Induction of Pluripotency by Defined Factors

good afternoon everyone it's not very many times that you remember where you were when you read a particular scientific paper I remember where I was when I read Takahashi and Yamanaka in August of 2006 I was at the beach I was supposed to be getting away from the scientific hubbub but I heard there was this paper that really ought to be looked at and there had been stories flying around in the rumor mill that a Japanese group had come up with a truly surprising and dramatic way to reprogram cells but the details weren't known at least not by me and now here was this paper in cell and as I read that paper I literally had the hair stand up on the back of my neck because I could see that this was transformative that what Shinya Yamanaka our speaker for today and his postdoc had figured out was really going to allow us to study development in ways that we could not previously have imagined from individuals whose phenotype we would be able to determine because they are individuals walking around at least that was my supposition now let me not get ahead because that paper described doing this in the mouse and it was another year after that in papers from Shinya Yamanaka and Jamie Thompson independently showing that this could also be done for human cells and so the era of the induced pluripotent stem cell was born on that particular strength of science produced by this remarkable scientist who were going to hear about hear from today Shinya Yamanaka has a fascinating scientific personal trajectory as well having been someone trained as a physician and as a surgeon an orthopedic surgeon and who in his typically modest way he describes as not having been a particularly successful orthopedic surgeon but that's not something I've heard from anyone except him and I suspect he was probably better than he had it's but it turned out not to be his calling and aren't we all the better for that I'm sure orthopedic surgeons are the worst for that but we're the better for that Shinya then went on to get a PhD did a postdoc at the Gladstone got interested in mouse models and development and in a series of elegant experiments after he had gone back to Japan came up with this strategy which I suspect he will outline for you that identified the way in which one could take a differentiated fibroblast and convince it to become pluripotent with the transfection of just four genes who would have guessed it was going to be that straightforward now of course it's not that straightforward and the whole field of IPS cells has been an incredibly active and exciting area and continues to be and in fact Shinya is here to help us tomorrow with a discussion we're having about IPS cell biology and the ways in which we might here at NIH in the intramural program make some additional contributions to the flowering of this field particularly in the ways in which it may affect translation so it is a great pleasure to be able to welcome this scientist to the podium today he has been honored in quite a number of ways receiving the Japan Society for the promotion of science prize and here in the US as you probably know he won the Lasker Award America's Nobel last fall and the Canada Gairdner international award as well and I think there are a few of us who would doubt the fact that he will enjoy a trip to Stockholm sometime in the not terribly distant future given the way in which this observation has emerged as perhaps one of the most exciting developments in science in the last decade he is currently going back and forth a bit between Japan and California about his main appointment is a director for the Center for IPS cell research and application at Kyoto University he continues to conduct groundbreaking eye PSL research with a couple dozen of researchers and students aiming to try to move this field in the direction of regenerative medicine so it is my great pleasure and privilege to ask you to help me to welcome to the podium professor Shinya Yamanaka nice good afternoon thank you very much Francis for the invitation and for the kind introduction I really appreciate and it's a great pleasure to be here today and it's a great honor I am so impressed that this lecture started on time this can never happen in UCSF so I'm so impressed so you may talk today I'd like to share with you the history and the potentials and some challenges of induced pluripotent stem cells so let me start with the history of myself so I did my postdoc in in the states in San Francisco in in the mid 90s at Gladstone Institute my boss at the time was Tom in a nightie and it's me and Tom and the main purpose of this slide is to show you that I had more picture I had more hairs in these days so during my postdoc training I worked on RNA editing of April lipoprotein B mRNA April B is one is the major protein component of LDL which is essential for plasma cholesterol or metabolism and two forms of a Povey protein is produced from a single Apple B gene by a mechanism named a Povey mRNA 18 which is a post-transcriptional modification of one sighting to editing this setting generates a new stop column in the center of a Povey mrna resulting in a SOTA form of a Povey protein named April be 48 this is a cycling deamination mediated by an enzyme which we designated April pick one so this is the hypothesis of Tom my boss at the time he thought that if we overexpress this enzyme a Bobeck one in diba of mice and human we should be able to lower plasma LDL cholesterol level so he expected that we may be able to use this gene in gene therapy to treat patients suffering from plasma high cholesterol and also atherosclerosis to prove his hypothesis this is our strategy we decided to generate transgenic Mouse Rhine overexpressing this a public one gene in liver specific manner so as just like many Japanese postdocs in NIH I worked very hard I worked seven days a week and experiments went very well I got these transgenic Mouse lying within six months but one day one of the technicians optom who was working with me are in the morning she came to me and she said something strange she told me that senior you are mice many of you mice are pregnant but they are male so I was I was confused I was a I was a physician so I knew only female can break can be pregnant so I went to the mouse facility and I saw my mice but they did look like being pregnant so I sacrified some of those male mice and I found instead of baby I found this huge liver in those mice so it turned out that a phobic one is a is an oncogene so we can never use this gene in gene therapy that we are crying so from this experiment I learned three important rules in science number one rule is that science is surprising it's it's difficult to predict it's very interesting that's probably why I'm still doing science the second rule is that we should never try new procedures new medicine new genes directly on patients it's too dangerous we should do animal studies the third rule which may be the most important rule is that we should not believe in your body's hypothesis so Tom very naturally he got disappointed by this result but I got very excited about this result I really wanted to study how in the world this RNA dating enzyme can cause dis ugly tumors and Tom was kind enough to allow me to continue studying these cancers in Gladstone Institute of cardiovascular disease so I was very grateful to him so I spent almost three years to understand what's going on in these tumors and I found that other another mRNA besides a pelvic I'm sorry besides a april-b is extensively edited in transgenic Mouse liver in normal liver in normal mice and normal human the specificity of a public one is extremely high one sighting in April P mRNA is the only one known target of a public one in normal situation however in transgenic man sleeper I found that this new gene which I designated not one is extensively edited by overexpressed a public one and this waiting without results in generation of numerous stop codons in the middle way of this not one mRNA I generated antibodies to this not one protein and I found that in transgenic Mouse liver not one protein almost disappeared so here's my hypothesis at the time I thought that loss of function of this new gene not one may be responsible autumn or genesee by a public one to prove my own hypothesis I decided to make not one knockout mice so I learned how to culture our CSS how to target not one gene and how to make chimeras by dr. Bob robot Ferris in at Gladstone Institute who is one of my best friends and again I worked very hard and experiments went very well I got my targeting construct within two months and I got targeted ESL's and I got comic mice but at that time my wife did something terrible she went back to Japan with my daughters leaving me behind so my boss Tom became very happy because I can work much harder welcome luck so I spend six more months in the States working probably too hard and I kind of gave up and I decided to go back to Japan but home again was kind enough to allow me to bring those climbing mice I brought three chimeric mice to Japan so he allowed me to continue this research not one research so I found that not one is essential for the re Mouse development not one now mutant mouse embryo died right after implantation as shown in this slide so because these mutant embryos are too small I cannot study the function of not one anymore using this mutant embryo so I decided to knock out not one in ESL's in embryonic stem cells because I knew that not one is highly expressed in ES cells as well so again experiments went very well I got not one - - now ESL's very quickly and I found that when undifferentiated on feeder cells not one now ES cells looked okay they proliferated okay and the morphology looked okay however when I try to induce differentiation by removing feeder cells I noticed market differences in that not one now ES cells could not differentiate at all even without penises as shown here so as you know ES cells have two important properties rapid proliferation and reportin see I found that not one is essential in promoting seed but not in rapid proliferation so up to this point ES cells simply to me a tool for generation of knockout mice but because of these unexpected results the role of not one I got very interested in the biology of ESL's itself so ESL's become a target of my research so I got very interested in the biology of mouse ESL's but after spending a few years in Japan after going back from the states I suffered from disease a mental disease the name of the disease is ta d this is an IH this is the center of Health in the world so everybody should know PA D but probably not because this is the name I named PA D stands for post America depression so it was a terrible depression so when I went back to Japan I only had three communiques mice but after two months I had fifty nice after six months I have 200 mice after one year I had 400 mice and I had to take care take care of those 100 mice all by myself I didn't have any money to hire technicians so I had to change kgs twice a week I have to wash all of all those cages by myself and the medical school I went back was was very good in clinics but at that time it does not so good in basic science so nobody I could understand what I was doing they they were not interested they were not interested in Mouse basic biology of Mouse ESL's so I was often told that Sheena used to do something more related to medicine so because of those reason I got very depressed and I was allowed to quit science but very likely to two things happened which rescued me from PID the first thing was the generation of human ES cells by Jamie Thomson because of this I learned that ES cells are very related to medicine by using yourself we may be able to treat those many patients suffering from various diseases and injuries by cell transplantation therapy however at the same time ES cells had many hurdles as well we have to worry about rejection after transplantation and we have to use human embryos so many people were and are still against the usage of human ES cells including your former president so he was strongly against the usage of human embryos so I from Jamie Thompson's work I learned that human yeast cells could be very useful but at the same time they have many many challenges as well the second event which rescued me from PID was my new appointment in another Institute of Science and Technology I was recruited as a as a PI as an associate professor so I got my first own laboratory so this institute is has very good scientific environment just like many US institutes and they have many good scientists many good they have good scientist scientific fundings and most importantly they have many many good graduate students however the system in this Institute for students is that there are like 20 laboratories in biomedical or Institute and every year they had 120 new graduate students so there is a competition between laboratories for new students and it's student choice it's not our choice students can select which lab they want to go so because as a new STI as a youngest P I and as a tourist PII I was not sure if any students get interested in my laboratory so I I thought I should have some very attractive very a good long term goal on my laboratory so that I can attract new students because of that I think I thought very hard and I made this a long-term goal of my laboratory so I try to make es like stem cells not from human embryos but from somatic cells like skin fibroblasts by means of reprogramming so in April in Japan school starts in April so in April in front of 120 new graduate students I thought that well this is the goal on my laboratory if this we can make it it's wonderful we can overcome the problems of human ear cells and we should be able to help many many patients of course I knew that this is very difficult it it would take 30 years 40 years or maybe forever of course I knew that but I didn't tell that to do those new students I only told my my dream so very luckily I got three students in my laboratory cousin takashi a kekkai ho and you see meat o Kesava so these are the very fast students in my lab and they are now a treasure oh my oh my life so with the students i we worked very hard this is my hypothesis i I thought that by introducing factors that maintain prepotency in mouse human ear cells into somatic cells we may be able to induce through potency based on this hypothesis we decided to understand how to reportin c is maintained in mouse ear cells and this is what we knew in 2000 when we started this project we knew that grief is essential in Mouse ear cells and in 1999 starts 3 was identified as an essential downstream target of live in Mouse ear cells to additional transcription factors sox2 and op 3 4 which are specifically expressed in Mouse ear cells also indispensable in the maintenance of potency in Mouse ear cells so these are very famous prayers however I did not study these very famous players because I thought it's too competitive as a very small new laboratory I thought we cannot come it with other bigger laboratories so instead of studying these famous players we try to identify new players which are not listed in this slide so you'll see me one of my first students she identified catered for as a downstream target of stat3 califo is also a transcription factor and we with Yoshimi 8co castle and some technicians in addition to care for we were able to identify some other factors which are listed in red by 2004 other laboratories were also working on 3d potency in mouse ear cells and they identified other factors which are shown in pink so by 2004 we had good numbers of factors that play important roles in the maintenance of potency so they are good candidates for all factors that can induce important C in somatic cells then we needed a simple and sensitive a/c system to evaluate this candidate and it turned out that one of the 24 or in the previous ride I had 24 candidates but it turned out that one of the 24 factors FEX 15 which we originally identified as ecad 3 it turned out that we can use this gene at such a such an asset system to evaluate these candidate factors this was done by a co another first student so we identified this gene as one of genes that are specifically expressed in ES cells a co found that F if X 15 is directly related by the two essential ESL transcription factors of three four and six - he has the sequence of of the FBX 15 and answer there are binding sites for acht 4 and 16 next to each other because of this this specific expression and because of this direct aggression by opt wall and socks - we predicted that this change to be important in Mouse ear cells and our Emil's development so we decided to make knockout mice or fp8 FBX 15 again you see me and my first technician Tomoko Jessica did this project instead of deleting simply deleting FBX 15 we decided to not clean the neomycin resistance gene into the FBX 15 locus so it's kind of a promoter trap strategy so you see me and Tomoko or did a very good job we got clinic mice and we also got heterozygous mice and I told you see me that we would not get homozygous mutant mice by crossing heterozygous mice because we believe believed that this gene should be essential in our Emil's development however we found that FBX 15 is not required in most virulent those knockout mice were just healthy I told Yoshi me that well well I knew this result but you should make knockout ESL's you cannot make nocardia cells this scene should be essential in ESL's there may be some overlapping gene in development but distance should be essential in ESL's so she tried to make a knockout homozygous mutant ESL's and she did we found that this gene is dispensable in ES cells they look just fine and they proliferate just fine I didn't have anything else to tell her so knockout mice were healthy and knockout be yourself but as healthy only Yoshimi became very unhappy so usually in such a case without any phenotypes we usually close knock out Mouse lines because it's a waste of time and waste of spaces however this is the very fast knockout Mouse line I generated back in Japan so it's it's very special to me so I couldn't just cross the line and I thought very hard any other usage of these mice and it turned out that these mice are extremely useful in evaluating those candidate factors first of all it's very easy to maintain these nice because homozygous mutant mice were just healthy because of neil knocked into the FV x 15 which is very specifically expressed in Mouse ESL's we found that somatic cells like fibroblasts derived these Nokia mice was sensitive to g418 because they don't express FBX 15 they don't express Neil by contrast we found that ES cells from these mice do Express FBX 15 so they do Express Neil and we found that these ES cells are resistant to very high concentration of g418 more than 12 milligrams per ml it's kind of waste of money but we tested but we could not kill these cells because of this finding we thought that which we expect that we should use these cells from these mice to evaluate those candidate factors so we isolated math and we confirmed that these Neph are sensitive to 0.8 then we introduced those candidate factors by means of retroviruses into umf and we expected that if some of these candidate factors do induce prepotency do you make es like status from Neph those cells should become g418 resistant we did not expect complete reprogramming complete conversion to years like status but even partial reprogramming should be sufficient to make these cells resistant to No concentration g418 that's what we expected so into year 7 4 I'm sorry in 2005 I moved to Kyoto University with these 24 candidate factors with this assay system and most importantly with some important people like kazoo and Tomoko Tomoko but however you see me who developed this assay system did not move with me she graduated and become a postdoc in some other laboratories so instead of you see me I asked castle cust Akashi to take over this project and to evaluate these 24 candidates by using this assay system well I knew this project was still very risky but very likely casually had his first paper in nature a few months ago so I thought he would be okay for the next two or three years without having any paper so that's why I asked him so he was very happy to know that he can take over this project and very naturally he tested one factor one by one and not too surprising surprisingly we he could not obtain any g418 positive colonies by expressing each of the 24 candidates as you can see here so oh I told Castle look Castle I told you this is a very good acid system because we don't have any false positive then cousin proposed a very stupid question he said Monica sensei I want to mix all the 24 retroviruses Wow I am very proud of myself who did not stop that experiment so he simply mixed the 24 little viruses and surprisingly he did get GOI resistant colonies I asked him to repeat this experiment again and again and he always got a 10 to 20 g418 resistant colonies he picked up these colonies and he found that these cells are very similar to Mencia cells in morphology in proliferation in gene expression and most importantly in promotin see so we found that some combination of the 24 candidates can actually induce potency in meth then the next important task was to narrow down factors to determine which combination out of the 24 for factors is actually required it was I thought it was very difficult because we didn't know how many out of 24 are required it can be 2 3 4 5 6 10 to 20 so we cannot we cannot simply try all the combinations it was too much even even for cars but then has proposed this time very genius experiment he he said he wanted to remove one factor out of the 20 volt factor one by one so for example in this case he removed the number one factor and transfect the remaining 23 factors so there are 24 combinations so oh then he transfected all the 24 factors he obtained like 30 g418 colonies however when he removed one factor this is what he obtained so we learned that by removing one of these four factors highlighted in bed he could not obtain any g418 wisdom coins well when he removed number 22 which is cynic he did get some g418 registered colonies but they did not look like yourself and when he picked up those colonies he could not maintain those self so we thought this number 22 Simic is also indispensable so of course he then transferred those four factors together into MF and confirmed that these four factors are essential to generate es like sales from meth so through these experiments in 2006 we were able to show that by introducing these 4 factors of 3 for 16 careful and cynic we can make es like stem cells from Mouse fibroblasts not only from meth but also from adult relative 5 of us so we designated these cells iPS cells induced reporting stem cells next year we and other sort that we can make human iPS cells with the same or slightly different combination now we others have shown that Simek is not essential we can make IPS cells without Simic but without Simic the efficiency is much much lower so we are now generating IPS cells from many patients suffering from various diseases all we need is a small procedures skin biopsy a grouping salt and in Spain they showed that we can make IPS cells even from a single hair follicle so in the future we may be able to avoid this small procedure however to some people like me a single hair means a lot so I'll do the skin biopsy but for some patients here may be more convenient but from at the moment from skin biopsy we can make skin fibroblasts and by introducing the three or four factors we can generate IPS cells and we can as IPS cells we can expand these cells as much as we want then after expansion we can induce directed differentiation into various types of cells including this kind of beating cardiac myocytes so these cells used to be skin cells just a few months ago but now they are beating so it was very very remarkable so when I first saw this beating heart I was very impressed by my heart very synchronized with this beating cells we have generated a cure cells from many many Japanese and other individuals to our surprise the efficiency of IPS cell generation is comparable regardless of the ages of donors so we can generate IPS cells even from like 80 year old with a comparable or efficiency with with no Nate so this is what we should be able to do with these patient specific IPS cells we could use these cells in toxicology making disease models drug screening a good good example of such applications in vitro application is QT prolonged syndrome which is a arrhythmia about diagnosed by elongated QT in EKG this is induced by many drugs many common drugs and some individuals are sensitive to many drugs some of them some of those patients have known gene mutation but in other patients we don't know about genetic mutation but there should be some genetic abnormalities in those sensitive individuals QT syndrome often results in this lethal arrhythmia so if this happens it's not it's terrible to patients and it's also all table to the drug company who developed that new mason's because they in many cases they have to withdraw these drugs so we really want to predict the occurrence of these QT syndrome and other cardiac toxicity the best way at the moment may be to ask those patients or sensitive individuals to do this kind of test to take the EKG and to apply new drug candidates but of course this is a very dangerous risky test this patient may undergo lethal arrhythmia so usually we cannot perform this kind of dangerous test but by using IPS cells all we have to ask is a small skin biopsy from this patient then we can make IPS cells we can expand those IPS cells and we can make beating cardiac myocytes and then we can test any new drugs on in a petri dish by using these IPS cell dr cardiac myocytes having the same genetic information with the sensitive patients so I hope this this kind of technology will be available or Barisan probably in this year in in the States and in Japan as well so of course in the future we would like to use these patient specific IPS cells in cell therapy and many papers have been published showing therapeutic potentials of both mouse and human iPS cells we have collaboration with keio university and we showed that mouse and actually human iPS cells derived neurospheres neural stem cells can be effective in spinal cord injury model mouse so if we use patient specific IPS cells in regenerative medicine we can avoid ethical issue regarding the usage of human embryos and also we can avoid immune rejection after transplantation however making IPS cells from each patient is very expensive and also very time-consuming in the case of spinal cord injury we have to transparent e IPS cell derived neural neural spheres within seven to ten days after the injury however it takes at least a month to make IPS cells it takes another month to expand and it takes another month months to make neural differentiation from IPS cells so we can never make it tool to resolve these challenges we are proposing to make IPS cell bank just like a blood bank so instead of patients in this IPS of bank we drag to make IPS cells from healthy frontiers how about HRA well we found we calculated that if we can identify 50 individuals having unique HLA homozygous alleles for the major three loci a B and D are those IPS cells would cover more than 90 percent of all the Japanese populations with the perfect match of the of the major three HRA Loki as a matter of fact we have identified a person who happens to be a medical student who has homozygous ETA REO and which is the most common type in Japanese population and we generated IPS cells from that person and we found that that single IPS cell line covers up to 20% of all the Japanese population with the perfect match of all the three loci so we are now asking enough funding to establish these IPS cell bank to the new to the government but now that we have the new Japanese government they are not so friendly to scientists so we are having a little bit of trouble so but to realize therapeutic applications of IPS cells in cell therapy we have to make sure that IPS cell IPS cells are safe as at least as safe as human ear cells so as you know NIH said yes to the first clinical trial to Jerome who will use ESL derived audio dendro sites to treat patient suffering from spinal cord injuries and I agreed that the highest hurdle for that approval was thus safety issue especially especially teratoma homogeneous t of transplanted cells so before transplantation we induce in vitro directed differentiation into like neural cells or gajendra sites or cardiac myocytes then we transplant these differentiating cells into either patience or animal models but if we still have some undifferentiated cells we will have teratomas we don't need like many undifferentiated cells if we have probably 10 or 100 undifferentiated cells in 1 million differentiated cells that is that are probably all we need to get teratoma so we compared I P as Mouse IPS cells and yeast cells in terms of teratoma propensity this was done by a graduate student Coco Yura so we induced neurospheres differentiation which was developed by dr. Hideo Cano of Keio University so Coco all generated primary neurospheres and then secondary news words from Mouse ES and IPS cells and she found that IPS cells are indistinguishable from yourself in neurosis fear formation and in differentiation into neurons astrocytes and or God and recites from neurospheres so ES cells and iPS cells comparable we can see can also made various specific types of neurons as well so again IPS cells were comparable to year cells she then labeled IPS cells with Venus and then transplant a transplanted secondary neurospheres into Mouse brains she found that those IPS cell derived neurospheres I can integrate it into Mouse brains and they can differentiate into mature neurons astrocytes and Olga and resides in people in mouths grains so again IPS cells are comparable to year cells in functional or differentiation in people email screen however after a month or two she found that some mice develop tumors after transplantation of IPS cell derived neurospheres we confirmed that these humans are turtleman's consisting of various types of tissues of three germ layers so in these experiments almost all IPS cells have nano GFP reporter so undifferentiated cells are green but after differentiation they are not green anymore usually we don't see any green cells in secondary neurospheres however we found that in some IPS cell clone we still see GFP positive cells in secondary neurospheres so she quantified the percentage of GFP positive cells in secondary neurospheres by flow or flow cytometry and this is result via cells there is almost no GFP positive cells in neurospheres from yourself we have many IPS cell independent ones like half of them equivalent to ES cells but in the remaining half we saw significant amount of GFP positive cells even in secondary neurospheres and there is a good this is very reasonable but there is a good correlation between the size of the teratomas and the percentages or gf the positive cells does positive strong correlation so the question was IPS cells are safe as ES cells and the answer is yes and no we found that some IPS cells some IPS chromes equivalent to ES cells so they are safe IPS cell clones but we also have unsafe IPS cell clones which have higher propensity to form teratomas there are two possibilities these are safe IPS cell groans even in these IPS unsafe IPS cell clones most of the cells are are good but they have a few bad cells within this distance groans the second possibility is that most of the cells in this but unsafe groans are refractory to differentiate for unknown reason so there are two possibilities but we found that even from unsafe IPS cells we can make good neuro differentiation and a suicide as well as Oliver Dental sites there is no significant differences between safe IPS cell clones and unsafe IPS cell clones so this means that this second possibility is likely the case so even in unsafe IPS cell clones there must be many good IPS cells but they also have some bad I P s l's to prove to test this facility we performed sub cloning experiment so we created these bad IPS cells at the cloning density at a very low density so after two weeks we observed many colonies and KOCO picked up individual colonies she expanded and they they almost they look all the same undifferentiated and of course they are positive for GFP because they are undifferentiated then she tried to she made secondary northfield from these subclones and we found that some sub groans like number 10 very good we have few GFP positive cells in secondary news fears however in some South groans like number 17 they are very bad we saw like more than 30% results still GFP positive so we have many good sub Crohn's and bad sad groans from a single unsafe IPs alone but by doing southern blot of in this case shocks to retroviruses socks to trans gene we confirmed that these good subclones and blood thud groans originated from same presenter so they they are very strong we then compared the expression of these good and bad clones by DNA microarray and we found the very similar but we did see some differences for example when we looked at the expression of h 19 which is a famous imprinted imprinting gene we found that we found that difference between good sub crohn's and bad seconds and sorry for this complicated slide that in stands for ESL's S stands for safe I T electron and we have three unsafe IPS er chrome and good subclones and bad subclones for each unsafe IPS alone so as you can see here in ESL's in save IPS cell chromes the expression level which 19 is his role by contrast in most of bad IPS cell clones the expression of h19 is very high by contrast in two of the three unsaved IPS clones we found that h19 is row lower in good subclones so this suggests that these differences between good sub groans and bad sub groans are caused by some kind of epigenetic differences so we also found that the origin of IPS cells have strong impact on the quality of IPS cells so again this shows the percentages of GFP positive cells in secondary neurospheres we have generated IPS cells from map and about five of us and also adult hepatocytes and we found that IPS cells from meth almost comparable to ES cells however many IPS from ttf not safe some IPS TTFN are safe so again the origin of IPS cell has a strong impact on the safeness of resulting IPS cell Chrome's so again we found that some IPS cell groans Mouse cyclists and clones are safe but some are not safe they have some minor perforation which probably has genetic abnormalities and also we found that Origen have strong impact on this phenomenon how about human iPS cells we have to remember that in most cases we establish VIP cells from adult fibroblasts which may be equivalent to mouse tilted fibroblasts also we don't have tails so all causal evaluated quality of human IDs cells so he induced differentiation of human iPS cells by moving feeders and by using five of rust culture conditions for five days and then after five days they he has receded 1.5 million cells per 100 mental dish with feeder cells and with ESL culture conditions and after 14 days we observed some airstrike colonies even after in vitro differentiation so he has tested many many independent human IPS cell phones in each dish represents independent IPS cell phone and in some this we can see only 10 to 20 years like colonies but in other dishes like this like this we saw more than one thousand years like colonies even after in vitro differentiation so this is a quantification of the previous result he repeated this assay three times for each clone so these different colors we present to triplicate results this is results of two independent ESL lines so they only produced a few years like colonies after in vitro differentiation some human ES cells are comparable to ES cells but the remaining IPS cells produced many es like colonies even after in vitro differentiation so just like Mouse I think some human yourself are not safe they should contain some minor population with minor operational cells which are refractory to differentiation so well this is the conclusion of my talk today in order to realize IPS cell based cell therapy we really have to establish uniform and complete the program in order to achieve this we already have to determine the best origin of human iPS cells and also we have to determine the best induction method best gene delivery method and best combination of factors to induce uniform and completely programming and I think what is more important is that we really have to have very good evaluation methods to evaluate quality and safe nests of human iPS cells before any applications not only cell therapy but also like drug discovery and ecology we really have to achieve these goals so I'd like to thank these young students and fellows in my laboratory this is my laboratory in Kyoto I have like 40 people including some strange we are students like this guy this is a boy boy student and also I have a small lab in San Francisco it's small but we all have begged people in San Francisco with this I'd like to finish my talk and thank you very much for your kind attention thank you for a wonderful presentation we have time for some questions there are microphones in the aisles so if you'd like to pose a question please approach the microphone and here we go yes thank you congratulations for the pioneering work and I have never heard such an excellent presentation with so many good jokes so the question I have is in the IPS cells the program did you use Mik or other transcription factors that will have reduce the chance of tumor formation yes so although I didn't show you but we found that committed mice from IPS cells having weak integration more than 50% was 60% of those mice developed tumors within one year after birth so we should not use meat retroviruses that's for sure so the question is in the other alternative method of embryonic stem cell formation the egg itself has some repressing factors which might be missing in this so you think because of this there might be higher chance of tumor formation that's a point we have been working very hard to identify other factors that can replace technique and we define some factors so we are now testing whether those factors can very safe compared to week one oh and it takes some time because we have to observe those mice fall along to at least a year thank you minutes just to follow up on that question in your screen for the factors and you have a good sa system did you find factors which have negative effect that is the suppress IPS generation from other factors could those factors be used in situations where we don't want other cells to self-renew so after identifying those four factors cause I did additional experimenting which he tested each of the remaining twenty factors plus the four factors and he did find that some factors do have negative effects those gold could those factors be useful for action yeah four years later yes thank you yeah hi a great talk just similar question on you know if you used episomal vectors that disappear from the cells after say reprogramming you think that will have an impact because you know one of the things in using Lenti or or some of the integrating vectors is you know you don't know if they will turn back on at some point or that's what's causing this so we did make mouse IPS cells with grass meat and we have two types of IPS cells one with plasmic integration so it's like a stable line right but the remaining we did not detect any plasmid integration so it's a transient expression so we found that we we made commitments from those types and we found that even with past plasmid if Mick trusting is integrated it's as dangerous as retroviruses right we observe very high incidence of tenacity however from IPS cells without integration we did not observe higher instances of domestic so I think it is Simek integration I think transient expression of Semak should be okay but we really have to do longer observation to make definite companies that only Simek that's affecting the tumor Janissary or you think factors are responsible as well well so far you know we observed those mice almost two years but we do not see increased origin st in mice having insertion of the remaining three factors look for sixteen and catered for so Civic is very dangerous but the other three factors seem to me okay thank you let me ask one of the questions so you showed elegantly the difference between safe and dangerous clones can be identified by doing the separate cloning out and they come from the same original retroviral a transformed cell it would seem that given the h19 results that as you said epigenetics may account for the differences so if you've done a systematic assay of the chromatin structure for instance or with DNA methylation to see whether you can identify a whole host of things that are different between good and bad clone well that's exactly what we are now working on but a student student coco who did this experiment just move to another laboratory so it's it's being slowed down well there will be a reception in the library all right now but let us thank our speaker again for a wonderful presentation you

Channel: NIH VideoCast

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Id: AD1sZU1yk-Y

Length: 69min 51sec (4191 seconds)

Published: Mon Nov 15 2010