 Thank you very much for the invitation or for the opportunity. I'm a human geneticist and I got into comparative genomics because we reasoned that it might be a good idea to look at differences between humans and non-human apes and other primates in order to understand what makes us human. We failed. We don't know what makes us human. We find a lot of interesting things along the road and it's been quite a journey. As I said, what really motivated us at the beginning is to try and understand the basis for adaptation of complex phenotypes in humans and those include adaptations that ultimately make us more susceptible to diseases. But we didn't know how to do that and we were committed to try to do that by studying humans and our close, extant evolutionary relatives, not go to mice or other model systems and that of course makes everything challenging in some respect because of the ethical and practical considerations of accessing humans and non-human apes. Back in 2003, we started simply comparing gene expression levels between species with the hope of understanding combined with the comparisons at the sequence level, understanding of some perspective on how gene expression changed, regulatory change and perhaps ultimately connected to some form of adaptation of complex phenotypes and we failed. Around 2007, when we couldn't really push it through to the complex phenotypes, we decided to take a step back and since we have now such great maps of differences in gene expression, transcript expression between species and we understand something about the evolutionary forces that shape those transitive expression even though everything we say about complex phenotypes is just at that point storytelling, we can go back and study regulatory mechanisms and that has been work that is still ongoing and work that continue to evolve as new technology evolves and new protocols evolve and the ability to visualize and characterize new regulatory mechanisms evolve and it's work that opened up a whole I think window into studies of regulatory variation within population between humans, the entire EQTL field and regulatory QTL field and I think that for me for us the journey was very satisfying and that interplay between comparative genomics with large effect size where causalities out the window to within species where effects are smaller but you can make more inference about causality using some form of mediation analysis was really gratifying but really the first time we were finally able to take one step towards some measure of complex phenotypes was when we studied changes in protein expression, still everything that had to do with complex phenotypes itself remains just storytelling and then in 2011 where finally in 2013 we established it we started working on adaptation in cell culture now that's a far cry from adaptation in vivo or anything you can do with actual model species and in this conference we're clearly at a disadvantage where a lot of you are working on actual organisms and animals in a multiple species and can perform experiments we are committed to working on humans and chimpanzees and species that we don't want to really touch and so everything is still in vitro but it's using a system that we couldn't use before which is the IPSEs so this is a legacy slide which for sentimental reasons and some bet I'm never changing but the numbers are outdated we now have close to 20 validated chimpanzees IPSEs and actually close to 200 human IPSEs and a lot of them are done exactly in the same technique using the same technique and they're really highly validated and of high quality we validate them using a large number of assays including of course making sure that they're fully pluripotent making sure that the clones that we choose represent the particular individuals from which the clone was derived routinely when we wake them up we check their carotide and and those are fluid and so a lot of time we have to just terminate some clones and go back to clones that are healthy and have healthy carotide at the beginning of this journey we used to do teratoma assays and thankfully the field moved away from those as a critical checkpoint of course we do eb's for every cell and actually we continue to do these eb's routinely to just make sure that the lines continue we have a lot of early stage line but even the late stage lines are continued to be fully pluripotent as you may have heard I worry a lot about batch effects and so we have studied at great length and at great detail the potential batch effects that might impact work with stem cells what I'm showing you here specifically is the relative impact of starting the IPSC generation from individuals that are from different populations so different population of species of chimpanzee what what what some or maybe most cold subspecies different populations in humans and we showed that whether you study gene expression gene emethylation and I didn't put other slides here on histone modification accessibility and so on the obviously the species effect is much more pronounced than the effect of the different population but you still have the effect of different population visualized which is a good thing because it means that the stem cell reprogramming maintains the genetic identity of the donor and whatever variation you see actually between individuals an interesting observation that we confirmed and and now using a large number of tissues is that there's less regular variation in IPSCs than in practically any other tissue that was studied terminal cell types here specifically you see the difference between fibroblast LCLs and IPSCs and the bifurcation the trees on the two sides are the different species and and in the IPSCs you can see you can see in the IPSCs there are there's just much less variation than in any other cell type and that is interesting of course because everything that intuitively we immediately associate with early developmental stages canalization and I'll kind of get back to it when I demonstrate two utilities of these IPSCs we spent a lot of time really a fair amount of time in our lab validating differences or studying differences between differentiated cells from these IPSCs and primary tissues and I'm gonna take a minute to explain how important that is and to acknowledge that this well not perfect is something that we think about and and does show that this model is useful so the the issue here is that we are doing in vitro work and we are doing in vitro work because we are unable to do in vivo work and when you're a human geneticist the two competing models that you have are always in vitro cell lines or go to mice or other model system that are acceptable depending on the phenotype that you're studying and mice are not humans and in vitro is not in vivo and you never can never get out of those types of criticisms they're all valid but the fact is that you're not going to experiment on humans and you're not going to experiment on chimps and so we are choosing the in vitro system we are choosing to work though not on immortalized cell lines that are more standard but at all those differentiated cells from IPSCs and we study at great detail the similarities and differences between the differentiated cells and the primary tissues that they're supposed to model to understand to what extent those systems in vitro systems are faithful to what extent they're useful and we optimize different protocols in order to create cells that are closer to the primary tissue so in this particular example we going for cardiomyocytes and we're using different protocols to generate cardiomyocytes and we're testing the gene expression the methylation other phenotypes in those cardiomyocytes comparing them to the same data collected from primary hearts sometimes from primary hearts from the same animals from which we actually created the IPSCs because this is this has been a long process as samples that I and my lab have been collected for actually almost 20 years and some of these chimps passed away and then we were able to collect their hearts not anymore the NIH has a moratorium on collection but we have a lot of those tissues in our freezers so I'm not going to ask you to read all this this is this is a large slide that shows that essentially cardiomyocytes are most similar to hearts but there's also a take home message is which is that you'll never confuse cardiomyocytes with hearts cardiomyocytes form their own clusters they don't look like anything else other than hearts but they also don't exactly look like hearts but when you actually look closely to figure out what are those are going to be differences between cardiomyocytes and hearts you discovered that the vast majority 70 75 percent of those genes can be explained by the culture in conditions there are not necessarily artifacts associated with the fact that this is an in vitro differentiated cardiomyocytes but for example differences that are associated with the fact that you don't have lipids in your culture in media and so a lot of the genes that respond to lipid metabolism are shut down and those the vitro cardiomyocytes you can perform experiment with primary cardiomyocytes culture the same way and you can get a little bit closer profile so the take home message here is that these systems are useful they're far from perfect but you can characterize the extent to which they're useful and the extent to which they might not give you faithful information so without in mind I want to I want to just tell you two quick projects without getting into too much details just provide examples of the utility of this system in comparative genomics and of course I chose two studies that you couldn't perform using frozen tissue samples or the standard immortalized cell lines the first study is a study of comparative of a developmental trajectory that of course is impossible to do other than in vitro in humans and chimp and the second study is really the reason we started this back in 2011 which is to study some phenotypes that are a little bit more complex than just study the molecular profile of a sample and I call this project the in vitro heart attack we're changing the oxygen level in the cardiomyocytes so here's the first part is a comparative study of endoderm differentiation we started with this honestly for no other reason that it was just the first differentiation protocol that worked in the lab for both species it's a three-day protocol it's easy of course since we its comparative genomics and we can't have batch effects we require that the cells will differentiate to the same endpoint using the exact same protocol and it's surprisingly difficult actually to have one protocol working with similar efficiency in both species and so as we develop more protocols that and today we have quite a few for hepatocytes for cardiomyocytes for chondrocytes for other cell types but the first one was was the definitive endoderm so it's a three-day protocol we did it in four IPSCs from chimps in four IPSCs from humans plus a couple of replicates and skipping nine months of QC I immediately will jump to the fact that most regulatory trajectories here are highly conserved in human and chimp and that was completely expected that definitive endoderm is such a fundamental basic tissue shared in all mammals and obviously highly conserved in human and chimp and so we expected that and that was actually in many ways good to see because that means that our IPSCs in human and chimps and the differentiation protocol performs equally what was surprising to us but I think also very rewarding is to observe this reduced within species variation in gene expression in primitive streak which is the samples in day one in observation that we didn't have in any other day so what you see here is the the p-values rejecting the nipals is that the f-test that that assumes that there is no differences in variation and these plots are a little more complicated to explain these rely on the pi zero estimate from one species given that you observe reduction in other species but the take-home message is that this pattern is highly shared so it's not that random genes have reduced variation within the species across individuals as the differentiation moves from day zero to day one these are genes that consistently have reduced variation in both humans and chimpanzees and so this idea that you really canalize this differentiation to a particular developmental state a particular network state that you need to hit roughly at the same point at the same time with hundreds of genes is something that we seen in both species and that that's not something that we've ever seen before and in fact it it really changes a little bit the way that we now think about analyzing these types of comparative data because we're no longer just looking at these changes in gene expression thinking well in one species it went up by this much in another species it went up by only this much so that's different we're now thinking of hitting a particular state a particular growthary state and if you're already there maybe you don't need to change much that doesn't mean that there's a difference between the species if you hit the same state that might mean that you were already where you're supposed to be and that state might be conserved and it's a little bit different way of of analyzing the data the second project I want to tell you about is really where I think most of this lead and we have three projects like that now in the lab but I chose to speak about this one because this one is the most mature in fact already published and so what we do here is we take kadiomyocytes and we grow them in neuromoxic oxygen conditions and so for those of you who don't do much cell culture or did cell culture only you know briefly during your training you're probably cultured cells in just atmospheric oxygen which is 21 percent that's not neuromoxic conditions that's high oxidative stress for your cells they're actually a which is a topic for another whole different conference or there are a large number of papers that got their results a little bit wrong because they measure everything in oxidative stress conditions so these cells start from neuromoxia by the way most of my lab obviously does it at oxygen levels that is atmospheric so i'm sure that we get that problem too but in this particular case since we have to give them heart attack we can't start with oxidative stress conditions so we start with eight to ten percent oxygen that's condition a we move them to one percent that's condition b we keep them there for six hours or and move them back to neuromoxic condition and we have two recuperation two conditions where they can stay in that high oxygen level eight percent before we measure the RNA as well again either six hours at sea or 24 hours at the end let me tell you immediately because you might wonder by 24 hours namely 24 hours after reperfusion the cells are nearly indistinguishable than what they had the starting point at a which means that this entire process was generating back the the cells we start with and you no longer or almost no longer have any indication of stress so uh reperfusion give them high oxidative stress before it goes back b of course is is lack of oxygen and that's kind of the you know fondly i refer to this as the heart attack now much of the of the of the response is conserved but you see in these plots are dust the dot plots of gene expression differences in chimpanzee on the y's and the humans on the x from a to b from b to c and from b to d and everything is or almost everything is near the diagonal you see something a little bit of a subset of genes in color that represents the differences between species in that response but most of it is conserved here are examples of conservation these are genes that are important because we know something about their function and they respond very similarly i gave this example mainly because of this gene where you can see that the actual level of expression sorry i thought that you see the the gene in the middle you can see that the actual level of expression between the species is quite different but the response response pattern across conditions is actually almost exactly the same now i'm not going to take you through this slide in great detail but tell you the take home message that that these genes with interspecies conserved response are actually unusual and they're important for human disease in many ways they're enriched among cvd g was there enriched among particular genes that are seen to participate in different disease process that affect the heart and they're depleted for ecu tails that are identified in g-tech so so those ecu tails that are basically high effect sizes shared among a large number of tissues those are not the conserved response genes for heart attack and reperfusion so they have a lot of signatures of of importance and and they create these they generate this list of genes that provide additional promising targets that for follow-up and this is not you know for the first time in my career this is not this hand waving uh you know these seem to be under selection let's follow them up we never will because i don't believe the selection in the first place these are actually gene that respond to particular medically relevant intervention in a conserved way between two species they're highly enriched in process that we care about these are real uh worthy targets of follow-up and and some of them we do uh there are obviously also differences and those are extremely interesting differences in response genes that respond particularly in chimp genes that respond particularly in human as you may know uh there is uh quite a lot of work about the differences between human and chimp in the way that the the two species experience cardiovascular disease and some of these genes are just you know the storytelling about them that what we know about them may fit right in to try to explain those phenotypic differences and so i think for the first time we have through this in vitro system some access towards these processes that we couldn't address using frozen tissues standard cell lines and clearly not anyway in vivo in these species so the eyepieces are really cool and you should have some we are sharing these eyepieces we have been sharing them for many years we can't share them overseas because of cites especially with the new rules it's it's practically impossible it's just impossible we tried but we are sharing them domestically we shared them with 26 labs already in the united states there are in fact more papers using panels of chimpanzee eyepieces that we created from other labs than from my own lab and we're pretty proud of that we're sharing them with no restriction limitations we don't need to be a collaborators we don't ask you what you do with them we don't care if you compete with us it's just the resources and that's how we treat it here's a you know quick and dirty summary that i'm kind of proud of of the type of topics that people use our eyepieces in their papers that's not papers from my group and and all it takes is an email and a couple of forms with your keys and we give you the cells i'll end by acknowledging your keys and southwest foundation and dri because i wouldn't have a career in comparative genomics if not for these primate centers hard work and continued commitment and and and collaborations with our scientists that rely on these samples and of course energy ri as well as energy message bi for for funding this type of work for so many years thank you