> 00:20:34,310 ADAM MARTIN: That's from a somatic zone. 322 00:20:34,310 --> 00:20:34,977 [VIDEO PLAYBACK] 323 00:20:34,977 --> 00:20:37,550 - And these pipettes are operated 324 00:20:37,550 --> 00:20:39,373 with a piezoelectric device. 325 00:20:39,373 --> 00:20:40,790 So you can't see it here, but it's 326 00:20:40,790 --> 00:20:43,610 like a little jackhammer going very quickly, 327 00:20:43,610 --> 00:20:47,000 (TRILLING SOUND) like Woody the Woodpecker, getting in there. 328 00:20:47,000 --> 00:20:47,583 [END PLAYBACK] 329 00:20:47,583 --> 00:20:49,083 ADAM MARTIN: I don't know if I would 330 00:20:49,083 --> 00:20:51,120 have used Woody the Woodpecker as an analogy, 331 00:20:51,120 --> 00:20:53,710 but that's basically the idea. 332 00:20:53,710 --> 00:20:57,110 So you can take a nucleus from a somatic cell 333 00:20:57,110 --> 00:21:01,340 and transplant it, if you will, into an oocyte and then 334 00:21:01,340 --> 00:21:06,380 determine whether you can get either a blastula 335 00:21:06,380 --> 00:21:13,430 or an entire organism from that differentiated cell's nucleus. 336 00:21:13,430 --> 00:21:17,030 So this is one result from Sir John Gurdon. 337 00:21:17,030 --> 00:21:21,710 And what the experiment was, in this case, 338 00:21:21,710 --> 00:21:27,900 was to take oocytes, or eggs, from this wild-type laevis frog 339 00:21:27,900 --> 00:21:35,040 and to transplant nuclei from donor frogs that are albino. 340 00:21:35,040 --> 00:21:37,610 So you can see this is an elegant experiment in that you 341 00:21:37,610 --> 00:21:40,160 can sort of track the origin of the nucleus, 342 00:21:40,160 --> 00:21:44,120 because it's genetically marked with this albino sort 343 00:21:44,120 --> 00:21:46,460 of phenotype. 344 00:21:46,460 --> 00:21:50,740 So you're getting a nuclei from-- 345 00:21:50,740 --> 00:21:55,060 you're getting nuclei from albino tadpoles. 346 00:21:55,060 --> 00:21:59,060 So these are differentiated cell nuclei. 347 00:21:59,060 --> 00:22:03,110 And they're transplanted into wild-type donor eggs. 348 00:22:03,110 --> 00:22:05,540 So normally, the wild-type frog would 349 00:22:05,540 --> 00:22:09,380 reproduce frogs that look like it-- that are non-albino. 350 00:22:09,380 --> 00:22:13,160 But in this experiment, Gurdon and his lab 351 00:22:13,160 --> 00:22:17,120 were able to get frogs that were albino. 352 00:22:17,120 --> 00:22:25,310 So these would be sort of clones of whatever albino tadpoles 353 00:22:25,310 --> 00:22:28,010 they got the nuclei from. 354 00:22:28,010 --> 00:22:32,720 So you see, in this case, it's the origin of the nucleus that 355 00:22:32,720 --> 00:22:36,920 is determining sort of the phenotype of these frogs. 356 00:22:36,920 --> 00:22:40,100 So that allowed them to show that the nucleus is getting 357 00:22:40,100 --> 00:22:43,610 reprogrammed from this albino tadpole 358 00:22:43,610 --> 00:22:47,180 and is able to still generate all of the cell types 359 00:22:47,180 --> 00:22:50,400 present in a normal organism. 360 00:22:50,400 --> 00:22:50,900 Yes. 361 00:22:50,900 --> 00:22:51,400 Brett. 362 00:22:51,400 --> 00:22:53,795 AUDIENCE: So this is an unfertilized egg 363 00:22:53,795 --> 00:22:55,280 that they were taking from the-- 364 00:22:55,280 --> 00:22:56,330 ADAM MARTIN: Yes. 365 00:22:56,330 --> 00:22:58,742 AUDIENCE: And so they're extracting all this DNA, 366 00:22:58,742 --> 00:23:01,732 then putting in a full set of DNA from the albinos. 367 00:23:01,732 --> 00:23:02,440 ADAM MARTIN: Yep. 368 00:23:02,440 --> 00:23:04,148 AUDIENCE: It would go on to differentiate 369 00:23:04,148 --> 00:23:05,090 a full set of DNA? 370 00:23:05,090 --> 00:23:05,840 ADAM MARTIN: Yeah. 371 00:23:05,840 --> 00:23:06,830 AUDIENCE: OK. 372 00:23:06,830 --> 00:23:07,580 ADAM MARTIN: Yeah. 373 00:23:07,580 --> 00:23:09,410 So, yeah, they're-- and actually, 374 00:23:09,410 --> 00:23:13,610 it was taking unfertilized eggs, which is the biggest trick. 375 00:23:13,610 --> 00:23:16,290 I think people had tried to do this in frogs before, 376 00:23:16,290 --> 00:23:19,850 and it failed because they were using fertilized eggs. 377 00:23:19,850 --> 00:23:24,650 And there's something about sort of the oocyte development that 378 00:23:24,650 --> 00:23:28,950 makes it better at reprogramming the nucleus. 379 00:23:28,950 --> 00:23:29,450 OK. 380 00:23:29,450 --> 00:23:31,730 So that's with frogs. 381 00:23:31,730 --> 00:23:35,150 So that experiment was actually done in the late 1950s, 382 00:23:35,150 --> 00:23:38,120 published in the early 1960s. 383 00:23:38,120 --> 00:23:43,520 And so it took another 40 years or so for the first mammal 384 00:23:43,520 --> 00:23:44,660 to be cloned. 385 00:23:44,660 --> 00:23:47,810 And you guys probably weren't even born yet. 386 00:23:47,810 --> 00:23:49,460 But for those of us who are older, 387 00:23:49,460 --> 00:23:51,080 we remember this because there was 388 00:23:51,080 --> 00:23:54,710 a big brouhaha over Dolly the sheep, which 389 00:23:54,710 --> 00:23:57,530 was the first cloned mammal. 390 00:23:57,530 --> 00:23:58,925 I believe that's Dolly there. 391 00:24:01,490 --> 00:24:04,850 So this is-- 392 00:24:04,850 --> 00:24:06,590 I'm not sure which one is Dolly. 393 00:24:06,590 --> 00:24:10,550 They're both the same type of sheep. 394 00:24:10,550 --> 00:24:14,270 So this was done by Ian Wilmut and his group. 395 00:24:14,270 --> 00:24:16,550 One thing I want to point out about this 396 00:24:16,550 --> 00:24:19,460 is it's incredibly inefficient. 397 00:24:19,460 --> 00:24:22,220 If you look over here, this Dolly 398 00:24:22,220 --> 00:24:27,700 resulted from over 400 oocytes having 399 00:24:27,700 --> 00:24:31,600 this sort of nuclear transplant take place. 400 00:24:31,600 --> 00:24:34,610 So it's not a very efficient process. 401 00:24:34,610 --> 00:24:39,100 And so that lack of efficiency is due to the fact 402 00:24:39,100 --> 00:24:44,320 that the nucleus is resisting getting reprogrammed. 403 00:24:44,320 --> 00:24:51,900 And this is a graph from one of John Gurdon's sort of-- 404 00:24:51,900 --> 00:24:55,600 he wrote a review article on sort of reprogramming 405 00:24:55,600 --> 00:24:57,550 after he won the Nobel Prize. 406 00:24:57,550 --> 00:24:58,870 This is from that. 407 00:24:58,870 --> 00:25:02,230 And what's plotted is sort of the frequency 408 00:25:02,230 --> 00:25:05,080 in which transfers results in sort 409 00:25:05,080 --> 00:25:08,950 of a differentiated organism. 410 00:25:08,950 --> 00:25:12,370 And what's on the x-axis is the stage 411 00:25:12,370 --> 00:25:15,970 of the cell that's used to transplant. 412 00:25:15,970 --> 00:25:20,230 And so what you see is that over the course of differentiation 413 00:25:20,230 --> 00:25:22,450 it gets harder and harder for the nucleus 414 00:25:22,450 --> 00:25:27,790 to get reprogrammed to create sort of all the cell types that 415 00:25:27,790 --> 00:25:32,670 are normally present in an adult animal. 416 00:25:32,670 --> 00:25:35,790 So nuclei do become more restricted 417 00:25:35,790 --> 00:25:39,360 in their ability to be reprogrammed over development. 418 00:25:39,360 --> 00:25:43,200 But the fact that any of them are able to be reprogrammed 419 00:25:43,200 --> 00:25:45,060 suggests that when they are-- 420 00:25:45,060 --> 00:25:47,030 that during differentiation there 421 00:25:47,030 --> 00:25:49,080 is not a loss of gene content. 422 00:25:52,550 --> 00:25:55,560 And John Gurdon has done a lot to characterize 423 00:25:55,560 --> 00:25:57,430 sort of the changes in the nucleus that 424 00:25:57,430 --> 00:26:01,150 happen during differentiation which sort of resist 425 00:26:01,150 --> 00:26:06,220 this reprogramming by the oocyte cytoplasm. 426 00:26:09,680 --> 00:26:10,180 All right. 427 00:26:10,180 --> 00:26:12,910 So mechanistically, what's happening? 428 00:26:12,910 --> 00:26:20,560 Well, one big-- one of the people 429 00:26:20,560 --> 00:26:24,970 who really showed what's going on there is Shinya Yamanaka. 430 00:26:24,970 --> 00:26:35,750 And what his work shows is that you can take just a few genes-- 431 00:26:35,750 --> 00:26:37,810 it turns out it is four-- 432 00:26:37,810 --> 00:26:41,380 and you can induce reprogramming by just 433 00:26:41,380 --> 00:26:47,380 expressing these four genes in an adult differentiated cell. 434 00:26:47,380 --> 00:26:56,640 So in this case you have a differentiated cell. 435 00:27:03,530 --> 00:27:06,600 And what he initially used we're fibroblasts, which are 436 00:27:06,600 --> 00:27:09,360 a type of differentiated cell. 437 00:27:09,360 --> 00:27:11,940 And what Yamanaka showed is that you 438 00:27:11,940 --> 00:27:15,900 can express four transcription factors, one 439 00:27:15,900 --> 00:27:18,660 of them being this factor here, Oct4, 440 00:27:18,660 --> 00:27:21,380 which is expressed in the inner cell mass 441 00:27:21,380 --> 00:27:23,655 and was shown to be involved in sort 442 00:27:23,655 --> 00:27:29,700 of maintaining pluripotency of these inner cell mass cells. 443 00:27:29,700 --> 00:27:34,060 So you could take Oct4 plus another transcription factor, 444 00:27:34,060 --> 00:27:37,290 which is important for pluripotency, plus two 445 00:27:37,290 --> 00:27:45,008 others and c-Myc. 446 00:27:45,008 --> 00:27:45,800 These are the four. 447 00:27:45,800 --> 00:27:48,320 So these are all transcription factors. 448 00:27:48,320 --> 00:27:50,930 And what they found is if you express these four 449 00:27:50,930 --> 00:27:55,550 transcription factors in a differentiated cell, 450 00:27:55,550 --> 00:28:01,280 you could get it to revert to a more pluripotent state. 451 00:28:01,280 --> 00:28:12,230 So this results in what is known as Induced Pluripotent Stem 452 00:28:12,230 --> 00:28:19,010 cells, or IPS cells, for short. 453 00:28:24,210 --> 00:28:28,380 And then like embryonic stem cells, 454 00:28:28,380 --> 00:28:33,930 here these cells can give rise to different cell types. 455 00:28:33,930 --> 00:28:39,030 And so one of the goals of this field of developmental biology 456 00:28:39,030 --> 00:28:45,720 and cell reprogramming is to use this technology 457 00:28:45,720 --> 00:28:49,630 to replace cells that are lost in patients. 458 00:28:49,630 --> 00:28:52,170 So this is known as regenerative medicine. 459 00:29:01,680 --> 00:29:06,840 And the idea of the theory of regenerative medicine 460 00:29:06,840 --> 00:29:15,420 is to take ES cells or induced pluripotent stem 461 00:29:15,420 --> 00:29:24,840 cells from patients and to be able to culture them in vitro-- 462 00:29:24,840 --> 00:29:26,205 so culture in vitro-- 463 00:29:32,140 --> 00:29:36,570 and then to be able to direct these cultured cells 464 00:29:36,570 --> 00:29:40,410 into different cell fates in order 465 00:29:40,410 --> 00:29:46,020 to use these cells possibly to transplant them back 466 00:29:46,020 --> 00:29:51,390 into an individual where maybe these cell types are dying. 467 00:29:51,390 --> 00:29:58,860 So you could then differentiate these cells using 468 00:29:58,860 --> 00:30:02,490 certain biochemical signals that you can add to the media 469 00:30:02,490 --> 00:30:05,370 in order to create different cell types, 470 00:30:05,370 --> 00:30:13,200 like neurons, muscle, maybe skin. 471 00:30:13,200 --> 00:30:20,010 And one example of this was shown by Shinya Yamanaka, where 472 00:30:20,010 --> 00:30:27,710 what they did was to take human fibroblast cells, 473 00:30:27,710 --> 00:30:29,900 put them back in the pluripotent state 474 00:30:29,900 --> 00:30:32,840 by expressing those four factors, 475 00:30:32,840 --> 00:30:35,270 and then get these cells to differentiate 476 00:30:35,270 --> 00:30:40,470 into cardiac tissue, so cardiac cells. 477 00:30:40,470 --> 00:30:41,480 Here's a movie. 478 00:30:41,480 --> 00:30:46,160 So these are from adult fibroblast cells. 479 00:30:46,160 --> 00:30:48,590 But now you can see they're beating sort 480 00:30:48,590 --> 00:30:51,380 of like a cardiac tissue would. 481 00:30:51,380 --> 00:30:56,940 So these were made into IPS stem cells, cultured in vitro, 482 00:30:56,940 --> 00:31:00,310 and then directed into a cardiac muscle fate. 483 00:31:03,500 --> 00:31:07,650 So the goal would then to be use these 484 00:31:07,650 --> 00:31:21,620 and to transplant them back into a patient, such 485 00:31:21,620 --> 00:31:23,390 that if a patient had, let's say, 486 00:31:23,390 --> 00:31:26,600 a neurodegenerative disease and was lacking 487 00:31:26,600 --> 00:31:28,700 a certain type of neuron, you could then 488 00:31:28,700 --> 00:31:33,170 take cells, start them on the path to neuronal development, 489 00:31:33,170 --> 00:31:36,200 and then transplant them back into that patient. 490 00:31:36,200 --> 00:31:42,260 And if these cells are derived from the DNA of that patient, 491 00:31:42,260 --> 00:31:46,130 then there won't be transplant rejection. 492 00:31:46,130 --> 00:31:49,460 Because what regulates transplant rejection 493 00:31:49,460 --> 00:31:54,530 is the major histocompatiblity complex locus. 494 00:31:54,530 --> 00:31:56,450 And it's polymorphic. 495 00:31:56,450 --> 00:31:59,810 But if you're taking a nucleus from the patient 496 00:31:59,810 --> 00:32:03,110 and then causing the cells to differentiate in vitro, 497 00:32:03,110 --> 00:32:05,120 you're taking the exact same-- 498 00:32:05,120 --> 00:32:07,340 you're taking clonal cells and putting them back 499 00:32:07,340 --> 00:32:09,380 in the same patient, such that they 500 00:32:09,380 --> 00:32:11,765 won't be rejected, ideally. 501 00:32:15,870 --> 00:32:16,370 All right. 502 00:32:16,370 --> 00:32:20,390 Now I want to try something with the remainder of our time. 503 00:32:20,390 --> 00:32:22,070 I want a group-- 504 00:32:22,070 --> 00:32:22,760 all right. 505 00:32:22,760 --> 00:32:24,320 Everyone on this side of the room 506 00:32:24,320 --> 00:32:27,530 over, I want you over here. 507 00:32:27,530 --> 00:32:29,880 And everyone on this side of the room, 508 00:32:29,880 --> 00:32:32,140 I want you on this side over here. 509 00:32:32,140 --> 00:32:33,200 OK. 510 00:32:33,200 --> 00:32:36,125 Maybe you guys can go over here so that we're more balanced. 511 00:32:39,360 --> 00:32:40,860 We're going to have a little debate. 512 00:32:51,340 --> 00:32:52,240 You can sit down. 513 00:32:52,240 --> 00:32:52,760 It's OK. 514 00:32:59,000 --> 00:33:01,150 Be in a position to talk to each other. 515 00:33:01,150 --> 00:33:04,354 I want you guys talking to each other. 516 00:33:04,354 --> 00:33:07,060 That was the goal of putting you close together. 517 00:33:07,060 --> 00:33:07,560 OK. 518 00:33:10,770 --> 00:33:12,480 So in the past couple weeks, there's 519 00:33:12,480 --> 00:33:16,200 been a little bit of a stir because there's 520 00:33:16,200 --> 00:33:19,800 a researcher in China who has claimed to have made 521 00:33:19,800 --> 00:33:22,470 the first gene-edited baby. 522 00:33:22,470 --> 00:33:24,570 Perhaps you have heard of this. 523 00:33:24,570 --> 00:33:27,630 You probably have because it's been all over the news. 524 00:33:27,630 --> 00:33:29,580 So I want you guys-- 525 00:33:29,580 --> 00:33:30,970 let's see. 526 00:33:30,970 --> 00:33:34,360 I want us to debate whether we should-- 527 00:33:34,360 --> 00:33:36,990 what are the advantages or disadvantages 528 00:33:36,990 --> 00:33:40,750 of gene editing or human cloning. 529 00:33:40,750 --> 00:33:42,420 You could talk about both. 530 00:33:42,420 --> 00:33:45,960 And then I want you guys to be able to present them to me. 531 00:33:45,960 --> 00:33:48,690 I'll write them down, and we'll have a discussion about it. 532 00:33:56,799 --> 00:34:00,220 Guys, continue this discussion sort of outside class. 533 00:34:00,220 --> 00:34:01,720 I think it's really interesting. 534 00:34:01,720 --> 00:34:03,670 And it's going to be something that you're 535 00:34:03,670 --> 00:34:07,710 going to hear a lot about in the coming years, I'm sure.