My name is Deepak Srivastava, and I direct the Gladstone Institute of Cardiovascular Disease and the Roddenberry Stem Cell Center at Gladstone, and I’m a professor at the University of California in San Francisco. Today I’d like to share with you a story that illustrates how we can begin to use knowledge we gained from studying developmental biology, so early embryonic development. To understand the causes of human diseases and even find new therapeutics for human diseases. And I’ll largely focus on diseases of the heart, but I’d like to highlight the fact that the principles I’ll share with you could be applied to many different organs, and many different human diseases. So I have a background as a pediatric cardiologist, and the disease that I take care of is illustrated here. Which is a child, newborn, who was born with a heart defect, where during embryonic development this baby’s heart just failed to form properly in the right 3-dimensional shape. And as a result, after birth the child had a number of problems and required very early surgery, as you can see here. And this type of problem is extremely common Heart defects occur in 1% of all live births worldwide And despite our best efforts at surgical intervention, which have now become quite good, it remains the leading non-infectious cause of death in the first year of life. So we have to understand this disease better, and in the future, hopefully treat them in a better way, both through preventative measures, but as well as treatments that could change the natural history of this disease over time. Now it’s been our hope that if we understood how nature normally forms a heart in an embryo, that it might inform us of this disease. But it also might inform us about a different kind of heart disease. And that’s the type of heart disease that occurs in adults, which is very different from what I just mentioned to you. So in adults, heart disease across the world now is the leading cause of death. Just within the United States, there are over 1 million people every year who have heart attacks, and because we’re doing better and better with saving those individuals, the problem has been that they are saved, but they have damage to their heart. And as a result of that damage, there are now 5-6 million people living in the United States who suffer from what we call heart failure, which is when the pump just because of the damage can’t pump well enough to support the circulation in the body. And if you look worldwide, that number is about 24 million. So it’s an enormous health problem, and right now we have very few approaches that can address this disease and none that are actually curative. Because at the end of the day, if you’ve lost heart muscle from damage, the only real way to fix that is to create new heart muscle. So to regenerate the heart, and right now we don’t have any ways to do that And what I’m going to show you is that possibly, by using the clues from nature of how it normally makes a heart, that in this situation we might be able to create new heart muscle for this disease. So the themes of what I’ll share with you today are: one, to understand how early cardiac cell fate decisions are made in the embryo as it’s first forming And that involves both understanding signaling pathways that impinge of these decisions, transcriptional networks that establish these very cell fates, and even translational networks that are embedded in these pathways using microRNAs that then titrate the right dose of these networks to exert the proper outcome. And the second thing that we’ll talk about, is how we can utilize the knowledge of these networks and overlay them on human genetic models to begin to understand the basis of human disease. And not only understand the genetics of those disease, but also understand the mechanisms using induced pluripotent stem cells in a dish. And finally, I’ll share with you how we can use these developmental biology networks to reprogram cells in situ in an organ for regenerative medicine So all of this work starts by going back to the basics of how does an organ form in utero? And for what I’m showing you here, these are the steps that we know of that are involved in a heart forming early during embryogenesis. And so we know that as early as two weeks after fertilization in the human embryo, there are cells like you see here that begin to align themselves

in this crescent shape. And already at this stage, before there are any organs in the body, these cells already know that they’re going to become heart muscle in the future. And even at this stage they’re already parsed in these pools of progenitors that I’ve highlighted here in either red, purple, or yellow. And these cells go on to form specific parts of the adult heart. And in particular, you can see that these yellow cells align themselves in this back of this tube that forms at about 3 weeks after fertilization This tube starts beating, and then these yellow cells come into the top and form what’s going to later become the right ventricle of the heart. And the red cells form the left ventricle. So already, we’re beginning to see that there are different pools of progenitor cells that will give rise to different chambers of the mature heart. And this begins to explain an observation we’ve had in pediatric cardiology for a long time, and that is that children are often born where there are defects in just one part of the heart but the rest of the heart is normal. So for instance, a child might be born missing their whole left ventricle, or missing their whole right ventricle and the rest of the heart is fine So now we can begin to understand why that might be Because you can imagine that there might be a mutation in a gene that was critical for these yellow cells to form, but not the red or vice versa, and if you have that mutation, you’d lose only that part of the heart but not the rest of the heart And so we’ve learned a lot about this process at the molecular level by using a variety of model organisms including fruit flies, zebrafish, chick embryos, and mouse embryos And collectively in our field, we now know quite a bit about the key genes that drive the events you see on this slide But animal models turned out to be very difficult to use to get at the earliest cell fate decisions that occur in the first two weeks of life, as I’ve mentioned earlier. And for understanding that process, we as a field have turned to the pluripotent stem cell system in a dish, where we can take pluripotent stem cells and guide them along a path in a stepwise fashion like you see here. Where they become mesodermal progenitors, and those mesodermal progenitors become these multipotent cardiac progenitors. And we can even mimic the different pools of progenitors in cells in a dish here So those red and yellow cells we can isolate in a dish, and the yellow cells are very similar to those in vivo, where they are multipotent. They can become cardiomyocytes, endothelial cells, or smooth muscle cells. And so we’ve utilized this system to work out the intricacies of how these step-wise decisions are made at the earliest time points, to tell an early cell that it’s going to become a cardiac progenitor and then a cardiac myocyte. Or an endothelial cell or a smooth muscle cell. And together, by using animal models and stem cells in a dish, we as a field have developed a quite deep understanding of the gene networks that are dictating cardiac cell fate and subsequent morphogenesis. So we know a lot and maybe know most of the players that are critical. And we’re still trying to figure out the exact mechanisms by which each of these critical central players are actually exerting their effect. And currently we now have the tools to do that in biology that we couldn’t even do actually just a few years ago. So, we’ve known enough to know what the key players are, and so we’ve begun to ask, are these gene networks that we’ve discovered the ones that are in fact disrupted in the setting of human disease? And the answer to that turns out to be as it’s evolving, is that yes, these are indeed the key genes that are disrupted So what I’m showing you here is a subset of the key gene networks that are involved in controlling the yellow cells, red cells, blue cells at the top, and most of the genes on this slide are transcription factors that are playing key roles And we can begin to arrange them in a hierarchical network Like what you see here. And what’s been satisfying is that each of these gene networks is hit one or more times, with mutations in humans. And I’ve indicated those mutations — those genes that are mutated by asterisks in this slide. And what I want to emphasize

here is that in every one of these cases, the gene mutations are actually heterozygous mutations. So only one of the two copies in the genome are mutated, and in many cases, the one that’s mutated is not even a complete loss of function mutation But maybe only a single amino acid has changed, so it’s a hypomorph We’ve only reduced the dosage of that key gene in its network a little bit. But that’s enough to get over the disease threshold And so what that tells us that the dosage of these networks is exquisitely controlled in a precise fashion. But it also suggests that if we can just increase the dosages of these networks a little bit, we might someday get over the disease threshold. And so the hurdle might not be so high, to prevent some of these diseases I just hope that our understanding of this process might actually lead to some preventative measures in the future Now our laboratory has, in specifically, identified years ago, mutations in a very important developmental gene called Notch1, that causes valve disorders. And we’ve also identified mutations in another transcription factor called Gata4 Which is very important for formation of the septum of the heart So the walls that divide the different chambers of the heart And I’m going to spend a little more time sharing with you some of the things we’ve learned recently about the mechanism by which this heterozygous mutation actually causes human disease. Because at the end of the day, just identifying the genes that are mutated doesn’t get us that much further to a therapy, we have to understand the mechanism by which these gene mutations cause disease. Before I do that, I want to also highlight the fact that we are now able to generate exomes of thousands of individuals with disease And in an effort to a National Heart Lung and Blood sponsored consortium, we and others have found that there are in fact a number of genes that are involved in congenital heart disease And in most cases, those mutations are also heterozygous mutations. And so, from the mutations I described to you before, and the ones that we’re beginning to find now through exome sequencing. As I mentioned, if we really want to understand the mechanism in a human cell, we’ve got to have human cells in front of us to study. And for many years, we tried to understand the mechanisms by making mouse models of these mutations. And we learned some things, but at the end of the day, most mice that were heterozygous for these mutations were fine. The threshold in mice turned out to be very different than in humans. And so the key advance that has allowed us to now begin to understand mechanism has been the discovery by our colleague here, Shinya Yamanaka, of induced pluripotent stem cells. So Dr. Yamanaka found that we could take fibroblasts or blood cells from an adult, and introduce just four critical genes. And those genes were enough to reprogram those cells into something that looked and behaved just like a human embryonic stem cell, in that they were pluripotent. He termed these iPSC cells, or induced pluripotent stem cells. And this discovery opened up the possibility that we might be able to take stem cells and make stem cells from a patient, and use those cells for regenerative medicine by transplanting them back into the patient because they’d be genetically identical And there are many efforts to do that, but that’s still years away. But what is happening now with this technology and I’ll share with you an example of this, is generating these cells from an individual with a genetic mutation, and then turning those pluripotent cells into the cell type that you want to study the disease. And then you can now have billions of cells in front of you that you can now study that has the genetic mutation that’s causing disease in the patient, and now you can finally begin to interrogate those cells. And really get at the nitty gritty of why did that mutation in that patient cause a problem in the cells that are now suffering from the disease. So this was a real breakthrough that allowed us to make the progress that I’ll share with you. So if you want to do that for any cell type, the first thing you have to do is be able to generate large numbers of the cells that are affected by disease So what you’re seeing here is a sheet of cells that used to be on somebody’s skin. But now have been transformed into a syncytium or a sheet of beating cells that are all beating

synchronously. And these are human iPSC derived cardiomyocytes. We’ve become very good at making these, and we can make billions of these cells with 90% pure cultures of these cells And so we’ve done this now for the human disease that I mentioned to you where we’ve had this mutation in this critical gene, GATA4, that’s absolutely essential for normal development in the heart and embryo. And so what you see here is the family that we took care of and studied some years ago that spans 5 generations Everybody who has this single amino acid switch from a glycine to serine at position 296 in GATA4, which is a transcription factor, has the disease. And while we had identified this gene mutation many years ago, this is an example of where we got stuck by just studying the mice. Because the mice that are heterozygous were pretty much okay and didn’t have a lot of the disease manifestations. But what we did think a few years ago was that this point mutation might be causing a problem in the ability of this protein to interact with another protein called TBX5. And the reason we thought that might be the case is because humans with mutations in TBX5 have the same problem that humans with GATA4 have Which is that they get holes in their heart, or septal defects And the amino acid that’s affected in this protein is shown here, and it sits right next to a zinc finger in GATA4 that’s very critical for protein-protein interactions. And so when we made this mutation of GATA4 and overexpressed this mutant protein in a non-cardiomyocyte years ago, we could show that GATA4 doesn’t in fact interact with TBX5, and this mutation breaks apart that interaction. But what we didn’t know is does that really happen on DNA in a human cardiomyocyte when they’re at their normal levels. And what I’ll show you is in fact they do, and we’ve been able to experimentally show that and understand why that would cause trouble So, this is a heart from one of the family members, that I showed you before, that does not have the GATA4 mutation, it’s like a normal heart. It’s an ultrasound where you see the left ventricle is here, the right ventricle is here. And these chambers are pumping normally. What we found is these patients with the GATA4 mutation not only had holes in their heart, but as some of the kids got older, they ended up having a different problem And here is an ultrasound of one of those kids who’s now a teenager And what I’d like you to appreciate is here in the right ventricle, you see a lot of white stuff here. That white stuff should not be there. It should be black, which is meaning an empty cavity full of blood. Instead, there’s a lot of muscle fibers that are invading into the cavity of this ventricular chamber And the heart isn’t pumping as hard as it should And so, this is a problem that’s often thought of as a problem reflecting immaturity of the muscle cells. And so we thought we might actually be able to see if that’s actually happening in their iPSC-derived cardiomyocytes. So, we’ve generated iPSC cells from these family members that are shown here in 8 individuals, four with the mutation and four without And we took skin biopsies from these individuals, transformed them into stem cells, and then into beating cardiomyocytes like I showed you earlier. And when we made these cells, which were done by two talented trainees in the laboratory, Yen-Sin Ang, a postdoc, and Renee Rivas, an MD PhD student They made these iPSC cells and looked at the RNA of these cardiomyocytes. And it turns out, if you make iPSC derived from all 8 of these individuals and try to compare mutant ones with the mutation and ones without, there’s a lot of noise in the system. Because each one is a little bit different And so, the real breakthrough that allowed us to get to the next level came with the advance in technology of gene editing So, by using CRISPR technology, we could go into these iPSC cells and just in the patients who have the mutation, correct that mutation so that everything else in the genome is the same except for this one nucleotide change And so we called these isogenic controls, and now when you compare the isogenic controls with the ones that

have a mutation, and everything else is the same, then everything displayed itself because the noise went away And so, we were then able to figure things out that I’ll show you in the next few slides. So the first thing we wanted to know was can we in fact recapitulate the problem of the muscle being immature and having trouble contracting in these cells even though they started on somebody else’s skin? And to do that, we used a system we developed in collaboration with Beth Pruitt’s lab, where we can grow these cardiomyocytes not in the dish like the culture I showed you before, but rather have each muscle cell in an individual well by itself in these patterned microwells that are indicated here. So here, each of these colored areas you see is a single cell in a well And these cells can beat and we can actually measure their force of contraction as individual cells. And so we’ve done that for a number of cells, so in this graph, each of these dots represents a single cell and the force they generate And in the red cells are the ones with the mutation compared to the ones without. And you can see that the force generation by these mutant cells is less than the ones that don’t have the mutation. So in fact, we’re able to recapitulate some aspect of this cardiac dysfunction in this artificial system. So we then looked at the transcriptome of these cells, since this is a transcription factor mutation, we wanted to know how the transcriptional output was different So what you’re seeing here is a heat map of the genes that are downregulated with this mutation and those that are upregulated. And what you’ll notice is that there are a number of genes, about a third of the genes were downregulated And if you look at the GO terms, of the gene ontology terms, they characterize the genes that are downregulated It looks like this. It’s enriched for genes involved in heart development, cardiac chamber morphogenesis, contraction So all the genes that should normally be turned on so this muscle cells knows it should beat vigorously and turn into a heart are decreased. And in contrast, if you look at the even larger number of genes that are apparently upregulated, and look at the GO terms there. This was really quite interesting There are a whole host of genes that are involved in vascular or endocardial development that are now up that should have been shut off. And so, somehow just because of this single heterozygous point mutation, there’s a whole host of genes that should’ve been shut off that now aren’t. So why is that? So, to answer that, we began to look at where do these genes TBX5 and GATA4 normally sit, and how might that disruption actually cause the problem? And so what I’m showing you here is the ChIP, chromatin immunoprecipitation, or ChIP-seq sequencing data of GATA4, where it sits on DNA, TBX5 and where it sits on DNA And a histone mark, Histone 3 K27 acetylation, which marks active enhancers. And what you’ll see is that in these areas here that are blue, which indicate where GATA4 normally sits on DNA, which each horizontal line representing a single genomic locus where GATA4 is binding to the DNA If you look at the comparable areas where TBX5 sits, about half of the places where GATA4 sits across the whole genome, it’s sitting there with GATA4. So these are really obligate partners throughout the genome. And it’s essential for those to interact at those sites. And if we look at this more broadly and look at the sites across the genome where these two proteins are co-bound In the wild type setting, there are about 2500 spots And it turns out, that in the setting of this point mutation, about half of those, indicated in blue here, are lost So with this mutation, at about half the sites where GATA4 normally sits on the DNA with TBX5, its partner, it no longer can, because of this point mutation. And if you go a step further and ask, what are these genes where this interaction is lost? What types of genes are they? And look at the GO terms of these, again, it’s those genes that are most important for contraction of the heart muscle, for the heart to form, for general heart defects to occur. And so this begins to explain why this point mutation in GATA4 is actually causing the

cardiomyopathy and the congenital heart defects at a very fundamental level of its inability to now control transcription in the proper way in the human genome. So this is an example that the power that this approach can bring to understanding the mechanism by which this disease occurs. Now, I’ve told you so far about how we can use this to understand why a muscle cell might not contract normally. But what about the 3-dimensional aspect of this disease, where you have holes in the heart? When we set out, we thought this might be really difficult because it’s a 2-dimensional system of these iPSC-derived cardiomyocytes, yet we’re trying to model a 3-dimensional — to understand a 3-dimensional problem What I’m going to share with you in the next couple of slides is how we believe that we’ve actually been able to decipher the mechanism of this 3-dimensional defect, despite having a 2-dimensional system, which we think bodes well for other form and other types of disease modeling for genetic defects. So to do that, I first need to give you a little bit of background in one aspect of developmental biology And that is that early during development, there is a signal that comes from the pulmonary endoderm, which when the heart’s forming early in an embryo, this pulmonary endoderm sits right behind the developing heart, right next to it And it secretes a very important morphogen called Sonic Hedgehog, which is involved in development of many different organs. The neighboring cardiac muscle has to receive that Sonic Hedgehog signal and in response to that Sonic Hedgehog signal, actually forms this atrial septum that you see here And the Sonic Hedgehog signal is received by a receptor on the muscle cells called Patched, and it leads to a series of intracellular events that are mediated by a transcription factor called GLI in the nucleus. And that then helps the muscle cell respond to this Sonic Hedgehog signal, and then grow this septum. And we know that if you delete Sonic Hedgehog from the pulmonary endoderm, you end up with mouse hearts that don’t have a septum. And if you delete GLI from the cardiac muscle cells, you also get holes in the heart in a mouse. And the reason I’m telling you this is that it turns out that in our iPSC-derived cardiomyocytes with this GATA4 mutation, we found that Sonic Hedgehog and the machinery in the cardiomyocyte to receive this Sonic Hedgehog signal was downregulated broadly. And in particular, the receptor PTCH1 and PTCH2 were severely downregulated. And the transcription mediators, GLI2 and GLI3 were also downregulated. So what we think is happening here is that in the setting of this mutation where GATA4 and TBX5 should normally sit at those regulatory regions to turn on PTCH and GLI, they don’t. And so now you get downregulation. And so even though the Sonic Hedgehog signal is coming from the developing lung, it can’t be received properly in the developing heart. Thus resulting in this defect. And so, what we think is going on in this setting based on what we’ve learned by going from the developmental biology, understanding the genetic mutation, and then modeling in iPSC-derived cells, is that normally GATA4 and TBX5 have to broadly across the genome sit at parts of the DNA to robustly turn on cardiac genes. And at the same time, they have to sit at other sites across the genome, like endothelial or cardiac genes, and actively shut off those regions by recruiting co-repressors. In data that I didn’t show you, what we found is that this is an active process and that has to happen for normal development. And in the setting of a human condition, where all you’ve done is made this protein unable to recruit its cofactor, that now we see a situation where there’s aberrant downregulation broadly of cardiac genes and aberrant upregulation of other genes that should be shut off and now aren’t shut off. Thus, affecting the function and behavior of those muscle cells So, what I hope you can tell from this, and this example could be applied to many, many diseases, is that by deeply interrogating a human cell in a dish that has the mutation that causes disease and using gene editing approaches, we can really get at the nitty gritty of why this disease occurs. And now we have cells in the dish that we can use

to screen for drugs that actually might reverse the problem that we’ve identified. And we’re actively doing that now So this is the end of the first part of my talk, related to how developmental biology can be used to understand the human disease. And in the second part, I will talk about how we can use developmental biology gene networks for regenerative medicine. Thank you very much

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