Stem cells and genome editing | Professor Janet Rossant FRS

thank you I was just talking earlier and said last week I was at a public lecture by Alan Alda the actor whose big mission today is about communicating science and one of the first things he said was get out from behind the podium so I am out [Laughter] and now I'm ready to tell you about some of my research but I've set this in a broader context I really want to talk about stem cells and genome editing which you hear a lot about in the public domain and in literature and I want to talk about the implications for these developing technologies for precision medicine so how they can be applied to human health but I'm going to set it and then try to set it actually in the context of some of my own research and my research over the years has been focused on this embryo here this is the mouse blastocyst and this is the stage of development that I first encountered when I was a graduate student with Richard Gardner in Cambridge with Martin Johnson and I fell in love with the blastocyst and over the years on and off I've been working on this stage of development sometimes going up and do other things because the tools weren't there but we keep coming back to this embryo and the mouse blastocyst at this stage here is the size of a speck of dust it's but about a hundred cells and it's just about to implant in the uterus so this this embryo then has three cell types and outer cell called the trophectoderm a group of cells at one end called the inner cell mass which contains two cell types at this point primitive endoderm on the surface and this little group of pink cells called the epic blast and the questions that we wanted to ask were what are these cells what do they give rise to we know that now from many studies from many people over many years from the 1970s to the present of more and more detail we know that reflected on gives rise to the major parts of a placenta the primitive endoderm to the yolk sac and this little group of cells are what we call pluripotent because they give rise to the entire fetus so that then was what I was working on before I when I was here and came in Oxford and Cambridge and then I left the UK in 1977 married a Canadian moved to Canada and started thinking about stem cells I met my stem cell heroes this is turn McCullough they are the people in Toronto who first identified stem cells in the Hamada poetic system and their concept that there was a self renewing cell that retained the capacity to make multiple cell types is really fundamental to how we think about all stem cells today self renewal and retaining potential and then that started me thinking and others as well could we make a stem cells from the blastocyst in particular of course people are particularly interested in whether this would make stem cells from the pluripotent cells and it's lovely in Toronto outside the Mars facility where again the foundation is we have a statue of till in the color and here's Jim with his statue and you can actually sit on there and have a discussion with your heroes so what we now know of course is that you can get stem cells from the mouse blastocyst the most famous being embryonic stem cells which derived from those epiblast cells now we tried to make them we were not the first to make them they were derived by Matt Kaufman and Martin Evans in Cambridge and Gail Martin in San Francisco but they are the cells that behave like these pluripotent epiblast cells they grow indefinitely in a dish expressed specific transcription factors you put them back into an embryo may contribute to all the cell types of the fetus but not at the center not to the yolk sac and my lab made trophoblast stem cells which continue to divide indefinitely we made Zen cells and you can see when you put those back in chimeras they do what they would have done in the embryo so we've captured that linear specification state in there through cell lines they make em they make ended and they make placenta and it's these cells of course that in the end have been the most famous the other ones are great we do lots of good thing with them but embryonic stem cells of course have this capacity apparently to make all cell types and mouse but will they really play a potent if they were then yourself alone should be able to make a mouse because that's what the epi blast in the blastocyst can do so my colleague unrest knowledge and I did a did a bit of genetic trickery in what I still think is my most interesting experiment ever because what we did was we said well if ES cells could make the fetus if we combine them with an embryo that can make trophoblast and ended in but can't make the EPI blast then maybe we can have a complementation system whereby the ES cells can actually take over the fetal compartment and that's what you see here it's a tetraploid embryo Green ES cells blue put them together entire fetus is es derived but more excitingly of course is if you let those go through and you're gonna get whole litters of mice that are perfectly viable they're all GFP expressing this time around and they are entirely derived from ES cells themselves have been grown for many generations in a petri dish and to me that is still a pretty amazing thing to see but it was those those papers where we demonstrated the true pluripotency of mousey yourself that really underlie then the concepts that if you can do this in the mouse then what have you could do it's in the human because now you'd have a stem cell line in which you have endless supply of cells to make every cell type in the body and potentially use it for regenerative medicine and of course again we know the history in 1998 Jamie Thomson in Wisconsin was the first to drive human embryonic stem cells same thing at whoops sorry as we do in the mouse we take take the blastocyst and derive these cell lines and indeed in cell culture human number and it's dense now differentiate into many many different cell types but what was more exciting still and really transformational and leading on to this sort of precision medicine connection that I want to talk about was the discovery by Shinya Yamanaka that you could actually do away with embryos and take adult cells both mouse and human skin cells blood cells other cell types and add back express in those cells some of the genes that we and others had shown were important for the pure potent state in the embryo and you could turn back the clock and turn adult cells into embryonic stem cells essentially these are called induced pluripotent stem cells and they can differentiate in culture and now this opens up the possibility of personalized stem cells and now we're into the beginnings of precision medicine because we could take cells from everybody in the room take them turn them into induced pluripotent stem cells and have an endless supply of cells to study your genetics but also potentially in the future to provide yourself they would allow you to be already treated for serious degenerative diseases so rejection free transplantation is a long-term goal here a long way off I would say the most important short-term goal is that these IPS cell allows to model human disease in the petri dish and think about screening for new therapies so of course as we know Shinya Yamanaka was awarded the Nobel Prize in 2012 and I always like this because this is sort of how science goes along with the person who first got me interested in developmental biology when I was an undergraduate not said John Gurdon who was a first of course to show that in frogs you could use nuclear transfer cloning to reprogram an adult nucleus back to the beginning of development so this is fundamental basic science but hugely influential going forward so these cells these pluripotent stem cells have rapidly opened up new vistas in regenerative medicine and people often say well you know - pluripotent stem cells we've heard a lot about them are they really moving forward to the clinic well the answer is I think yes just really in the last two or three years I've become convinced that we are seeing some of these discoveries from pluripotent cells moving towards the clinics and this is just a quick list of what's going on now around the world and the diseases and syndromes and degenerative problems that have been addressed with potential sales from pluripotent cells spinal cord injury macular degeneration Parkinson's there's an international consortium including people here in the UK that's very close to a phase one trial you using dopamine neurons diabetes insulin producing cells and exciting but very difficult using ES cells to generate real heart muscle to repair and that's going on again in good preclinical studies so just to say I've been changing my slides on the run here because actually just last week this announcement came out that in Japan this is the first clinical use of IPs derived dopamine neurons to treat Parkinson's patients so the first patient has been treated with these cells in Japan so things are really moving forward and we're going to see these treatments moving through clinical trials and hopefully into the clinic but as I said really the biggest use of IPS cells right now is this concept of being able to study people's disease model disease in a dish and so we got involved in this and this is because I at the Hospital for Sick Children so I'm going to give my research here as an example of what you can do with IPS cells - mortal disease and help develop therapies so cystic fibrosis a single gene disease recessive disease causes it's the gene was cloned in Toronto at SickKids in 1989 it encodes a chloride channel CFTR chloride channel and over the years since 1989 until very recently improvements in survival for kids with cystic fibrosis were all related to improvements in treatments of the symptoms but not addressing the protein recently there are drug treatments based on repackaging the CFTR channel and improving its function that are in the clinic however they're not they don't work on all patients even with the same mutation and they're very expensive so there's a big desire to really have a way of telling which patient is going to respond to which drug so a personalized test before you give the drug to the patient and so what we've been doing the lab which is really work by a research associate Amy Wong is taking embryonic stem cells and then IPS cells and CF patients and driving them towards differentiating into ciliated cfd are expressing lung epithelial cells and then using those long epithelial cells to test drugs so just here show you proof of principle at the top we see that we have wild-type ES cells tight Junction epithelium expressing see after you're on a cell surface CF mutant cells no CF on the surface TFT are on the surface treat them with drug CFTR comes back on the surface so we can measure the response now we've transformed this into a multi world plate system so you now can take individual patients and look at their response to an agonist of the CFTR protein and you see here's two patients same mutation different patients one response to the drug one does not so we're seeing now these individual responses and of course now what we're doing is really comparing the cell responses in culture with our colleagues work in the clinic who are seeing the patients and their responses to drugs so this is really moving forward through this large-scale study that brings together many groups across the hospital to really essentially try to get to a point where we get the right drug to the right patient and again this is kind of approach is going on worldwide with many different diseases and even in cystic fibrosis there's an even shorter term assay that's been developed by Hans clava sand bed big man in the Netherlands where they take primary cultures from the gut and make little gut organized so organizers are a new way of beginning to have new models for human disease what this what we see at the top here is these little sissies little gut organized again treated with the agonist they swell because the CFTR channel functions the CF mutant cells don't swell treat them with drug and they swell and they're actually now beginning to use this clinically to test which patients to receive which treatment so this is really them moving towards precision medicine so we're looking at personalized drug therapies based on modeling disease in addition across different diseases we're also seeing stem cell derived fara peas but really this is all very well if we know that patients have a genetic disease like cystic fibrosis why are we still struggling to try to replace their cells with stem cell therapies or to treat the patients with drugs that they're going to have to take for the rest of their lives if we know what the gene is why don't we just fix the gene so gene therapy gene replacement gene correction can we do that well of course you know what I'm going to say is that we have a new transformative agent here another game-changer after stem cells the game-changer is really CRISPR cast 9 gene editing and this is a tool that was first discovered in bacteria it's how bacteria fight off viruses and it was discovered by people working on yogurt because if your bacteria dies it gets infected by virus you're in big trouble and so the companies put a lot of effort into understanding how bacteria fight viruses and they do it via this nucleus the cast 9 nucleus which cuts DNA mix the doubles trying to cut very precise positions determined by the binding of this guide RNA and now as people have worked through this and turned it into a tool that can grow out of bacteria into men yourself you can essentially design the guide to direct that cut to wherever you want in the genome of whatever organism you want and so it has two three actually there's a missing piece they're three very important characteristics that one has to bear in mind it's very specific more than most other kinds of approaches very efficient and should have said cheap so there are other nucleo systems out there but the CRISPR cast name is cheap and the founders of this technology Jennifer Doudna Manuel Fangio Fong Jiang George church members have really democratized it made this available to everyone worldwide you can genetically modified plants animals bacteria whatever but in the context of having personalized therapies we can start now really thinking about somatic gene therapy not just adding genes but potentially really manipulating the genome within enough cells in the body to have a potential cure for a disease and this is again moving extremely fast forward in the clinic in many different areas this slide just gives you an idea of just some of the different diseases for which people are thinking about ways that we might use what we will call somatic gene therapy editing the genes within the person who has a disease and they really cover a wide range so cystic fibrosis is certainly one where you might imagine getting an adenovirus into the lung and fixing repairing the gene in enough cells to recover function you can think about haemophilia these these are also moving to the clinic where you would take again add more virus and correct the hemophilia mutation in enough cells to get enough function people talk about in the long run maybe you should go in and knock out pcsk9 and adjust your cholesterol levels skin skin diseases epidermolysis bullosa and these I think you're really interesting once in the blood systems probably the ones that are moving fast because there you can take out the cells edit them outside the body and put them back so sickle-cell disease in beta thalassemia the idea here being not actually to edit and repair the mutation but to reactivate the fetal globin gene so little provide enough function to allow the patient's to really have a much more normal existence and again these are moving rapidly to clinical trials many companies and many organizations around the world really pushing this hoard at the somatic cell level so all good but oh let me just show you this my favorite of where this is moving recent work from Eric Olsen's lab suggesting that you may even be able to do this for Duchenne muscular dystrophy which I had always thought was a a target that would be impossible because every muscle and the boys bodies are degenerating so you'd would you be able to get enough gene repair across a whole body to be able to at least delay on the progression of the disease and again not yet in humans but in mice and in dogs this is recent work in dogs in a preclinical model Olson's lab has been able to show that they can repair sort of make repair this this mutation here that causes an out of frame they can cut it out the me frame put the gene back in in frame and get enough really quite amazing recovery not every cell of course but using a V virus they're getting across different muscles in the body this potential repair so we're seeing this really attacking diseases that I would have thought were impossible only a few years ago so if we can headed out genes and patients with disease why would we do that so each stage you know why would we give do drug therapies if we can collect the gene if we can correct the gene the patient who has a disease why don't we edit it out before they get the disease so this brings up the issue of germline gene editing so we've all seen these kind of pictures since they design a baby we're using CRISPR cos nine in the early embryo you could in British potentially removing the Cystic Fibrosis mutation repairing Huntington's disease other single gene deep defects could be repaired in the early embryo and then though all the cells of the baby and the next generations would have that repaired gene so this concept then of germline gene editing has really got a lot of publicity and I did I'm I'll show you later I had to change my slides again so but why am i interested in this I'm going to take this back to my research so thinking about gene editing in embryos the first place you go and test all these things out of course it's in the mouse embryo so we're back to the mouse so can we gene edit the mouse embryo how efficient is it and why would we want to do that so I want to tell you a little bit about my science now back to the blastocyst so I'm going to give you a quick survey of how the mouse blastocyst is to put together and why we want to be able to do more gene editing to develop better tools to understand how a mouse blastocyst is put together so we are still struggling with understanding how you get from an egg to the blastocyst and the dynamics of the process involved we do that because we still want to understand how pluripotency is set aside in the embryo and we hope to use that to make better stem cells either directly from embryos or by induced pluripotent c and we need better tools for gene editing we've been able to genetically manipulate the mouse embryo for many years by slow tedious processes where we actually do it in embryonic stem cells make chimeras breed the chimeras breed the chimeras to make Mouse lines and then we can do our experiments if we could do it so efficiently that we could do it right in the embryo then immediately we have the mice with the mutations and the alterations we want and the speed and the complexity of experiments we can do increases dramatically so why do we want to do that and what we want to do let me tell you a little bit just two slides really on what we know about how the blastocyst is actually put together so we know that there are key linear specific transcription factors for all three cell types that are affected on the epiblast and the primitive endoderm we know they're necessary if you knock them out some of them are sufficient to change cell fate but what we have spent more time struggling with still I would say is to understand what's upstream what turns on those transcription factors we assume that there must be some signals going on as the embryo develops with cell to a blastocyst that signals the onset of this first cell decision in the Snowmass vs. trophectoderm and the next epoch blasts and proved who ended them so what is going on if we just look at what the expression of these proteins here this is serious - yes a transcription factor that's required for the trophectoderm in the inner cell mass here we've got got assets which is a primitive endoderm factor enamel which is a epiblast factor and they're all in a bit of a mess here we've got double positive single positive so clearly and everything we know says that this is a gradual segregation of the lineages so what's going on well we think that the first cell decision between the inner cell mass and trophectoderm is this is summarizes everything we know almost everything we know to date and what we know is that actually the first decision depends on the outside cells being different from the inside cells and the outside cells express CDX - because this curl activator protein called Yap is in the nucleus and drives downstream transcription in the inside self yeah this phosphorylated doesn't enter and instead of trophectoderm markers we turn on epiblast markers like socks - its phosphorylated by the Sirians raining kinase laughs which is in this complex on the cell membrane and in the outside cells because they're apq polarized this complex is torn apart and therefore Yap can enter in yes so we think it's a hippo certainly we think there are mechanical signals cell polarity we still have to understand the dynamics of what's going on here but at least we have a an idea of what's leading up to the final lineage fate so what about the next one well I showed you already that as you go from making an inner cell mass and the trophectoderm to making the primitive endoderm and every blast you start with a kind of mishmash of cells double positive single positive and eventually they sort themselves out a different process from just the the morphogenesis of the Moriya and the blastocyst there's a cell sorting going on here we think we know that a different signaling pathway is important here and it depends on fgf signaling and so cells that receive fgf for turn on downstream genes like garlis 6th and become primitive endoderm cells that don't express the receptor and don't turn on the downstream pathways become epiblast what we don't really understand we know the downstream responses here we don't understand what's going on here it's just a stochastic process is it a dynamic process all that other signals are important here and how the cells actually segregate them out themselves out of the blastocyst stage so we still have questions we always have questions in science it seems so these are just some of the things we want to understand what we're trying to explore in the lab my Ladin other labs as well the roles of contractility polarity position signaling and can we actually monitor this in real time this is a complex process we want to study and be able to visualize the processes that are occurring the embryo can you monitor transitions in transcription factor can we disrupt the pathway it's very specifically and this is why we keep whenever we have these questions in the lab there's always question well we need more different mouse lines we particularly need to have more fluorescent tag fusion alleles and other synthetic biology wheels so we can watch the dynamic process so this is where two postdocs in the lab been goo and Esther I took this on and said well Chris Burke hath 9 is great but it doesn't work very well when injected with mouse embryo into the zygote to do the kind of in search big insertions of fluorescent tags and other things that we want to see and so they asked well why is that and they with a bit of lateral thinking they found they worked out or maybe they were just lucky who knows but basically whoops where most people have been injecting into the zygote stage let's think the single cell they worked out that the best time to do this was probably at a two cell stage which has a longer g2 period and homologous recombination which is what we have to do to actually get the insertion of the the fluorescent alleles into the cut made by cast nine is bound to occur largely during g2 this is also a period at which the the chromatin is extremely open because this is like gothic genome activation anyway whatever the reason I have to say we don't really know if those those are rational rationalizations whether the real mechanism the bottom line is this works and works very efficiently so what we were making the first place a fluorescent fusion alleles for all the different genes that we want to study in the early embryo and this is how it's done people I will show this because people are always surprised it means that both cells or the two cell stage yes quite easy really you just turn it around we get all lots lots of emails about this whole procedure of course and there's always this question it's okay if I can just inject one it's like well why don't you end it both doubles your chance so that's that's the procedure and here were just the first experiments were done nothing very fancy here for those of you know about gene editing and the tools were using messenger cast a message a single guide RNA circular DNA template was sort of one KB arms and just the first experiments then two of our favorite jeans socks two and got a six going from zygote injection got a six not bad actually but at the two cell stage then we really go up from very low to about thirty percent and doing this this approach wasn't quite good enough for bin so he added another twist on this to make it more efficient in this case what he did was to follow some work have been done by Chinese grouping culture where they added streptavidin to the cast iron protein and in vitam elated the template the homologous recap the pair template they were going to use so this just helps everything come together the PCR template and the enzyme get brought in association and this now increases your efficiency again so what we see here is that we're something like goddess six which is our favorite genes it's so easy to target we're up to close to a hundred percent efficiency a hundred percent of those who are examining at the blastocyst carry the insertion allele and in fact we've had live mice born that a homozygous insertion so this is now moving to extremely high efficiencies so with this we have been able to generate a sort of catalog we have epic glass markers to affected own primitive endoderm others all of them now going through and again bearing in efficiency between 10 and 50 percent and up to a hundred percent so now we are showing that this can be an efficiencies that start you thinking about germline editing so just to show you a couple of pretty pictures what we're seeing here is an image of the embryo going from the moral apply the green is seedy it's too tagged and then you'll see the red that sucks - coming up in the inside cells but you'll see they never seem to overlap so we do think that CDX - and socks - really are balanced out as shown in that model in terms of the the role of Hippo signaling pathway this is just we're now combining wall markers so let's have three at once this is now in collaboration with our friends with a light sheet microscopy - Lelia and now you're seeing the detail of analysis now you're seeing the white which is a seed yet - being segregated and you're seeing within the inner cell mass again this complex very dynamic process that eventually leads to a segregation of the endoderm and epiblast but once you start looking at this you see it's not that simple so more analysis ahead certainly for this so there's lots of things we can do in the mouse with it and we and others are obviously gonna do this it just moves us forward and mouse genetics new revolution in Mouse genetics all that ESL stuff that we did for many years those cells are sitting in freezers everybody is doing with gene editing we can use synthetic biology tools we can we think we can do genetic screens right in the embryo by efficiently making stop codons in in the genes we can have multiple reporters not just in the embryo but in other tissues as well maybe the people who are looking at the cell at this might want to tag every gene so we could look at that in different ways and of course then the question becomes can you extend it to non-human primates or indeed to humans so I am NOT saying that the approach is we're doing in the mouse the - cell injection it's necessarily going to be something you want to move to the human because even at the most efficient approach that we have we are not at 100% efficiency if you want to genetically correct a mutation in a human embryo you need a hundred percent efficiency every time and you need to be sure that it's safe and you have no off target effects so but it does mean that it's so different from where we were just a few years ago that the concept of doing human German germline editing has is a realistic possibility everything is improving every day new approaches are coming out to improve the efficiency of this kind of gene editing so it would allow individuals who have genetically related children without passing on a known risk of a genetic disease has potential we wanted to to alter the predisposition to diseases like cancer heart disease Alzheimer's and of course enhancement back to that picture of the designer baby one of them was you know improved athletic ability I think we could all think of a few genes the one could edit the one would know would actually do that so enhancement is not out of the question in terms of the possibilities then these of course raise all the sort of ethical concerns that we as scientists in society have to start discussing where do you draw the line well I was part of the first international summit on human gene editing in 2015 it's me on stage which came out with this statement that basically said it would be irresponsible to proceed with clinical use of germline editing until we know it's safe and efficient we need to know the risks and potential benefits and there's a broad societal consensus and whatever that means about the appropriateness and regulatory oversight at present these criteria have not been met after that meeting the National Academies of science was one of the many working groups that around the world have considered this that Naas spent a year with many many stakeholders I was part of that process as well and came out with a very influential document that I have summarized pages and pages into a sort of traffic light system the conclusion was that Germa that genome gene editing for basic research because it is a hugely important research tool if I've just shown you the mouse embryo should be done in human cells and in human embryos and here in this Institute Cathy knockin is using gene editing to study the same kind of questions in the human embryo that we're addressing in the mouse embryo that's the medically and editing to treat disease has huge potential and that would have no particular different regulatory concerns than current gene therapy protocols might even be safer germline editing I put a amber here means which means not a complete ban but think very carefully don't mind adding to treat serious genetic disease where no alternative exists and all the safety issues have been addressed so not a no but very high bar to pass before that should go forward and then I read to either somatic or germline editing for enhancement purposes now define define serious genetic disease define enhancement these are the issues so that was those that was the call from from that group and I think over the last year or so since that's come out if anything worldwide including here in the UK there has been various surveys of the public and so on to say that people are beginning to be a little more understanding that general energy might be considered one day so the immediate sort of you know gut response oh no we shouldn't do that has softened but nobody has really is really proposing that were anywhere near safety and efficacy to move this to the clinic at that time so you know what I'm going to say now as I'm yesterday so that's why I got in my talk I'm an actor add a slide so just yesterday we heard that Jonghyun he in China has claimed to produce twins in which he used in editing just the way I showed you in the mouth to make mutations in the ccr5 gene which is the receptor for the HIV virus which in series should prevent those babies be infected with HIV it's pretty pretty clear that this is this contravenes everything I just said about germline editing as in the NES report this is not a serious genetic disease there isn't you are if there's an enhancement this is definitely an enhancement in fact potentially dangerous enhancement because loss of that protein also makes you more susceptible to other viral infections so we don't know the consequences even if it's true and it's not correcting a serious genetic disease and it's unclear exactly what ethical permissions he had his University has certainly disowned him and is calling for an international investigation and the hospitals in which he claimed to have had ethical review don't seem to agree and experiments were done in other hospitals and this is really difficult it's particularly difficult for our Chinese friends because you saw in that first genome editing summit that actually on the stage was was challenging pi who was who was one of the Chinese leaders and the Chinese Academy so this everybody has been working on an international consensus here and so a Chinese scientists feel this is really obviously something that they want to object to so those are there is an announcement signed by Chinese scientists which basically says this is unethical it contravenes everything that we've been working for and that China needs to be sure that they can send in regulations to prevent this happening in the future but there it is and so it occurs exactly the same time so I'm here and not at this meeting so just as well and that's the second international summit on human daily living is taking place as we speak in Hong Kong and that doesn't seem to me to be a coincidence that this report was put out at the same time as its meeting so the question is we get a new consensus emerging from this meeting can we move forward because this is going right against everything that anyone around the world is working for and it happened in China it can happen anywhere it's very hard to completely control what is as I said an efficient easy and cheap approach to modifying the genome so that's it we've gone from the blastocyst to stem cells to editing to ethics but it all begins and ends with the blastocyst and I just have to thank for some of the research that of my own research they talked about Amy Wong for the lung work as the post I've been good for the to cell editing our collaborators at Genelia for the imaging and our collaborators at Karolinska for the some of the RNA sequence single-cell analysis that is part of what we do to understand how the mouse embryo develops so thank you [Applause]

Channel: acmedsci

Views: 227

Rating: 5 out of 5

Keywords: academy of medical sciences, health, janet, rossant, sickkids, toronto, canada, expert, lecture, mouse, embryo, blastocyst, stem, cells, pluripotent, regenerative medicine, medicine, research, talk, stem cell, therapies, gene, correction, CRISPR, cas9, editing, ethics, medical, duchenne, cystic fibrosis

Id: D0CEfzMXgy4

Length: 40min 3sec (2403 seconds)

Published: Mon Dec 10 2018