2012 Nobel Lectures in Physiology or Medicine

but before we begin I would like to ask you all to bring out your cell phones and make sure that they are turned off thank you for your cooperation now let me introduce Professor urban Lee and all share of the Nobel Committee and professor at the Department of cell and molecular biology here at Karolinska Institutet and professor Lee and all will introduce the Nobel laureates and give the motivation for the price thank you the laureates distinguished guests friends and colleagues I'm privileged and honored to stand here today to introduce the two Nobel laureates in Physiology or medicine 2012 dr. John garden and Shinya Yamanaka together these two scientists have fundamentally changed our review of the cellular differentiation process and how we view two specialized cells in our bodies they've shown that the differentiated cells state was not caused in stone but that a dormant potential rejuvenation could in fact be unchained in these cells this is not only made us view cells in the new light but also open new avenues to gain insights into disease and in the future to improve diagnosis and hopefully develop new therapies to either two incurable diseases the achievements also provide a prime example of our fundamental discoveries rooted in basic science can profoundly impact on or attempting to say rejuvenate many areas of medical research to put their discoveries in context let us briefly revisit how we came to learn about differentiated cells the journey from a fertilized egg to an adult individual has always fascinated mankind and an earlier count on developmental processes date back to Aristotle us when he temporarily left philosophy to go to the Greek island of Lesbos and together with T of Rastus ponder on botany zoology and generally on the wonders of life for a long time then progress was more limited but with the advent of the first microscopes in the 1600s this allowed objects in the size of a cell actually to be observed and Robert Hooke one of the pioneers he looked at a section cut out from piece of cork and you observed structures that he thought resembled miniature versions of amongst chamber and hence he called these structures cells and the modern cell theory and the concept that a cell is the building block of all living forms that was established in eighteen hundreds by scientists like Theodor Schwann matthias schleiden and rudolf virchow and as histology evolved and microscopes and staining techniques got better it gradually became clear that our bodies contained many different types of specialized cells each with a unique morphology and here Ramon or Kyle's beautiful drawings of cells in the nervous system comes mind in the light of this the notion that a differentiated cell was an endpoint the final stop on a developmental journey grew stronger and this notion was reinforced in the early days of selling molecular biology but at the different cell types not only looked different they also were endowed with different compositions of proteins and later on also RNAs furthermore with the discovery of mobile genetic elements by Barbara McClintock it's inconceivable that a genetic material in the cell would be irreversibly altered during cellular differentiation there were also entropy based models often portrayed as a mountain landscape where the differentiated cell stage was conceived to be the most energetically stable state and the differentiated cells would peacefully be resting in the mountain valleys put shortly the dogma was building that the differentiated else could not be persuaded to make a round trip back to their original immature state and such was the scene when a John John Gurdon took on this question discouraged early on by a teacher to venture into science but equipped with a sharp intellect curiosity and I've been told an aptitude for doing things in a small scale such as building three mastered sailing ships in walnut shells John Gurdon asked if a differentiated cell nucleus could restart development if it was placed in the right environment they enucleated egg cell and this work carried out in the Frog Xenopus laevis began in the life late 1950s and culminated in the landmark publication in 1962 and this discovery really ran counter to the prevailing view that cell the cell differentiation state was irreversible but John Gordon was right and based on his findings somatic cell nuclear transfer has since been expanded also to mammals notably in the form of Dolly the sheep John Gurdon originally trained would make a fish bear at the University of Oxford where he received the PhD in 1960 after stay at Caltech from 1960 to 1963 he returned to Oxford to then later moved to Cambridge University in 1972 from 1989 and onwards has been with a an institute in Cambridge that since 2004 carries his name the Gurdon Institute John Gurdon has received numerous awards including the Wolf Prize in 1989 and the Lasker medical research award in 2009 he was elected fellow of the Royal Society in 1971 and knighted in 1995 such would rightfully be Sir John and with this as a background please join me in welcoming John Gurdon who will present his Nobel lecture under the title the egg and the nucleus a battle for supremacy John should be alright Julian so first let me thank Aban lindahl for his very fun introduction and then say that this is of course a very special occasion for me and I mean or masti grateful to the committee for Physiology and medicine for this unique and immense privilege of finding myself here today so I've chosen to use this title for my talk because I think the interaction between ergo nucleus leading eventually to reprogramming can really be summarized as a battle between the egg and the nucleus and at the moment I would say the egg is doing very well but the nucleus is slightly better and we need to realize the full potential we need to give the egg a little more help to try and reduce the remarkable resistance of the nucleus to being reprogrammed so these are the subjects I will talk about a bit of background explain the history of the subject and then talk about the egg how it is trying to reprogram a nucleus and then talk about the nucleus how it's trying to defend itself by being reprogrammed and finally a few words about prospects so I always think one should start with a question and in my case the question was all this time ago whether all cells in the body have the same sets of genes simple enough question the originators of this whole field were Robert Briggs and Thomas King and they had great success in 1952 in succeeding for the first time in transplanting a somatic nucleus to an enucleated egg and getting normal development from it however when they tried to do the same experiment with the nucleus of a more specialized cell or actually just an embryo little bit later on they found that there was no successful normal development at all by the time they took nuclei from the early embryo there was no success in obtaining normal development so of course that led to the idea that as cells differentiate they might lose the capacity of their nucleus to be reprogrammed might be lost irreversibly well I then started work after years later and was advised to work with the South African Frog Xenopus laevis is this one and we were I was lucky at an early stage to get an apparently normal frog from the nucleus of a reasonably advanced embryo this is the the first adult we had historically it was a frog of good quality it lived for about 20 years and it had about 4000 offspring so it was an indication that you can transform the nucleus without generating any adverse effects now a key step in this history was my supervisor Michael fish Berg who in a ingenious way discovered a genetic marker which was essential for proving that a transplanted nucleus on its own will give rise to a normal embryo and this new a new clear marker involves counting nucleoli in a cell you had to be able to distinguish between 1 and 2 and in these cells there is one nuclei in each of the nuclei representing as it turned out the site of synthesis of ribosomal RNA and in the wild-type which was called a to nucleus stream there are two nuclei in nearly all cells the one nucleus Newton's was a deletion of ribosomal genes mm-hmm and it was immensely valuable so that one could mark the nuclei being transplanted and show that the resulting embryos always had one nucleolus in our nuclei and never had two so with this background the examples work led like briggs and king the success rate went down the more advanced were the embryos from which nuclei were taken nevertheless even when you took swimming larvae their cells to a significant extent were able to produce entirely normal normal embryos and that then reopened the question of whether as cells differentiate their nuclei might still be able to generate normal development so looking again ahead a bit we started using the intestine of a feeding larva so it's intestine cells were functional and in this case we could use the GFP marker to mark these nuclei transplant the nuclei to eggs and when you do that you sometimes get normal embryos but more often you get these partially Bria's with normal cells at one end and a complete failure of development at the other end of the embryo now these embryos will die however they die within about a day however it's possible to take these cells and re transplant a nucleus from those into another set of eggs many of which will then develop normally without this making a defective embryo it's also possible to take a few of these cells which you may remember a GFP marked and Transplant them as a graft to a normal host embryo like this you can see the the graft stuck onto the normal embryo and when these developed they can form normal entirely normal embryos and all of these green cells each one is a single muscle cell nicely oriented orientated with respect to the host cells which are do not carry the marker they are black and it shows that the functional muscle can readily be derived from the nucleus of an intestine cell so that was be the origin of much of this work and when we summarize that put that together we can add up the success rate of the so called first nuclear transfers and add to that results obtained by the serial second nuclear transfers and also include grafts made in the way that I've described and when you do that the success rate with which you can obtain for example functional muscle and nerve from intestine is around 30% so that says that the cytoplasm is pretty good at redirecting the nucleus of a muscle cell so that instead of continuing its life is intestine ik and the cytoplasm of the egg can switch it back to the beginning of life and send it out in the direction of muscle and nerve but at this point this success rate of 30% also means that 70% of the embryos do not achieve that and so in terms of this conflict or battle we would say the nucleus on the whole is winning cytoplasm does pretty well but but most of the nuclei are unwilling to be changed back to redirect their differentiation so at this point I should acknowledge the work of will Moton Keith Campbell who in 1997 were able to do nuclear transfers in in the lam in in sheep an interesting question of course is why there was a gap of some 40 years or so between the early frog experiments and the sheep experiments and the short answer is that the people working with mammals mostly with mice early on did not actually follow exactly what was done with the frogs for some reason they chose to use fertilized eggs which were extremely difficult to deal with and enrollment and keith campbell chose to do exactly what had worked with frogs and obtained these wonderful results with sheep so in summary then the results with the frog and mammals are remarkably similar in each case you get about a 30 percent success rate if you take mammals reaching birth or Xenopus reaching tempo stages and as you take nuclear for more advanced stages the success rate goes right down so that's a measure again of this resistance of the nuclei defending themselves against the onslaught of the egg trying to make them change direction now I'll now refer to a remarkable experiment of James Byrne who was a student of mine but he didn't do this work with us he did it later so he took monkeys skin and eggs from another monkey did the nuclear transfer or cloning as it's called made the embryonic stem cells brilliant discovery by Martin Evans and was then able to increase the cell number and add factors to make these cells now turn into beating heart and if impatient this will probably show the beating heart cells from the monkey skin it's a sheet of these thousands of cells beating remarkably well in Indus these cardiomyocytes because the problem is when you transplant those cells to a heart they do not end present technology integrate well but that was a very elegant demonstration of what can be done now briefly let me mention this phenomenon or epigenetic memory which in effect shows that a transplanted nucleus will very often remember where it came from even though the egg is making its switch fate now in this case one was taking a muscle cell transplanting its nucleus and make an embryo and then separate the embryo into the future neuroectodermal nerve and skin cells take away the future muscle bit and separate the ended on bit and rather amazingly the future nerve and skin cells or gut cells continue to express muscle genes to a surprisingly large extent start with muscle go through and go into the nerve lineage but nevertheless those cells remember somehow that they came from muscle we had reason to think this might have to do with a particular protein called histone h3 point 3 and so we were able to use a mutation in that gene to essentially compete out the normal histone h3 point 3 which is present in the egg and show that when you did this you eliminated this memory sort of being 50% also it went down to just a few percent so that indicated that histone h3 point 3 is one of the factors the eggs you eggs use to increase transcription and when you transplant a nucleus that is already committed to one fate the egg enforces continuation of that expression which makes it very hard to reprogram it so this is another reflection of the amazing defense that the nucleus of a specialized cell will put up as it were to resist the reprogramming activities of the egg so in summary then the epigenetic memory as we call it is reflects the activity of this variant histone 83.3 which promotes transcription of active genes and the egg cytoplasm can reverse that but only with about fifty thirty to fifty percent efficiency so that then makes one turn to how one can start to analyze this conflict of interest a bit further and I'm now going to talk about these cells called first meiotic prophase our sites which are the progenitors of the egg now this simplifies the analysis to a considerable extent because in the experiments that I've just been talking about you transplant a nucleus and you get an embryo but before that happens there are numerous cell divisions and replications before the embryo can form an adult frog and this is where the where the reprogramming happens where the egg is switching the nucleus back now that complicates analysis very considerably because when this happens there is incomplete DNA replication and this damages the somatic nuclei so that many of them are no longer capable of following normal development to overcome that one can use these egg progenitors which we call oo sites they contain a large object called the germinal vesicle and one can inject multiple nuclei into this and this our site will continue life it has no DNA no DNA location occurs but intense transcription and after a couple of days or so these nuclei have now switched they have now been reversed rejuvenated and made to express embryo genes so you don't get new cells but you've undone a large part of the reversal process we can understand this by realizing that the egg progenitor or our site starts life as a germ cell and then it gradually forms the mature our site or egg during this time the it's nucleus can builds up a large reserve of these reprogramming factors and only when the our site eventually becomes an egg in what seems like a flesh just a moment the egg will form and will be fertilized normally make cells and already you start getting different cell types the red blue and yellow so the other site is preparing itself for a long period of time like a hundred days building up these reprogramming factors ready so that when fertilization or nuclear transfer happens it can immediately embarked on normal development that's why the our site is such a valuable source of these reprogramming factors the other site looks like this for the Frog it's very big cell about a millimeter in diameter and the its nucleus the germinal vesicle is also very large and the germinal vesicle contains this reserve of reprogramming components indicated in red and these are distributed to the egg when the oversight changes into an egg and then to the embryo and they are necessary for normal development so these are the key reprogramming factors that are needed to switch the somatic cell backwards in development and if you look at gene expression this works fine so here are these nuclei which are adult nuclei and no more ones work fine if you use rapidly dividing cells like HeLa within a day they have already started expressing the key pluripotency factors which we'll hear more about later on the other hand when you use nuclear specialized cells like Mouse Imus they are significantly resistant they resist the programming factors of the OA site so this system enables us to begin to analyze what is really happening so this led to the oversight transcription assays I call it and the major features of this turning out to be very useful is that Yukon transplants many nuclei several hundred makes analyses easier the new transcripts the pluripotency gene transcripts are accumulate nicely with time most notably the genes that are being activated undergo recurrent transcription continually many hundred times a day and that cannot be done in other systems and lastly the these injections into our site show this resistance or defense that were interested in so now I turn to the transcriptional activation in other words what is it that the egg is doing that is somehow able to a fair extent to rejuvenate a dot nuclei and send them back to the beginning of development so I see it as the egg attacking this somatic nucleus trying to force it backwards and so how does this happen in normal life the sperm is a tiny cell and after in in mammals it forms this enormous male pronucleus it undergoes a massive expansion because the egg forces that and so indeed when you transplant nuclei somatic nuclei that happens also here are these few hundred transplanted nuclei and in a short while they form these balloons big vesicles with a huge d condensation or opening up of the genome said the genes are now accessible to factors from the egg and part of this process is seen by the uptake of special molecules of the egg which in this experiment have been marked in red and we start by transplanting nuclei in which the the adult type linka histones are shown in green and when you follow this process in time one can see that the the green molecules disappear and the oocytes own red molecules take over to a remarkable extent almost all these transplanted nuclei have taken up the red special histone linker histone of the our site the few that happened the green ones are interesting there are ones where when we transplanted a nucleus by mistake we transplanted a whole cell if you transplant a whole cell nothing can get into it you have to transplant the nucleus not the cell for the eggs components to gain access to that nucleus and this process happens surprisingly quickly this is in in about two or three hours this whole process will happen and that's amazingly fast for a somatic nucleus that might expect to spend the rest of its life as a specialized cell within this short time it can be the whole reversal process can begin due to these special histones obtained contained in the in the our site and soon after that there's another chromosomal protein called histone h3 point-three which we've mentioned also invades the incoming nuclei and one can now follow this in some detail by looking at the different forms of RNA polymerase which also enters these nuclei as well so these are the transplanted nuclei 24 hours and 48 hours of to transfer and even the elongating polymerase 2 is successful in gaining access to these transplanted nuclei at the same time the nuclei lose some of the components the RNA polymerase brought in by the donor nuclei disappears and the tata binding protein of somatic cells disappears and is replaced by the tata binding protein of the oocytes so these are the switches which are happening very quickly and they arise largely because the these components are so abundant in the other site that they force their way into the nucleus just by mass action such a huge abundance they invade the nucleus and fill it up with these components and all these components are necessary for the reprogramming here you can see that the different forms of RNA polymerase 2 follow each other within a short time and rush into the nucleus to start transcribing the genes which have been opened up by the special histone that we just mentioned another interesting thing is that the RNA polymerase which is needed to read these genes transcribe these genes has to come from the OE site so you can do an experiment with alpha amanitin which kills the oocytes polymerase 2 and if you use nuclei which are resistant of this drug they shown in the blue are quite unable to be reprogrammed or gene switched on but the OA site polymerase is if it's killed will not allow that normally it allows it as shown by the substantial increase in expression of this gene so an important question is where does the selectivity arise so why is it that the a site or egg can switch some genes on and others not and this depends entirely on the cell type the the original state of the genes and the nuclei being introduced and it shows her that a gene which is very easily activated called sox2 transcription start site that gene will take up the special histone that Limor ace and it will be transcribed well now globin genes are not able to be reprogrammed by the other site they're resistant to it they defend themselves against this egg activities and nevertheless they absorb they can't help absorbing this special linker histone which over everything which opens everything up on the other hand are completely unable to respond to the polymerase and so they are not expressed so it in a straights the actual level at which the this resistance or defense is operating it's you can open genes up but the actual machinery needed to read the genes cannot work if the nucleus from the adult cell is exerting its defense so it will not allow these OSI factors to operate and so we can begin to put together these events which are needed for the site to have its reprogramming activity and this is these are some of them there's no doubt a few others but it gives us an indication of how the our site or egg attacks the somatic nucleus by overloading it with these abundant components which enter the nucleus and try to reverse it try to get it to go backwards in development it's quite useful to know what these are because they are necessary without them there would be no rejuvenation or reversal and these are some of the components which are needed for this special process so now we turn to resistance or as I call it defense by the nucleus and I would argue that this is in many ways the most important part of the whole process of reprogramming why is it that the nucleus is able to defend itself against this enormous influx of components offered by thee by the egg and in the work that will shortly hear about the famous IPS work the success rate of reversal is normally very low originally it was maybe one in a thousand or 10,000 cells so most cells have an extraordinary defense resistance to reprogramming and I would argue that if we could really understand that we could assist the egg or other components in rejuvenating nuclei and make the whole process of reprogramming considerably more efficient so what are what is the basis of this resistance how do nuclei defend themselves against the reprogramming components of the egg in one case we know something about this and you will know that in female mammals one of the two x-chromosomes has no gene activity the genes of all repressed they call it X inactive now this turns out to be true if you take nuclei from adult cells like mouse embryonic fibroblasts they make no response even in three days through a reprogramming by the egg on the other hand similarly inactive X chromosomes from the epiblast it's an earlier stage of embryos are very well reprogrammed so the two states of this inactive X illustrate the difference between them and hence what it is tell us something about the component that is responsible for this resistant state of the adult nuclei now the short answer to this is that it's been possible to identify this component and it's called macro h-2a and you can do experiments where you eliminate that resistance factor and when you do that you then turn on pluripotency jeans or tan socks even in the adult cells very well so this macro h-2a helps to explain helps to explain the resistance to reprogramming it's one of the molecules which has to be eliminated from nuclei to enable them to be reprogrammed so in conclusion this macro h2 is a good example of a molecule that marks embryonic differentiation and it acts as a epigenetic resistance factor to nuclear reprogramming so that leads us to now ask well what is what kind of reprogramming is this do do we see most genes in the genome being reprogrammed or is that a rare and specialized situation and when you analyze all the genes in the genome you find that the reprogramming or gene selection 48 hours after nuclear answer is highly selective so we can see that genes which were often the donor nuclei shown her low level or nil level of expression but half of them just stay off a few more were expressed to the high rate and they stay on after nuclear transfer and a few others when they're after nuclear transfer they go from an odd state to an off stage and a smaller number less than 10% are actually activated and those are the include the pluripotency genes so it means that the our site is not making a universal activation of all genes it's quite selective it will choose the kind of genes it wants to be to be reactivated a useful guide to the level at which this reprogramming happens so in conclusion the resistance or defense or the nucleus is both gene-specific and cell type-specific so there are two ways forward at this point one is to start analyzing the chromatin the molecules that sit on genes and control their activity and we'll talk about that first so the way forward here is to change these modifications genes have histones and the histones have components on them which regulate the expression of the genes and so it's possible to inject messenger rna's on on first and then you transplant nucleon the second day and then re isolate the transplanting nuclei on day three and analyze what has happened and when you overexpress by message injection these different D methylases or ubiquity ubiquitin raises it has a substantial effect it's possible to rarely remove these components on the transplanted nuclei and so see what effect they have and this shows that this is quite effective so normally you have high level of methylation on these nuclei if you overexpress the d methylase you essentially remove all those so that's a good way of changing these components and hence determining what effect they have as a matter of fact he takes me back to one of my few experiments that I really found worked amazingly well when I a long time ago I wanted to try injecting messenger RNA into eggs and it's a proper a project which would never have had any grant support at all because it was known that if you take eggs they are full or ribonuclease absolutely stuffed full and if you add right the nucleus to message it destroys it immediately nevertheless it was the kind of thing you think well we better just try it so we injected this precious message which had been exhaustively purified by people into the egg and amazingly it worked perfectly so why should that be and I suppose the lesson you learn is that when you inject something into an egg you do it almost no damage at all you don't break open all these nucleus containing components and message injection has turned out to be an extraordinarily useful technique and I reflect on the fact that if I had not had just enough funding to try it anyway the extremity never have been done and so how lucky it is when you sometimes have a little bit of extra cash to try something a little little exotic so when you do these experiments though they are quite effective you can see that here we have the heterochromatin one which is a nuclei when you demethylate the lice in it they completely disappear and as a control this doesn't work with another particular form of heterochromatin one and this this approach is proving useful because it's now we've begun to identify some of the components that actually create resistance and there's a particular enzyme called d ubiquity neighs which takes the ubiquitin away from histones and you can see that when you do this at time zero there is no increase in gene activity two days later it's very strongly activated and that doesn't work if you use D mythili's so we're beginning to identify those components on the chromosomal proteins that confer resistance or defense on the nuclei and I'm sure there's a great deal of more work to be done in this area but it's one way forward and it's some really only possible to do that if you have these very favorable cells which can be injected with almost anything you like so that begins to answer this question but it doesn't entirely answer the whole problem because it turns out that even if you remove almost all proteins from a nucleus from the chromosomes from nucleus nevertheless the nucleus still has defense so that's rather amazing and one would say well in that case how sure are you that this defense isn't something like the methylation of DNA well known to prevent genes being reprogrammed and that's easily disproved in many cases because you can remove all of the proteins from a nucleus every single one but leaving of course the methylation of DNA and when you do that the the defense disappears so you've taken something away which was creating this defense so what could that be and the problem then is to find a way of removing components progressively from a nucleus while you can still keep it alive enough to be transplanted and this was done finally by finding a way of wrapping the nuclei in a kind of cage so that you can take molecules away from them and nevertheless they do not explode and they can be injected and the first obvious test here is to ask about the non-coding RNA is that what it could be that gives this defense what you can do is to remove almost all of the RNA by digesting it away down to about some five orders of make a hundred thousand times less RNA take it away wash it all a Dornan a back and so the RNA is still functional but so then you have RNA depleted nuclei and when you do this you find that it makes no difference so here we are with opt for gene or Sox to gene and you take away the RNA or you don't you get exactly the same resistance to reprogram it because the slow rate it goes up so that told us that conveniently you can more or less eliminate all of the non-coding RNA as a defense mechanism of the nucleus what about proteins well you can remove almost all the protein by washing the new car in high salton in triton and how they are almost all gone and yet when you've done that you find that all of the most obvious proteins that could give defense to a nucleus and as a list of them here have been removed either completely or almost completely nevertheless the resistance persists this this bar shows of unlike normal ear cells which respond very well these ones don't and so there is something sticking to those genes which we don't know about and which constitutes the ultimate level of defense against reprogramming and that's the direction which this work is now taking so I'll leave that point there and in a way summarize the the point of this talk which is to say the egg is designed to transform the sperm which normally enters it at fertilization into an embryo active nucleus and the egg is amazingly good at that it does it in 99% of cases the egg will transform this minut completely switched off sperm into complete activity so that's what the egg is specially designed to do and it's pretty good at doing it on the other hand it has a struggle when you give it the nucleus of a specialized cell it it does a pretty good job about 30 percent success rate but it can't do can't do the job completely on the other hand the nucleus of a specialized cell is designed for different purpose it's designed to keep everything very stable and you will know that we don't have brain cells in our liver or we don't have skin cells in our muscle so once cells embark on a pathway they remain very stable and that's really why the nucleus builds up this defense against change of course if the defense is completely broken down there's a problem of cancer and other things so the nucleus is designed to maintain its pattern of expression and try to resist any change now the way forward from there will of course ultimately be I think to identify nuclear defense components remove them and try to facilitate or make more efficient this reversal or rejuvenation of the nucleus back to an embryonic state but the that will take time and I'm sure the more immediate advance will be through these brilliant methods that Shinya Yamanaka has developed which we'll hear about shortly so that's I think where that is and as I say the prospect must be - for us to try to defeat resistance and win efficient cell replacement so I'll just acknowledge the people who've been helping me in this current work and I would like to take the opportunity of mentioning a number of those who throughout my career have given enormous help and worked with me Donald Brown was one of my first colleagues and Ron Lasky has been with me for something in one way on another something like 35 years helping and guiding all the way along Doug Melton edit Roberta's Lawrence Korn Marv Wiccans allen coleman Chris Graham and John Nolan or all early colleagues with me at the time the early experiments were done and particularly I'd like to thank two people who started life as a technician when I had no no colleagues no graduate students at all had one technician and these are the two ladies who used to relentlessly collect eggs and ultraviolet irradiate them and hand them to me for nuclear transfer and it's a special pleasure that they are with us here and the presentation today has particularly centred on work done by James Byrne and steena Simonson Recaro and Astro and who are both from this country thank you 1962 was a remarkable year sorry not only for John Gordon's landmark paper but the world also witnessed the birth of Shinya Yamanaka and equipped with an MD from Kobe University and in PhD from Osaka City University Shinya Yamanaka first considered a career as an orthopedic surgeon but a post doctor's day at the Gladstone institutes in San Francisco open his eyes for the world of Celle molecular biology and after returning to Japan and the narrow University he became intrigued by stem cells and in particular by embryonic stem cells that were discovered by Martin Evans who's sitting here in the audience today because embryonic stem cells can maintain their immature state and pluripotency during extensive in-vitro culturing and Shinya Yamanaka was interested me interested in unraveling the basis for how this pure potency was retained and he made very important discoveries and contributed to disappearing the underpinning transcriptional circuitry description factory circuitry for example by cloning the gene Nanog however even bolder more courageous and far-reaching ideas were entertained by Shinya Yamanaka would it in fact be possible to reinstate pluripotency in an intact fully differentiated cell and at that time this was considered a utopian dream by most scientists or at least that that would be a very complex road to come even close to that goal but in 2006 after relocating to Kyoto University Shinya Yamanaka published a paper that spellbound the scientific community introduction of only four transcription factors could convert a differentiated cell to what became known as the IPS cell and in only six years this remarkable discovery that the differentiated state could be unlocked and converted to IPS cells that has really taken the scientific world by storm and the technology is now used in thousands of laboratories worldwide including several at this University and it's used to address important questions for example to model and understand diseases incompletely in other ways Shinya Yamanaka received his MD from Kobe University in 1987 and a PhD from Osaka City University in 1993 his currently professor and director for serie the center for IPS cell research and application in Kyoto and also senior investigator at the Gladstone institutes in San Francisco Shinya Yamanaka has received numerous awards including the Lasker medical research award and the Wolf Prize in 2009 he was elected member of the National Academy of Sciences in the United States in 2012 and with this as a background please join me in welcoming Shinya Yamanaka who will present his nobel lecture under the title induction of pluripotency by defined factors pleased junior well thank you very much professor Ivan Lendl for your very kind introduction it is a great great honor for me to be selected as one of the laureates of the Nobel Prize in Physiology or medicine this year I am grateful to the Nobel foundation and chlorine Institute for her for having chosen me as one of laureates actually on October 7th just one day before the announcement I met the president of Korean Institute professor Warworld Harrington in Kyoto she chaired a session which I attended when I said goodbye to her after the session I thought she winked at me I was not sure but I'm sure now she did so I feel especially honored to share this award with Sajal garden because for us a garden initiated the field the field of nuclear reprogramming without his similar work done around the year I was born I could have never been here today so thank you very much John and I whispering a lot for your brilliant head both inside and outside so in my talk today I'd like to talk on three topics first I'd like to introduce to you my early days in science as a scientist then I'd rather like to discuss about the researchers of my own and by others that led to the generation of IPS cells by finally I learn like to discuss the potential of IPS cells so let me start with this topic my early days in science I have to say I was extremely lucky in two ways when I was a graduate student and I was a postdoctoral fellow the fast track was that I had multiple opportunities to observe totally unexpected results that brought me to completely new projects the other luck was that I was able to work under two great mentors so in the next few minutes I'd like to share with you with my experiences when I was a student and the postdoc so as you heard from Professor endo I started my career as an off-speed ik sergeant but within a year or two I realized that I was not talented at all in surgery I also realized that even a talented surgeon talented doctor cannot help very patients suffering from intractable diseases and injuries because of those two reasons I decided to change my career from a surgeon to a scientist I entered a graduate school in Osaka City University and I majored in pharmacology I studied the regression of blood pressure mainly in talks the mentor at the time was dr. Kazuki Veera so this is the very first experiment I performed as a graduate student in Osaka still University we knew that the intervenors injection of the basal active molecule freight rate activating factor path into docks caused a transit decrease in blood pressure transit hypotension dr. Miura hypothesized that this transit decrease in blood pressure is mediated by another molecule from boxin 80 so he asked me to prove his hypothesis the experiment was very simple i pre treated dogs ways an inhibitor option box and a tube if doctors dr. Mira's hypothesis is correct this should be that we this is what we should observe so we shouldn't observe any decrease in blood pressure if dr. Mira's hypothesis is correct so this experiment was supposed to be a very simple experiment suitable for a beginner suitable for failed sergeant but but I observed was something totally unexpected in the beginning it looks like the from box on a 2 inhibitor showed any effect so I observed a normal pattern of hypotension just kind of boring result but after a few minutes I observed this kind of a very profound and prolonged decrease in blood pressure when I looked at this I got so excited that I rushed into dr. Mira's office and take him to the talk although this result was totally against his hypothesis dr. Muller got also very excited so for the next for the following two years I examined the molecular not molecule have the mechanisms of this unexpected result and that began my thesis after 2 years so then I decided to continue my training as a scientist in the United States I became a poster at grab some Institute of cardiovascular diseases in San Francisco my mentor at the time was dr. Thomas hilarity this is dr. hilarities hypothesis he was very interested in one gene a public one which he thought important in cholesterol regulation hypothesized that forced expression of a phobic one this gene in dipper with lower plasma cholesterol level so by using a public one he thought he may be able to prevent atherosclerosis so he asked me to prove his hypothesis by making transgenic mice over expressing a phobic one in dipper so I worked very hard day and night and I was able to generate transgenic mice expressing high amount of a public one and not liberal specific manner one day in early morning a technician working with me rushed into me she was taking taking care these mice and she said something strange to me she said Shinya your mice many opium eyes are pregnant but they're male so I hope confused so I went to the animal facility to check the mice and I found she was right many of my mice both male and female they looked like they're pregnant so I father looked into that was going on and I found this totally unexpected result it does not babies it was this huge dipper cancers so it turned out that this gene a phobic one is a very potent uncle we can never use this in treating Pasteur sclerosis it's via crime so very naturally doctor in authority got disappointed but I got very very excited and in a sense to me writing got also excited too he encouraged me to continue working on this gym a public one so I became only one researcher we worked on liver cancer in grad so Institute of cardiovascular diseases but again doc tannerite kept encouraging me kept helping me so I'm very very grateful to him so again in a sense I had two types of great teachers great mentors in my early days as a scientist the one great teacher is a real teacher dr. Meara and Doc tonality who encouraged them despite the results which were against their own hypothesis the other great teacher of mine was is nature itself who gave me totally unexpected results and who which or who brought me the completely new project I have been trying to be a good mentor as dr. Meara and dr. Dain energy that I found is very difficult but I keep I do my best so let me move on to the second topic of my talk today I would like to discuss about researches of myself and by others that were to IPS cells so my own research that led to IPS cells has studied from this unexpected result from this unexpected liver cancer actually young Boram is here with me he is also a postdoc opto minerality so in a snow samo is unexpected result so I try to understand the cause of this unexpected cancer clearance and I was able to identify one gene which I one new gene which I designated not one novel a phobic one target number one very straightforward name so fo great one is an enzyme I identified not one as a new substrate new target of a big one and from its sequence I predicted that a not one function as a tumor suppressor gene and not one I thought not one is responsible for the tumor for the cancer I observed in April back one transient mice so I generated knockout mice and also knockout year cells to prove my own hypothesis but here again I obtained totally unexpected result it turned out that not one is essential for prepotency of embryonic stem cells ESL's while I was working on that one I had to go back to Japan I really wanted to stay at grad so I really wanted to stay in the standing in the United States but because of various issues I had to go back to Japan but before talking about my life in Japan let me discuss about embryonic stem cells as all you know embryonic stem cells ES cells are first derived from mouse embryos in 1981 by doctors Sir Martin Evans and also by dr. Gail Martin ES cells have two important properties the first one is the rapid proliferation is there a motor in um oh my god I I promise I didn't do this by hell purpose immortality the other important I'm sorry for yourself the other important property is important see their ability to differentiate into virtually all types of somatic cells and germ cells that exist in the body and it turned out that not one is a sensor for potency when I generated not one no called ESL's then can proliferate normally but they can no longer differentiate so that one is essential for potency this unexpected results again brought me changed my project from cancer to stem cells to ES cells but again when I was working on that one when I was working on that one deficient ESL's I had to go back to Japan and I suffered from a disease which I designated dat well you may wonder what it is ple actually I coined the word PID stands for post America depression so you know I had such a great time in San Francisco at Gladstone in many ways after coming back to Japan I was depressed I was very badly depressed that I was about to could quit from science but very luckily two events happened in my life that rescued me from PID the first event was the generation of human ear cells by dr. Jamie Thompson in Wisconsin before his success we only had mouse embryonic stem cells I was just using mouse embryonic stem cells I was often told by my colleagues here that Mouse cells may be interesting that uses something more related to human disease human medicine so that was one reason for my PhD but because of his success it turned out that ear cells are very related to human medicine because human ear cells have the to share the two important properties with Mouse ear cells we can expand human ear cells as much as we want and then we can make various types of human somatic cells from human ear cells such as open edge of neuron neural stem cells cardiac cells and so on then we should use these human cells to treat patients suffering from various diseases and injuries such as Parkinson disease and spinal cord injuries and so on so again it turned out that ESL's themselves could help patients not loss patients but human patients so when I first read the science paper by dr. Jamie Thompson I I got so excited I still remember that moment the second event which rescues Me from PFD was my promotion to Nara in 1999 I became a principal investigator in another Institute of Science and Technology so I had my own laboratory for the first time when I was 37 years old as you can see here this Institute has a beautiful campus they have many good faculty members they have many good equipments they have many not many but some technicians who can help me with my mice and are the most importantly they have many talented graduate students so because of these two events I was able to recover overcome my PID and I was able to continue my science because I got my own laboratory for the first time I thought I should make a clear vision or a long-term goal or my own laboratory and I made this as the long-term goal or vision of my lab I want to make years like stem cells not from embryos but from somatic cells from patient's own cells by doing this we should be able to overcome the moral eschol harder of human yourselves because we have to use human embryos to generate human yourself many people are against the usage of human ES cells against the generation of human yourselves and the usage of human ES cells but if we can make years life cells from adult patients on somatic cells we can avoid this harder Mauro huddle I thought at least in theory we can do this we can convert without cells back into the embryonic state because of two scientific findings reported by previous researchers to scientific strings established by previous researchers the vast scientific stream plus is of course nuclear reprogramming studied by Sir John garden as you heard from in stock sajin garden clearly demonstrated that somatic cells can be reprogrammed back into the embryonic state the other scientific stream this factor mediated self a conversion which was initiated by dr. Harold Winthrop who showed that a single transcription factor my OD can convert fibroblasts into muscles in mice so because of these two scientific strings I thought there should be factors that can reprogram somatic cells back into the embryonic state by identifying such reprogramming factors we should we should be able to make ears like sim cells directly from so much however I had no idea how many factors were required it could be just one it could be ten it could be 100 or even more so I had no idea how long it would take to identify these reprogramming factors it could take ten years twenty years thirty years or even longer but they luckily it didn't take that wrong it took us six years before we identified these four factors in 2006 we were able to report this finding by introducing the combination of only full transcription factors or three ball socks to Simek and k4 into Mouse skin fibroblasts we can actually convert somatic cells into es like stem cells which I designated induced pluripotent stem cells iPS cells in the following the year 2007 2007 we and others have shown that the same factor combination can make human iPS cells so I want to use just one slide to show you how we identified those four four factors first we collected 24 candidate factors with a great help of my students and technicians among them is three members of my laboratory data essential work we also established a simple and sensitive estate system to evaluate is 24 candidate factors to members of my lab yozma talk Sabha and Tomoko saga did this work actually we generated a knockout mice and knockout ESL's aubergine which I thought the sensor in Mouse ESL's and in mass re development however nothing happened to those knockout mice and knockout ESL's North wise knockout mice were just happy knockout ESL's were just happy only my student your see me began happy but that but it turns out these mas provided us a very useful system as a system to evaluate these 24 candidates so this is another reason we should never give up then by using this simple estate system very sensitive as a system another member of my lab Casa Takahashi he did animatable job to identify the four factors out of the 24 so it was in the days three young researchers who generated IPS cells Yosemite oxalá and Kostas takahashi well my very first graduate students Tomoko Sokka was it my first technician without devoted work done by these three we could have never generated IPS cells at least in my own laboratory so I'm very very grateful to these young researchers so finally I'd like to discuss about potential or IPS cells there are two major medical and pharmaceutical applications of IPS cells the first one is pharmaceutical applications by generating IPS cells from patients we should be able to develop disease models then we can perform drug screening so this is a pharmaceutical in vitro application of IPS cells the other application is in vivo vacation is cell therapy or the regenerative medicine so let me first discuss about pharmaceutical application of IPS cells by using motor neuron disease as you know motor neuron is a specific type of nerve that transmits the signal from the brain to other muscles in these patients suffering from motor neuron disease there are motor neurons get degenerated and eventually done so they will become unable to move they cannot use the muscles however they can still think they can still see they can hear that they become unable to move and in the end they become unable to breathe so it's a very very sad situation for patients and for their family members despite numerous efforts by numerous scientists we still don't have effective drugs what is patients suffering from water neuron disease primarily because we don't have a good disease model to perform drug screening for many diseases we have animal models by which we can make we can perform drug screening and by which we have identified many drugs which are effective on both animal models and on human patients we do have mouse models for motor neuron disease like ALS many drugs have been developed using the animal models many drugs have fun to be effective home nice but very sadly all of those medicine drugs turned out to be not effective on human patients so for motor neuron disease for people we do have to use human cells to perform drug screening however it is impossible to obtain sufficient amount of motor neurons from patients by biopsy to perform drug screening so that has been the limitation but now with this technology IPS cell technology many researchers all over the places in the world they have been generating IPS cells from patients suffering from motor neuron diseases and other intractable diseases then they have succeeded in making a large amount of motor neurons having the same genetic information with patients then now we have an opportunity to generate disease models using patients on motor neurons and we now have a great opportunity to perform drug screening using patient's own cells one of my colleagues in Kyoto dr. Han Giza you know where he has generated IPS cells from both patients like errors and other motor neuron diseases as well as healthy control individuals as you know motor neurons and other neurons have these kinds of projections that Nesser for signal translate signal or or transaction from brain to muscles doctor I know have shown that motor neurons from patients have significantly shorter projections than control motor neurons so we are now using this phenotype to perform drug screening we have to screen thousands of drug candidates so we have been using this kind of robotics system to identify drug candidates for patients suffering from motor neuron disease and other intractable diseases let me move on to the other application namely cell therapy or also known as regenerative medicine so just like human in your cells we can expand the number of human iPS cells as much as we want then we can make a large amount of various types of human cells from IPS cells including dopaminergic neurons retinal cells cardiac cells neural progenitor cells bright red then we should be able to use these cells to treat patients suffering from Parkinson disease macular degeneration cardiac failure spinal cord injury and preterite efficiency in Japan and in some other countries researchers are conducting preclinical studies to prove the efficacy and safe Ness of these IPS cell mediated cell therapy next Monday December 10th I'm going to receive the Nobel Prize when we have for many researchers scientists who have contributed to the generation of IPS cells and who have contributed to the very rapid progress of this technology as I described IPS cells became possible with the three free existing scientific streams the first stream has nuclear reprogramming again it was initiated by dr. Gordon Sir John Gurdon then dr. sir young women succeeded in somatic groaning in mammals in sheep for the first time in 1997 in 2001 dr. Tucker's tada demonstrated that Mouse ES cells have reprogramming factors so this dream scientific stream was essential for me to initiate the other work the second important stream is again factor mediated cell rate conversion initiated by dr. wine fraud who unfortunately passed away when he was still very young the third essential stream is ESL's embryonic stem cells initiate initiated by Sir Martin Evans in 1981 dr. Austin Smith and others have been identifying many factors that play important laws them very potency including all three for so in 1998 Jamie comes dr. Thompson succeeded in generating human embryonic stem cells so all of these works was sensor were indispensable for the generation of like yourselves now from IPS cells new scientific streams have already announced in 2007 dr. love yes provided the first proof of concept of IPS cell based cell therapy in mouse models in 2009 dr. George Daley and dr. Kevin Egan generated IPS cells from patients for the last time I have to mention here that there is another string which has emerged from IPS cells in 2008 dr. Douglas Melton showed that cell fates can be directly converted in people in during mice by introducing just a small number of transcription factors this method is now called direct reprogramming which have been which has been followed by many researchers more recently in 2012 this year dr. Deepak Shri bustable provided the first proof of concept of therapy based on direct reprogramming in mice so I really hope that in the very near future these technologies will help patients either in cell therapy or in drug discovery I also hope that some of these researchers will visit another Nobel Prize for their works in the near future so finally I am going to thank many of my colleagues of my parents in two places one the first place is in code Center for IPS cell research and application established two years ago we now have more than 250 people working in this building and grateful to all of them for their hard work I am also grateful to coat University for their continuous support to our Institute and to our science our president dr. Hughes Matsumoto is with us today let me also thank to Gladstone Institute in San Francisco since 2007 I have a small group in addition to Kyoto at Gladstone in San Francisco compared to Kyoto it's a very small group but they are all talented brilliant and independent people so oh I visit Gladstone almost every four or five or six weeks my bigger visit to San Francisco till Gladstone always activates my science so I would like to thank dr. Bob maybe and also dr. Deepak Srivastava who are also with us today for this great opportunity finally also with me today are my family member I have my wife my mother-in-law my daughter's my brother-in-law my sister well my own mother is in Stockholm but she cannot make to this hole today well she said see your English is terrible so I won't understand well as as you know science being the scientist is full Joy's but at the same time it's rope stress so without continuous support from my family I could have never been here today so I'm very very grateful to my family well I miss my father who passed away 25 years ago and my father-in-law who passed away earlier this year I hope my group they are enjoying this moment together somewhere well it was my father who talked me into medicine and it was my father-in-law who showed me how a doctor should be my father-in-law was a doctor for a long time so I really wanted I really need need to bring our technology IPS cell technology until drinks into patients I really have to do that before I meet both of my fathers in the field in the near future so thank you very much for your kind attention to some talk before concluding the novella lectures for 2012 I would now like to ask both the Nobel laureates to step forward please please dr. Gordon I am wait where do we have the promenade oh there you are and a big hat well thank you very much for those outstanding lectures and now can I ask everybody to take your seats because I would like you to stay in your seats and let our honored guests leave first and thereafter everybody can leave so thank you very much for for your cooperation here so let us have the guests of honors leaving first and then the rest of us can leave thank you very much you you

Channel: Nobel Prize

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Keywords: Nobel Prize, Nobel Lecture, Sir John B. Gurdon, Shinya Yamanaka, Nobel Prize in Medicine, Nobel Laureates, Nobel Prize winners

Id: FMVEZaJLNOg

Length: 105min 51sec (6351 seconds)

Published: Fri Dec 07 2012