 Hello, everybody. Welcome to the Ethics and Research and Biotechnology series sponsored by the Center for Bioethics at Harvard Medical School. This is a monthly seminar series that explores issues at the intersection of ethics, technology, and bioscience, all with an eye to our practical approaches and policies and ethical responsibilities. I'm your host, Nsu Ken. I'm director of research ethics and senior lecturer at Harvard Medical School Center for Bioethics. And I'm professor of bioethics and philosophy in the Department of Bioethics at Case Western Reserve University School of Medicine. Now, if you're new to this series, I do have a few points to make about logistics. So first of all, thank you so much for joining us online. This event is being recorded and it's being live streamed via Facebook. The event video will later be posted on the Center for Bioethics Facebook page and on YouTube pages if you want to return to this later. You can submit questions at any time during our discussion by using the Q&A function. Don't use the chat function. Use the Q&A function for your questions. And we'll try to get to as many of those as we can at the end of our presentation. If you have any technical issues that come up, then you can use the chat feature to send a message to the panelists and staff that might try to help you with that. If you have any interest in upcoming events, you can visit the website for the Center for Bioethics. And with that now, I'd like to introduce our speaker for today. Our co-presenter is Hiro Mitsu, Hiro Nagauchi. He's professor in the Department of Genetics at Stanford Institute for Stem Cell Biology and Regenerative Medicine. He's professor of stem cell therapy in the Institute of Medical Science at the University of Tokyo. He is a renowned expert in hematopoietic stem cells and in the topics that we will cover today. I do want to make a few disclosures before we get started. First is that the International Society for Stem Cell Research is revising the guidelines for stem cell research, including chimera research. And I'm the chair of the Chimera Research Committee and Dr. Nagauchi is also a member of that committee. I'm also the co-PI on an NIH funded project looking at the ethics of human animal chimera research in collaboration with scholars at the Hastings Center. So what I expressed today are not views that necessarily correspond to those of my colleagues. One more alert for our viewers. There are slides with pictures of animals that you might pretty much expect to see in a scientific publication, a little bit of dissection, some organs. So I just wanted to give a little warning about that. Again, nothing you would not see in a scientific publication, but there are some images of rodents. So with that, I would like to turn it over to Dr. Nagauchi, hero. The topic for today is growing transplantable human organs in livestock. So I'd like to turn it over now and please take us through some of your work. I hope you can see my slides. Yes. Good. Thank you, Mr. for your introduction and good morning, good afternoon. My name is Ciro Nagauchi and I've been working on organogenesis project. And that's what I'm going to talk about from now. So I have two laboratories, one in the University of Tokyo, Tokyo, Japan and another in Stanford University. These two laboratories are working together, cooperatively trying to translate discoveries of basic sciences into the clinic. And I will tell you why I have two laboratories later in my talk. And in vivo organ generation project is the one I started as a sort of a side project about 12 years ago. And as in soon introduced, my major field of research is hematopoietic stem cells. So I'd like to start with a little bit of background. As you know, organ transplantation is the only cure for those with end stage organ transplantation. Because however, because of the shortage of donor organs, number of patients waiting for that transplantation is increasing as you can see here. In the United States alone, 20 patients die each day waiting for a transplant. And because of this absolute lack of donor organs, there's even a black market selling organs. And this is not a minor business. Actually more than 10,000 organs were sold in 2010. So this is a worldwide ethical issue ethic. So this is the current situation for organ transplantation. And as you can see, these people must have donated, I mean sold one of the kidneys because we can see the operation scar here. So although this is the only cure for the end stage organ failure, but there are several issues here. The biggest issue is of course, as I mentioned, shortage of donor organs. There's even, there's illegal organ trafficking. And also immunological rejection is another issue because organ transplantation is usually from somebody else. So we have to give patients immunosuppressant throughout life after transplantation. But these two issues could be solved if we can generate transplantable organs from patients' own stem cells. Then there should be no immunological issues involved. As you know, now we have IPS cells. In addition to ES cells, IPS cells can be generated from the patient. So we should be able to provide the patient's own cells organs, tissues. However, current therapy targeting diseases that can be treated by cell therapy. But with a cell therapy, you cannot help people with end stage kidney failure or liver failure and so on. So we need organs, not just cells, but obviously it's not easy to generate organs in a culture dish because it has a 3D structure and it has many different cell types involved in the organ. So my idea is to use in people environment to grow human organs because it should have everything, every environment necessary to grow organs. But of course we cannot do this in human. So we are thinking of making human animal chimeras, particularly livestock animals to grow human organs from IPS cells obtained from derived from the patient. Then we can solve these issues, shortage of donor organs and immunological rejections. So I think the audience about the IPS technology but let me explain a little bit about chimeras. A chimera is not a monster, it's an animal that has two or more different populations of genetically distinct cells. The important thing is chimera is not a genetic mixture, but just a mixture of cells. So we do not touch genes and chimeras cannot expand as chimeras. They only have two different jam cells but never a hybrid mixture. So the easiest example is partial chimera. For example, patients after blood transfusion or bone marrow transplantation, organ transplantation, they have somebody else's cells inside the body. So there are chimeras, a number of examples for partial chimeras. But what I'm going to talk about is a little different. It's a systemic chimera when two very early embryos are aggregated or mixed together very early in development because both of them have a capability to differentiate to become into different cell types. The chimera has two types of cells in all tissues and organs. This is one example. This is the rat mouse chimera from the left. It's a wild type mouse. And the right mouse is the wild type rat. And these two in the middle, rat to mouse for mouse to the chimeras. So it is obvious just by the cold color, they are a mixture of cells. So these are the chimeras that I'm going to talk about. I guess there's no need to talk about IPS cells. So I just tell you how we can, how we make chimeras. We, for example, if we want to make mouse to rat chimeras, we prepare mouse pre-important stem cells could be IPS cells or ES cells. We also prepare brass early embryos for rats. We call it the brassed system stage embryo, which is about three to four days after conception. So this rat brass is a small cavity. This is still a cluster of maybe a hundred cells. And this portion becomes the body and the other part will become a placenta. So anyway, we inject mouse IPS cells into the cavity of this brass system. And then this is how we do it. This is the brass rat brass system. And we are injecting mouse pre-portal stem cells through this tiny pipette. You can see these cells being injected. And 24 hours later, if we mark the injected the ps pre-portal stem cells with red, then you can see mixture of red and also host cells. Then we transfer this chymidic embryo to a foster mother. And an important thing is the host foster mother and host brasses has to be the same species. Otherwise, they cannot accept and maintain pregnancy. So in this case, we use the rat foster mother. And then three weeks later, we see birth of chymidas and many of them grow into adult and they look like this. So mouse to rat chymidas. So this is how we make inter-species chymidas. So now you know IPS cell technology and also how to make chymidas. So our future goal is this. We're trying to generate human organs in livestock animals. Suppose this is a patient with end stage heart failure. And then we generate IPS cells from this patient and we inject them into the brass system of livestock animals. But one idea that we made is we use organogenesis disabled animals. In this case, for example, we can make a pig that cannot form a heart. So that when we make a chymera, if we can make a chymera, this pig chymidic pig should have human cells everywhere. But in a heart because a host pig cannot make a heart. Heart should be all derived from patients IPS cells. So once the heart has become appropriate size, then we can take this heart out and transplant back to this patient. Although this heart was generated, formed in a pig environment, the cells are all from patients own IPS cells derived. So, you know, essentially this transplantation should be autologous transplantation that should not require any lifelong immunosuppression patients own heart. So this is the idea. And this whole idea is called blast cyst organ complementation. So we, this sounds like a scientific, you know, science fiction like story, but we have a lot of proof of concept data using rodents. Our first experiment was to use kidney deficient mouse. We knocked out cell one gene. So these mice cannot make kidneys and they die soon after birth. So here I thought, you know, that the space niche for kidneys is open. So if we make Chimera by injecting wild-type pre-potent stem cells like ES cells, IPS cells, these cells can form kidneys. They're normal pre-potent stem cells. So if we make a Chimera, the cells derived from normal pre-potent stem cells should use this space and develop kidneys. So, you know, these kidneys should be made of cells only derived from injected IPS cells. So this is what I initially thought and when I actually performed this experiment, it worked. So this is a cell one, no cut mass, no kidneys, but once in this case, GFP-marked IPS cells were injected into the brass cyst and two, three weeks later, when we looked at the embryo for neonates, we could see a generation of kidneys like this and brother was inflated by urine indicating that these kidneys are functional. And under the, you know, fluorescent microscope, you can see it's a systemic Chimera. So we see some fluorescence everywhere, but you know, if you look at kidneys, they're very, very bright for GFP indicating that most of the cells, as we expected, are derived from injected IPS cells. So this is our first demonstration of proof of concept of this idea. And this is not just for kidneys. We tried a number of other organs like pancreas, thymus, kidneys, liver, cells, blood, and more recently, brain, lungs, polyploid, gland, and germ cells as well. So it works at least in mouse to mouse Chimeras. But as you know, we cannot use human to make our organs. So we have to eventually use animals. It's gonna be a genomic interspecies brass cyst complementation. So we try to obtain proof of concept that we can make organs in genogenic for other species environment. So we try to test this using mouse and rat. They're both rodents, but different species. Rat is 10 times bigger than mouse. They have different number of chromosomes. There are many differences between the two. So we generated rat ES cells, rat IPS cells, and we performed experiments like this. So here we try to generate rat pancreas in mouse. Sounds like a crazy idea. But we try to see whether mouse can generate rat pancreas in this case. So we used PDX1G knockout mouse. PDX1 is necessary for the development of pancreas. So these mice cannot form pancreas. They die soon after birth because of the pancreatic insufficiency. So we injected wild type, normal rat IPS cells into the PDX1G knockout mouse embryo. And as we expected, we were able to generate mouse rat chimeras. And we were able to see rat pancreas in these chimeras. And because these mice could survive and grow into adult food, it means that these rat pancreas must be functioning right in mouse environment. However, interestingly, as I mentioned, in this case, we have to use mouse as a mouse salivate mother. The size of the chimeras was a mouse size. And so was the rat pancreas. So although cells were from rat cells, the pancreas was mouse size, very interesting. Now unfortunately, this mouse size rat pancreas was too small to transplant back to rat. As I said, rat is 10 times bigger than mouse. So we were not able to prove that this pancreas could be transplanted without any rejection. So we tried the opposite experiment. Now we try to generate mouse pancreas in rats. So we did exactly the reverse experiment. We generated PDX and knockout rat and we injected normal wild-type mouse IPSS. And as you can see now, as we expected, we could see rat size rat to mouse chimeras. And we were able to find mouse pancreas, this huge chimeras. But this side, rat size mouse pancreas was too, obviously, too large, too big to transplant back to mouse. So this is the size of the mouse pancreas generated in rat. This is the normal mouse pancreas. This is a wild-type normal rat pancreas. So you can see how big this mouse pancreas is. This is too big to transplant as a whole organ. But as you know, we do iodide cell transplantation for type 1 diabetes patients. Iodide is a cluster of cells in the pancreas, composed of beta cells or other cell types that produce various hormones. And most importantly, inseam produced by beta cells. So people isolate iris and transplant to the diabetic patients. So we sort of mimic that therapy. So we prepared iris from this mouse pancreas generated in rats and transplanted 100 iris, only 100 iris per mouse to the drug-induced mice to see if, whether they are rejected or not rejected, but if they are able to normalize or treat diabetes. So these are the two mouse two rat chimeras. And these are the pancreas. So we prepared iris from these pancreas and transplanted to the diabetic mice. This accesses the blood glucose levels and all of them because the stripe-induced diabetic mice they have very high blood glucose levels initially. But after a transplantation of 100 iris, within 60 days the blood glucose levels were normalized. Sort of very effective. And you know, even over a year, we are able to maintain a normal blood glucose levels in those recipients. And when we took out the graft by removing one of the kidneys, then the blood glucose levels went up very high, indicating that those transplanted 100 iris are responsible for this cure for the diabetes. And most importantly, there were some rat cells remaining in the graft, but they're completely removed because of immunocompetent recipients. And also we didn't use any long-term immunosuppressant, only for the first five days just to avoid acute rejection. But after that, no immunosuppressant necessary, indicating that these are truly self-idates and no need for immunosuppression. So this is indeed proof of concept data for ultimate although it was done in rodents. So the conclusion here is the exogenous in vivo environment provided near normal physiological developmental cues with proper epigenetic exchange to form a truly functional programs. And also, if it's a self-autologous iris, we only need very small number of iris to treat diabetes. 100-mouse iris is less than 2% of the iris that wild-type mouse has. So these two things we were able to learn. Now the question is, obviously rat and mouse are too small to provide human organs. So we have to move to larger animals. So as a host, potential host, we thought pigs and sheep are good because they have similar organ size, physiology, and also anatomy to humans. In addition, these livestock animals, they grow very fast within a year. They become large enough to provide human organs. So we generated by transgenic and also somatic cell nuclear transfer technology. We generated apancreatic pigs that cannot make pancreas. And we injected exogenous orange color labeled transgenic pig stem cells. We used a breast mare, so kind of ears-like cells to prove that breast complementation also works in large animals like pigs. And indeed it worked and some of them grew into adult food. And they're entirely normal for apancreatic function, including blood glucose levels. So at this point, this was like 2013 that we published these later, we are able to inject human IPS cells to the apancreatic, pancreas genesis disabled embryos. However, at that time, implantations of human animal, ademix embryos was prohibited by the guidelines in Japan. So although we have all the proof of concept data and also the materials to inject like apancreatic pigs, we're not able to test or perform experiments. So that's why I moved to Stanford University because here it was possible, it is possible. So we try to continue our study here in the US. However, after I moved to Stanford, NIH set restrictions on Chimera research. So they stopped funding these research. So it was disappointing. Now, Insou has a comment on this. That's right, thank you so much. I just wanted to expand a little bit on this point. This is an important point about the American context. Back in September of 2015, the NIH put what's called the funding moratorium on certain kinds of stem cell-based Chimera research. So in particular here, they don't allow the funding for research that involves the transfer of human pluripotent stem cells into non-human vertebrate embryos that are at the pre-gastrolation stage. So pre-implantation embryos. And they really call that, for example, non-human primate blastocyst as a no-go zone for this. This moratorium is still in place today. There's still no funding for the type of work at the NIH level for what HERO would like to do. There has been a proposal to have a steering committee form that would provide advice to Francis Collins on a case-by-case basis for protocols, but to my knowledge, there is no such steering committee that has formed. I want to contrast this with England. In the UK, there's what's called the HFEA, the Human Furalization Embryology Authority, which gives all licenses for human embryo research. And interestingly enough, they have a category that are called Admixt Embryo, defined as an animal non-human embryo that has human stem cells put into it. And the HFEA governs that type of work only when the human contribution, quote-unquote, predominates. It's not really clear what they mean by predominates. Is it the fractional percentage of human cells that predominates, or is it maybe where the cells migrate? Maybe they go to a particularly important region, like the central nervous system. And so the human contribution predominates in that sense. It's not very clear. But in the UK, at least there has been an attempt she tried to deal with this type of research and put it under review under their current system. But getting back to the NIH, it's very interesting because you have to know that there is no other moratorium on other forms of human and non-human mixing. So for example, there's no funding moratorium on the genetic humanization of mice and other lab animals. There's no funding moratorium on other very similar types of research. For example, the transfer of human glial progenitor cells into the neonatal brains of mice. So one might ask, well, why is that? Can you go to the next slide, please, Harold? So it appears that the overriding concern is a real unease about the process of biological humanization, biological humanization, leading to a radical sort of moral humanization, that there's gonna be an overlap between biological humanization and what might call moral humanization. And so what we have here in the photo is, what may be one sense of morally significant humanization would be appearance. There have been actually some bioethicists that I'm aware of who've raised a concern that it would be deeply troubling if there were a sheep that had a human face, or in this case, a pig with a human-like face. I really think that concerns like that need a real science reality check, because in order to get that kind of outcome, you would have to make chimera modifications to all three germ layers, mess germ after germ end germ, and only limit that chimeraization of all three germ layers just to the face. And I don't think that that's possible. I think you would have to do probably genome editing instead to get something like this. So I don't think it's actually possible to get these kinds of outcomes, but certainly the public and others have quite an active imagination. So that might be one concern. What about pig with a human brain or non-human animal with a human-like brain? What's the concern there? The concern might be some human-like cognition. What about pigs with human germ cells, sperm and eggs? Again, I think the idea there is that the concern that there could be an inadvertent fertilization of that between a chimeric animal and non-chimeric animal, you end up with a human-animal hybrid. Again, nobody wants that. And there are sort of guidelines right now about not breeding chimeric animals that could have germ cell formation. We are gonna talk a little bit later in this presentation about specific strategies to avoid widespread uncontrolled unwanted chimerism. So I'll save that for a little bit later, but I wanna leave you with one thought before I turn it back to Hiro. Here's one thought I want you to think about. If moral humanization really means something like the appearance of uniquely human cognitive traits, uniquely human mental experiences, then I think that particular worry lies more in the area of science fiction and science possibility. Take for example, a 100% human brain in a newborn. 100% human, that newborn brain is not gonna have typical human cognitive like traits or higher functioning if it doesn't also have interaction with society, interaction with caregivers, it doesn't learn a language, it doesn't actually get treated like a human being. So there is no inevitable property of human cells, I would say that gets you without any doubt human cognition or human-like cognition. So with that thought, I wanna just leave it there and I wanna return now back to the science and I'll turn it back to Hiro Mitsu. Take it away. Okay, thanks. So I was not able to get NIH funding, but luckily I had funding from Southern California Institute of Regenerative Medicine. They're more generous. So I was able to continue working on this project. And one of the first things I did is to create a pancreatic sheep because sheep study, sheep embryology is not available in Japan, but here at UC Davis, for example, they have experts on this. So we this time used CRISPR technology to make straight PDX and knockout, it worked. And then we also started to make human sheep chymidic embryos in this photo. We are injecting TD tomato, which is the red color labeling, TD tomato labeled human IPS cells into E5 sheep embryo. Of course, we have all the approval from the Stanford University and also at UC Davis. So it took some time that we started to do this kind of making human animal, human sheep chymidus. And 24 hours later in culture, we were able to see human IPS cells still there and well mixed with the sheep cells, looks like. Then our collaborator, Professor Pablo Ross and his team is injecting these chymidic human sheep chymidic embryos into the uterus of the foster sheep. This is the uterus, this is sheep, so it's sheep mother and he's injecting these chymidic embryos into the uterus. And then three weeks later we recovered all these embryos and in this case, I think nine out of 20 sheep chymidic embryos showed more than one human cell in 100,000 sheep cells. Even, so this is a very small number compared with the red mouse chymidus. So somehow the human sheep and also some other group has shown as human big chymidus is far more difficult to make compared with red mouse chymidus that we have shown. So at this point, our real challenge is to overcome this interspecies compatibility, we call it xenogenic barrier. Human IPS cells minimally contributed to human sheep chymidus and that must be some kind of barrier to prevent efficient chymidus formation. So we continue to, we go back to rodent studies and analyze carefully what happens to chymidic embryos after transfer. So we generated rodent to mouse chymidia and followed their fate after transfer. So this is the chymidism of each embryo after transplantation. So at the 9.5, we see many embryos with high blood pressure that chymidism. But around E15, E11.5, all these embryos are gone by E14.5, only a very low chymidism embryos surviving. This is in contrast to the mouse to mouse chymidus where even at E15.5, more than 25% chymidism, most embryos have average 25%. We also tested the embryonic survival rate and in the case of rat ESOs to mouse, rat to mouse chymidus, the survival rate goes on very quickly after around E10. This is in contrast to the mouse to mouse chymidus, more than almost 80% of them survive even at E15.5. So clearly inter-species chymelas have a problem, particularly with a high degree of chymidism, they are prone to greater incidence of intrauterine death or some sort of malformation. To further confirm this idea of genetic, or evolutionary distance may be involved, we try to make chymidus between mouse and prairie ball, or you call it ball mouse, they are one of the most distant species within rodents from a mouse. So the mouse rat is diverse about 25 million years ago, whereas mouse and prairie ball, they are diverse 44 million years. So we generated IPS cells from whole mice, two independent lines, and then we injected them to mouse brushes and see how they behave or how they make chymidus. So two independent cell lines using two independent experiments. In both cases, we do see for chymidus at earlier stage like E10.5, but E13.5, the number of chymidus decreases, as you can see, and at birth, the number further decreased to less than 3%. So this is much less efficient than mouse to rat, rat to mouse chymidus. So chymidus generation was between mouse and ball mouse was possible, but much less efficient. So these are the mouse ball chymidus, the wild type mouse, so a little weird looking chymidus, but they could survive. So it appears that this general barrier is probably the result, as a result of evolutionary distance. So mouse rat is right here, chicken quail, these chymidus are being reported, and also we tested this mouse prairie ball, which is around here. So we are able to make chymidus relatively easily if it's less than maybe 50 million years of diversions. But now we have to make chymidus between human pig, human sheep, human mouse. This is about more than 90, 90 million years of diversions. So this is a little difficult to overcome. So to further understand what happens if we use higher evolutionary more up on top of the higher closer to a human being. So, but obviously we cannot make human monkey chymidus. So we decided to test what happens if we try to make non-human primate, non-human primate chymidus. So mouse rat diverged about 24 million years ago. Human and chimpanzee are much closer, only six billion years of diversions between these two species, very close to human. So the same is true for rhesus macaque and pig-tailed macaque. They are very close in terms of evolutionary distance. So we cannot use human, we can use chimpanzee IPS cells. IPS cells, so technology is so convenient, we can make IPS cells from various species quite easy. So we obtain some blood from the chimpanzee and then we established also other people established chimpanzee IPS cells. So we use these chimpanzee IPS cells but also IPS cells in these different subspecies of macaque. We try to see how they behave when they try to, when we try to make chymidus among these non-human primate species. The experiment is extremely difficult, obviously. We can generate IPS cells from pig-tailed macaque, chimpanzee, and we can label these cells with TD tomato. And we transfected BCL2, this is the antibiotic gene. And we know from our rodent studies that BCL2 helps to increase the climates of survival of the injected IPS cells. Then we injected these cells into the macaque embryos. This is in collaboration with the scientists at UC Davis. And we cultured for 48 hours and see how they behave. The culture of these non-human primate embryos very difficult and it's not easy to maintain them and cultured for more than 48 hours. So we analyze in this preliminary experiments, we analyze the 48 hours after the initiation of culture. And we have different experiments, but here we try to see how they behave. They mean human-human or human-chimp co-culture. And then they developed into muscle cells and these human-human-human chimps that both differentiated well and cooperated functionally. Co-operated functionally, right? So human and chimps, although there's some difference in the culture conditions, but they behave very in a very similar manner. So again, we're very close. However, if we co-culture mouse-mouse human and like mouse-mouse, mouse-human, they do not impregnate very well. As you can see, they go independently. They do not, they tend not to mix together. We also analyze the, you know, chimerism 48 hours between these NHP and HP chimeras. As you can see, you know, it's much better than mouse-fraily bone, mouse-rat chimeras. So in some cases, more than 90% of them show chimerism, particularly with the help of PCL-2. So they do well at using this very short time culture period. But we have to, of course, see observe much longer, but there are some technical difficulties. Hopefully we're able to transfer these back to the foster mother to do in vivo experiment. So anyway, these interesting and important findings for our future potential work to overcome xenogenic barrier. So meanwhile, it almost took 10 years to revise the guideline. But finally, Japanese government lifted the ban on human animal chimera research. So we, over the years, we discussed, I discussed with many people and it appears that, by this time, it was like two years ago, we know that human animal chimera is not easy to make. So they, I think they do not, they realize that it's not necessary to worry too much about the humanization of the pig or sheep or any animals when we make human animal chimeras. So now I got formal approval to do human animal chimera research last year. And we started to the injection of human IPS cells into pigs and rats and mice and so on. But also, meanwhile, my collaborators also generated a number of organogenesis disabled pigs, not just pancreas, also kidneys, blood and vessels, liver, and even the double no-cut meaning all the pancreas, blood and vessels are lucky. So we should be able to, if it works, make human pancreas also the blood and vessels are from IPS, human derived. And we're starting injection of IPS, human IPS cells and pig embryos. And in some cases, we see good integration of human cells into the pig cells. But eventually they lose their contribution. So we still need to overcome. We need to understand, manipulate this xenogenic barrier between human and pig. So in order to, over the years, I realized that as in Sue mentioned, many people concern about the generation of animal with human brain cells or jam cells to avoid, but it is very difficult to define what percentage of human cells present in animal brain is okay, and it's very difficult. So one idea that we have is to completely to make an IPS cell lines that cannot at all contribute to human forebrain or jam cells. So just to test this idea, we knocked out OTX2 and PRDN14. These genes are important for the generation of brain and also jam cells. But when we try to make pancreas, these cells were able to make pancreas, functional pancreas, but we didn't see any contribution of IPSC derived cells in these five minutes. So essentially, if we use this, similar to this double knockout IPS line human, then we do not need to worry about generation of, some of the ambiguous animals pigs with human brain or human jam cells. So this is one approach to minimize to reduce the people's ethical and social concern. So we are planning to use this kind of IPS cells for our future research on human animal violence. So I think Insou has a comment on this. Sure, thank you. So I wanted to just address a little bit of what we know or don't know about public attitudes regarding this type of research. So I mentioned that the NIH had the moratorium, they still have the moratorium in place right around the time that they proposed this idea of a steering committee. They had a public comment period where people could go to the website and enter their thoughts or their ideas or comments in an online portal and they got thousands of responses. Now, I don't really know how informative that is and getting a sense of what public attitudes are because in many, many cases, thousands of these cases, you had essentially the same comment cut and pasted onto the comment field. So we're not really sure how representative that is of actual individuals. And a lot of the comments had to do very broadly with things about, you shouldn't be playing God or this is a central research in general or embryo research in general is wrong. Nothing really specifically addressing this type of research itself. So the NIH comment period, I personally think is not very informative for a gauge on where social views are. There was this paper that we have up here on the screen that was published recently by a group and what they claimed was that about 59% of the American public can personally accept pig organ chimeras. Now, what's interesting about this article is that if you look at the title, it's not specific to human pig organ chimeras. It seems to speak more to just in general human animal chimera research. So this was called out actually by a few colleagues who are my colleagues at the Hastings Center. They criticized this article for being not representative in terms of the sample used with respect to the age of the respondents, the geography and the gender didn't address what other types of chimeras and besides the pig organ chimeras. So I think those are pretty legitimate concerns are about this paper. Now, the authors did respond. They did respond by saying that they acknowledged these limitations in the paper itself. That's what they claimed. And they wanted really just to get a general sense of public attitudes about this kind of research. So I think there's a lot more work to do in understanding public attitudes. The authors of this study claim that the public attitudes had greater acceptance in Japan from this type of work. But I think this calls for a need for more information about public attitudes and what to do with that. Next slide please. So where are we right now with the International Society guidelines? We're in the middle of uprising now. I can really just speak to the current version that's live now, which is the 2016 version. I will say that in the absence of any kind of real hard data on where the public sits on this topic, both in the US and out of the US, we had to pretty much proceed as we did back in 2016 of just really focusing mainly on animal welfare. As Hiro had pointed out and if I've tried to point out, the level of chimerism we're actually seeing in animal models with human cells is really pretty low. This is quite challenging to get widespread chimerism of the type that people are afraid of. So what we've really settled for quite a while now is in the guidelines, attending to issues around animal welfare of modified animals. I think it's a supreme arrogance to think that once you transfer human cells, especially human brain cells into animals, somehow you're going to enhance them and you're gonna make them better, right? But the reality is if anything, modified animals have a radical lowering of their animal welfare and disequilibrium and other deficits. So we really wanted researchers and regulators to be attentive to that. But this is an ongoing issue. As I said, we're currently revising these guidelines and we do have to pay particular attention to the type of experiments that Hiro and his colleagues and others are pursuing. We have to attend to chimeric embryo work, which really was not addressed in the previous guidelines. And the work with non-humor primates and livestock animals there actually has to be quite a bit of additional guidance for investigators about how to work with these animals and to meet their unique needs. So look forward to the guidelines that are, we hope, going to come out sometime in April, sometime in the spring. And there's plenty more to talk about. Let me turn it back over to Hiro. Go ahead. OK, so to wrap up my talk, there's a conclusion too. So xenogenic system, IPS cells contribute to chimeric's much less efficiency. So there's a xenobarrier. And so what is the mechanism for this? There could be a number of reasons, but mostly dependent on the evolutionary divergence or the evolutionary distance between two species. These could be differences in ligand receptor interactions. They may have lower affinity, for example. This could be cell, there could be cell intrinsic aspects such as doubling time, differentiation rate, and so on. And also, there may be some affinity differences in adhesion molecules between two different species. There may be also involvement of innate or acquired immunity to prevent generation of interspecies chimeras. So in conclusion, I think we need to understand and manipulate better the xenobarrier. This is the key to the generation of human organs in livestock animals. So our future direction is, of course, we need to overcome the xenobarrier that exists between human and animals. I think it's also important to keep good balance between medical needs and social consensus. So we try to keep the transparency of our research. And also, we try to gain understanding of research content and medical usefulness of this type of research. Also, once we succeeded, we need to provide safety by excluding possibility of infection or tumor development and so on. So that's all about what I want to say. And these are the famous of the people who contributed to this project and also the funding agencies. So I stop here and I get back to insert. OK, great. Thank you so much. So we've had some questions come in. And so while I'm going through those, let me start with a question that's come in by Bob Trug. And it's actually the same question I had. As you may know, the church lab here at Harvard Medical School is interested in genetically modifying pig organs to make them compatible with humans for transplantation. Can you speak to that strategy? What are your thoughts on that? And are there advantages or disadvantages compared to the strategy that you're pursuing? Yeah, that's a very good question, I think. Actually, I was trained as an immunologist. I may be a dropout immunologist. I still think that, you know, immune system is very precise. And it's not just a major histocompatibility complex, MHC or HLA, that determines the, you know, self or non-self. So I think the approach humanizing pigs to avoid immunological rejection is fine for some organs like liver, because liver is tolerable relatively. But I think eventually even liver will be rejected and probably patient has to have, you know, immunosuppressants for a lifetime, I guess. So it could provide a good bridging therapy until the donor organ becomes available. But eventually I think it's not an ideal situation. It's okay, it's needed, but it's not an absolute answer to this problem. Whereas in my case, it's a little different. So we are making human organs in pigs. So in this case, we have to worry about rejection by the pig, including xenobarrier, but once, you know, generated, this organ is autologous. So it should provide, you know, more or less a complete cure for the patient. So that makes a difference. Thank you. So as an immunologist, let me just ask you one of the questions I had. How confident are you actually that after all the manipulations and growing in the pig model, for example, a human patient-derived IPS cell organ will actually be compatible? Are there any concerns about like surface markers changing or anything like that, that will make it a little bit more foreign to the person from which the IPS cells came from? Yeah. So, you know, once generated, it should be okay. But, you know, of course, those organ, organs generated in pigs should have some pig cells, you know, sort of coexisting or contaminating. But as I showed you in the rat mass experiments, you know, we are transmitting these into the immunocompetent recipients. It may, you know, we may need to do some immunosuppression initially. But, you know, since they're immunocompetent, eventually they will eliminate all the pig cells. So, you know, so eventually they will, the patients will not require any immunosuppression. That is my hope that, you know, the data from the rodent studies. A few more technical questions have come in. I mean, let me just get these two first. And then I think I'll address some of the ethical questions that maybe you and I could speak to. But another technical question kind of along these lines is, how do you actually get all the cell types you need in, you know, a pancreas or a kidney? That's not going to have pig contribution. I mean, you know, some, I assume that there are other germ lineages involved, right? So, if you do your knockout animal, and then you rescue it with the human derived cells, doesn't that pretty much follow just one germ lineage? How do you get all the cell types you need? Yeah. So, pancreas is a relatively simple organ, you know, developmentally. So, if we knock out PDX1, all the pancreatic cells in deficient, so we are able to replace it with the IPS cell derived cells. But of course, you know, best cells, hematopoietic cells, or fibroblasts, there are many cell types still. They are not under the influence of PDX1. So, that's why, you know, we made a double knockout mice, I mean pigs first, where, you know, best cells, blood and pancreas are all deficient. So, you know, at least making, you know, several combination knockouts, we should be able to replace most of it. It may not be all, but as I said, eventually, once transplanted, you know, human cells will somehow replace, eliminate pig cells and replace by his own. That's my optimistic. We saw also recently just published, we found another approach to do organogenesis in, in xerogenic system. So, this is a little different from a blasted complementation. And this system may provide even better replacement of the human organs, tissues and cells. So, you know, technology is developing. Hopefully we can provide better for complete human transplantable organs. Right. A really nice question came in. I think this is really for both of us. Other than the humanization or the moral humanization concerns about this type of work. Do you see other ethical issues, ethical concerns about growing human organs in animals? Of course, the second most common criticism is animal welfare. You know, how you can kill pigs just to generate human organs. But, you know, for this, you know, I can argue that, you know, as I said, every day, 20 people die waiting for donor organs. So if, ideally, if we sacrifice 20 pigs, if it works, then we can save those people and just think about how many pigs we are sacrificing for the food. This is almost negligible number of pigs. And I think, you know, from the medical viewpoint, I think this approach is, I think, rational. I mean, we can, you know, argue against that kind of, you know, criticism. Yeah. Yeah, I must say from my own point of view, there's an important distinction between the use of livestock animals for food and those for medical purposes such as this. This may not change anybody's mind, but I want to just point out this distinction. And that is, suppose in a world where we have these human organ chimeras for medical therapies, they're actually going to be treated as medical products. You, I would, I would think that the housing and the care and even the euthanasia of these animals would be more along the lines of like a non-survival surgery. And, and pretty, pretty, I wouldn't, it's not free range, but a pretty more comfortable environment than factory farming or some of the much more horrific scenarios that people are well aware of that happens in the, in the, you know, the food industry. So one might not maybe imagine or have a clear picture of exactly what that facility might look like, but that's actually some of the issues that we're touching on on our guidelines. What we're thinking about, you know, what recommendations do we have for housing and care of these animals, even if they're just used at the research phase, you don't want to complicate your research results by having stressed out animals and having, you know, unsafe work environments for staff and for, for the animals themselves. So I don't know if that's going to change anybody's mind, but you really have to realize that I think the conditions for raising organ chimeras are going to be quite different. Like I said, it's going to be a non-survival surgery for organ retrieval. And, you know, maybe that might matter for some people thinking about those differences. Okay. Someone asked, did you ever, did you inject mouse ESLs into monkey embryos? What type, what other type of cells did you put into monkey embryos? And did the mouse, did mouse ESLs, does that work or did you just stick to? I think we have done that experiment because we can do it without any approval. So, but I think it didn't work. I don't remember precisely. That's a, because we, the reason why we performed that is we know that mouse ESLs are truly naïve or chimera forming ESLs. So we wanted to see if it works in monkeys or some other species. But I don't think it worked well. So we tried, but it didn't work. We didn't, you know, do, we didn't spend so much time on that because now we're working on human IPSS. Yeah. And somebody else asked, even if the technology is feasible, would you be able to grow an organ suit quickly enough to help a patient? Yeah. As I said, you know, these livestock animals have been improved and they grow very fast. Within 10 months, they become over 100 kilos. So we have, I think we have, depending on the, you know, situation, but for those with, for example, heart kidneys, we have, you know, artificial organs to support their life for a while. So depending on the organs, but we think we can, if it works, we should be able to prepare organs within 10 months or so. Yeah. I think where some of the attendees are really quite fascinated in thinking about is the possibility that you can actually, once you are able to generate organs in this fashion, that there could be other, other social problems sort of that arise. So one issue might be, you know, currently in our very constrained way, we try to have an organ, you know, a transplantation waiting list and there's no, you know, money involved, but if you're actually able to grow organs in the, in the kind of like in the commercial environment, right? Do we need to have, and have you thought about any idea of like what, what selection would look like and how you could actually barely, you know, get organs into people who maybe on, on the current waiting lists are ranked very high, but maybe because they don't, they can't, you know, have access to researchers who can use their ITS cells or something like that, that they actually, the priorities get a little bit messed up. So do you know what distribution would look like under this kind of, this, this future and have you thought about any of that? That's an interesting question. You know, first of all, I should, as I mentioned, you know, there's a black market. Yeah, actually 10% of transplantation performed worldwide using, you know, those organs from black market. It's clearly, you know, for the rich people, I would say. And even, even, even though, even after transplantation they have to have, you know, suppressant throughout their life, it's a life from, you know, immunosuppression, which is very costly too. So it's a very expensive, you know, treatment at the moment. And people might think, you know, pigs using pigs is expensive, but actually it's not, you know, a pig costs only 300, 400 of us because, you know, we, you know, sacrifice many, many pigs, billions of pigs for food. So once, you know, we establish the system, the cost should be much cheaper overall compared with the current, you know, artificial, you know, organs and immunosuppressants. So, yeah. So once then, so that means probably initially we have to think about how we triage the patients. But once the system is established, it should provide relatively inexpensive way. So, yeah, only for the initial, maybe three, four years, we may have to triage patients. But, you know, as I said, they grow very fast. So we should be able to, you know, help many patients relatively short time. And not worrying about the, you know, price cost involved. Right. So, so your work focuses on the transfer of human cells and trying to develop, trying to try to fill in the missing organ of interest. But obviously you also have to work equally hard, maybe with another group in developing these organogenesis disabled animals. I noticed that in your slides that you've, you've had pancreas disabled, you've had various other organs, but I didn't see heart disabled. Is that a particularly challenging one? Because your other slides show that in the concept phase, it was all hearts, right? And I think people are kind of like really fascinated with heart transplant. Maybe it's because of the cultural baggage around hearts. But, but yeah, how difficult is making these organogenesis disabled animals and who's working on that? And that has to, I'm sure have to go in parallel with the work you're focused on. Interestingly, you know, for example, in the US, the cardiology is very well advanced. Many good cardiac cardiologists, a cardiac surgeon here. However, the basic science of heart development is not that advanced, I would say, compared with organs like pancreas, kidneys, liver. I think it's a little more, you know, complex the development of heart is. So we may have to knock out, probably because heart is a very important fundamental organ, you know, it starts, development starts early and involves several different transcription factors. And we still need to know much more about development of heart and molecular terms. So that's why, although I'm very, very much interested in the generations of heart, you know, this approach, but we still need some more basic science to do this. Yeah. So this next question might be a little bit tricky to for you to answer just in principle, but do you see any undesired co-effects or unpredictable unpredictable effects in the process that you're pursuing? That's that's what, you know, I'm constantly thinking about. Mostly, I don't concern much about, you know, human-like pigs. But as in the COVID-19, you know, I worry about, you know, theosis, some, you know, viral, new virus coming out of this type of, you know, human, animal, kind of. So that's one thing I worry about. But this is, you know, I think the chart is group's contribution. Now we can screen sequence whole genome pigs and look for any potential, you know, viral-like, you know, sequences and we can knock it out. So, you know, we may have some, you know, potential initially. But once this system start to work, I focus my, all my attention to the safety of this type of therapy. And technically, I think it's possible. Yeah. Have you given, here's another question. Have you given some thought to the patenting that underlies these technologies and whether some of that could become a barrier to people's access later? I have some patents, basic patents on this. But, you know, the patent has different meanings. You know, it makes me rich, hopefully, but that's not the only purpose. You know, I can also prevent other people, you know, use this technology and make money. So as long as I have the patent, I can control, you know, I can avoid, you know, unnecessarily capitalistic involvement in this kind of treatment. So I'm hoping that, you know, I don't, it doesn't need to be my group, but somebody will contribute to the work and hopefully provide this form of therapy with minimum cost. I know this medical cost is extremely high in this country. I don't want to add to that. So yeah, that's my idea. So you said that recently the Japanese government changed their regulations around this type of work. Do you think, do you find that attitudes are different in Japan around this work than in the U.S., as far as you can tell? What's your feeling on kind of any cultural differences, anything like that? Well, over the years, almost 10 years, you know, my work about research has been, you know, published. And also, you know, the media, newspapers, TVs, they also, you know, talk about, broadcast it or talk about our research. So people realize that, you know, initially it was kind of a shocking research for them, but now they realize what's going on. I tried to, you know, maintain the transparency of our research and also keep telling them what we are trying to do. So I think I've got better understanding of the people about what we are doing. And also the government has realized that, you know, human animal kaimera is not like making a monster and high contribution of human cells is not so easy. So those are the things the government has changed their attitude. Also people have also got a little better understanding what we are trying to do. Yeah, so you've been at it for quite a while now. What do you think is the factor or factors that have really contributed to the slower pace of what you're hoping to do? Is it funding issues? Are there scientific technical barriers? What are kind of like the biggest reasons why, you know, the work is taking a while? You know, Japan is based on the bureaucratic system. So the officials of bureaucrats, they want to avoid any bad marks disclaimer. So this 100% disclaimer is possible. They do not want to change. The current situation is very conservative. So I think that's the basic reason. And once they realize that some other, you know, scientists have made human animal kaimeras and almost no contribution of human cells, I think they thought, you know, it's okay, it's safe to change the guidelines. So they're not interested in introducing new treatments or new technologies. They just want to be conservative, not to be blamed. But it's a general attitude of Japanese, you know, bureaucrats. So since coming to the United States and working under CERN funding, do you find that CERN funding, this is the California Institute for Regenerative Medicine, this is state bond money that's devoted to stem-solid regenerative medicine? I just heard the audience to know. So you guys, CERN funding, do you find that that is a pretty good substitute for NIH funding or is there anything you kind of, you know, wish, any reason that you might want to wish for the moratorium at the NIH to be over that you can apply for NIH funding? I think, you know, from my experience, CERN funding is, I would say, better overall because they're more generous, faster, and for the, they don't require too many things. And I'm not very much fond of NIH review system. You know, if I depend totally on NIH grant, I cannot do any innovative research because NIH grant proposal requires almost done, you know, a very incremental sort of project. So, and also recently, the money, use of the money, grant money, is very much restricted to the purpose of the proposal. So we cannot do, you know, any trials or just how can I say, use this money to do anything else. I cannot do any innovative or just challenging experiments if I solely dependent on NIH grant whereas some is more flexible, I hope, and they take more challenging proposals. So that's something I like. Great. So I noticed that your work primarily uses pig models. What about sheep? So are there, do you think there will be advantages to using sheep at one stage of research and then pigs at another? I'm curious about why, you know, sometimes in your graph, you show both sheep and pigs, and we actually do talk at the ISACR about use of both animal models. Why did you gravitate toward pig use? Well, I didn't, you know, talk much about it, but, you know, from my experience, this xenogenic barrier is, you know, also they show variation organ to organ. So in other words, mouse rat chimeras, when rat cells is injected into mouse blood system, or even between mouse, say, mouse and rat, somehow interesting, if we make chimera, for example, the blood cells tend to be mouse dominant, because some other, you know, organs, rat cells tend to become dominant. So there's some, you know, organs, probably some organ-specific side kinds, some developmental cues involved in this xenogenic chimera. So unless we try different combinations of species, it's hard to tell, you know, which one makes better human organ. So if possible, I'd like to try many different species, but, you know, sheep and pigs are probably the only reasonable animals to try, because it requires a certain level of embryogenesis technology, and those technologies are not available besides pig and sheep, in a reasonable level. So that's why, so I think it's, you know, it's worth trying sheep as well, not just pigs. Yeah, so you maintain two labs, right? Do they do different work? What, why do you have two? Yeah, well, that's an interesting question. We work together, and we have the same ultimate goal, that is to make organs, human organs in livestock, but, you know, we have different technologies, and so, you know, although every week we have a meeting and discuss the progress, but, you know, of course we have different projects, depending on the laboratories, but we work together and try to avoid overlap and try to help each other. So that's how it works. Do you ever have conversations with your lab members and your postdocs about career development? I mean, I would think that some people may be a little concerned in your lab about, you know, if there is ever sort of bad press around this kind of research or public disapproval that their career trajectories could suffer. Do you ever talk to them about, you know, these other concerns about, you know, they're starting off in their scientific career, they're part of your lab, and this is, for some people, very controversial research. I was curious about how that mentoring goes. Well, I don't think, you know, they have, they're worried about it, much about it, and I think they understand that, you know, this is like a, what do you call it, moon shot experiment, very challenging experiment. I don't think any postdocs or graduate students can achieve this in three or four years. So it's a, you know, long range project. So, you know, however, the, although, you know, students, postdocs are working on this project, they can always find some interesting, you know, findings, discoveries. So people are publishing, as you know, we publish many papers. We haven't succeeded in making a human organ, because, yeah, but, you know, on the way, just like Apollo 11 project, we have, you know, internet, GPS, many, you know, bi-products from this, you know, long shot, big, you know, project. So I think they're enjoying it, so we, including myself, do not worry about those things. We may be too optimistic, but they are publishing good papers and getting good, you know, positions. So I don't think it's really a big issue here in my lab. Yeah, I'm curious. So when you get your articles peer reviewed, your manuscripts peer reviewed, what generally do the reviewers say? I mean, as full disclosure, I've never reviewed any of your work at the manuscript phase. Sometimes I get asked to do bi-ethics reviews. So I'm wondering, do you ever get bi-ethics reviews, or what are some of the reviewers typically like focused on? I'm curious about that. Yeah, that's an interesting question. So maybe seven, eight years ago, even our first set of paper, I think editors, some skeptical about what we're doing. And to tell you the truth, I wasn't trained as a developmental biologist. So I think I've made, you know, some warnings or technologies for the development of biologists. I was so naive. So they corrected some of our use of our language, some of our timeologies. I mean, the reviewers, the editors, they were somewhat skeptical. And some editors were, you know, worried about the, you know, virus issues, or this cannot be accepted socially or ethically. That kind of, you know, reviews or comments I used to get. You know, making camera is much better understood. And so I don't get any of those ethical or social comments, at least by the editors, scientific editors, and the reviewers. The good thing. Okay, so I think I have time for one more question. And this comes again from Bob Trug. And this is a really nice segue to, by the way, for the audience, our next presentation next month. But could you comment on the greater urgency of this type of work for Japan, where their brain death is a little bit more controversial and organs from brain dead donors are much more limited. Is there, is, do you see kind of a special, a special connection or interest there in Japan for these kinds of reasons? Yeah, again, you know, the culture society in Japan, a little different from, you know, Western, maybe US, European countries. But we still do not believe in brain death. But now it's, it's okay getting better. There's still very limited number of transplantation based on brain death organs. So, yeah, I think, you know, this is one issue, but also medical economy is a big thing, because we have more than 300,000 patients on hemodialysis, which is very expensive. And in Japan, everything is under national insurance. And this really becoming an issue for the medical economy viewpoint. So I think there's a big demand to, to generate, you know, transplantable kidneys, for example. And now the same is true. We have some Japan has technology to make good artificial kidneys, or at least the artificial heart. So the many number of patients on artificial heart is increasing dramatically. And this is very, very expensive, much more expensive than hemodialysis. And they, their life expectancy is like two or three years. So for those patients, real stress. So, yeah, there's a demand to provide donor organs for those patients in Japan. Well, thank you so much. I think I'm going to conclude with that then. So I thank you for sharing your time and your expertise with us. For the audience, I wanted to also announce that we have another session on March 26, Friday with Nana Sestan from the Yale School of Medicine. And you'll see if you go to that talk, how connected it is to what we just finished with here. I'd like to thank Ashley Troutman and Angela Alberti for all the logistics for this series. And I'd like to thank you, our audience for joining us. I hope you have a great weekend. Thank you. Goodbye.