Growing Transplantable Human Organs in Livestock

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, I.C. 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, uh, 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. Okay. 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 Hiromitsu, Hiro Nakaguchi. He's Professor in the Department of Genetics, the Stanford Institute for Stem Cell Biology and Regenerative Medicine. He is 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. Nakaguchi 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 express 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. Nakaguchi. Hiro, um, the, uh, topic for today is growing transplantable human organs in livestock. So, I'd like to turn it over now and please take us through, uh, some of your work. I hope you can see my slides. Yes. Good.

Thank you for your introduction and good morning, good afternoon. My name is Hiro Nakaguchi, and I've been working on organogenesis project, that's what I'm going to talk about from that. 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, you know, sciences into the clinic. And I will tell you why I have two laboratories later in my talk. And in vivo program generation project is the one I started as a sort of a side project about 12 years ago. And as Insung introduced, my major field of research is hematopoietic stem cells. So, I would 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. However, because of the shortage of donor organs, a number of patients waiting on the, when 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, you know, business. Actually, more than 10,000 organs were sold in 2010. So, this is a worldwide ethical issue, I think.

So, this is the current situation for organ transplantation. As you can see, these people must have donated, I mean, sold one of the kidneys because we can see this, you know, 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 immunosuppressants 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. Or iPS cells can be generated from the patient. So, we should be able to provide a patient's own cells, organs, tissues. However, current therapies are targeting, you know, diseases that can be treated by cell therapy. But, you know, with cell therapy, you cannot help people with end-stage kidney failure or liver failure and so on. So, we need to, 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 stereotypes involved in the, in the organ.

So, uh, my idea is to use in vivo environment to grow human organs because it should have everything, every environment necessary to grow organs. But of course, we cannot do this in humans. So, we are thinking of making human-animal chimeras, particularly livestock animals, to grow human organs from iPS cells obtained from the patient. Then we can solve these issues: shortage of donor organs and immunological rejections.

So, I think you, the audience, know 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, uh, different populations of genetically distinct cells. The important thing is, chimera is not a genetic mixture, but just a mixture of cells. So, we didn't touch, we do not touch genes. And chimeras cannot expand as chimeras. They only have, you know, two different germ 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, you know, somebody else's cells inside the body. So, there are chimeras. A number of examples for for partial chimeras. But what I'm talking, uh, about 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 stereotypes. That chimera has two types of cells in all tissues and organs. This is one example. This is a rat-mouse chimera. From the left, it's a wild-type mouse, and the rightmost is a wild-type blood rat. And these two in the middle are rat-to-mouse chimeras. So, it is obvious, just by the 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 pluripotent stem cells. It could be iPS cells or ES cells. We also prepare, uh, rat early embryos. The rat, we call it the rat blastocyst stage embryo, which is about three to four days after conception. So, this rat blastocyst is a small cavity. This is still a, you know, cluster of maybe 100 cells. And this portion becomes the body, and the other part, you know, will become a placenta. So, anyway, we inject mouse iPS cells into the cavity of this blastocyst. And then, this is how we do it. This is the blastocyst rat blastocyst, and we are injecting mouse pluripotent stem cells through this tiny, you know, pipette. And you can see these cells being injected. And 24 hours later, if we mark the, uh, injected ES pluripotent stem cells with red, then you can see a mixture of red and also host cells. Then we transfer these chimera embryos to a foster mother. And an important thing is, the whole foster mother and host blastocyst has to be the same species, otherwise they cannot accept and maintain pregnancy. So, in this case, we used a rat foster mother. And then three weeks later, we see birth of chimeras. And they, many of them grow into adults and they look like this. It's a mouse-to-rat chimera. So, this is how we make interspecies chimeras.

Now, you know iPS cell technology and also, you know, how to make chimeras. 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 blastocyst of our livestock animals. But, you know, one, you know, idea that we made is the, we use organogenesis-disabled animals. In this case, for example, we can make a pig that cannot form a heart. So, that when they make, we make a chimera, this pig chimeric pig should have human cells everywhere, but in a heart, because the host pig cannot make a heart. Heart should be all derived from, you know, patient's iPS cells. So, once the heart is, you know, 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 the patient's own iPS cells. So, you know, essentially this transplantation should be autologous transplantation, should not require any lifelong immunosuppression. Patient's own heart. So, this is the idea. And this, just this whole idea is called blastocyst complementation.

So, 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 a kidney-deficient mouse. We knocked out Salu1 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 a chimera by injecting a wild-type pluripotent stem cells, like ES cells, iPS cells, these cells can form kidneys. They are normal pluripotent stem cells. So, if we make a chimera, the cells derived from normal pluripotent stem cells should use this space and develop kidneys. So, this is what I initially thought. And when actually performed this experiment, it worked. So, this is the Salu1 knockout mouse, no kidneys. But once, in this case, GFP-marked iPS cells were injected into the blastocyst, and two, three weeks later, when we looked at the embryo or neonates, we could see a generation of kidneys like this. And bladder 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 are 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, you know, 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, vessels, blood, and more recently, brain, lungs, 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 going to be a xenogeneic interspecies blastocyst complementation. So, we try to obtain a proof of concept that we can make organs in xenogeneic or other, you know, 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, but 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 a rat pancreas in mouse. Sounds like a crazy idea, but we try to see whether, you know, blastocyst can generate rat pancreas in this case. So, we used Pdx1 gene 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 Pdx1 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. Because these mice could survive and grow into adulthood, it means that these pancreas, rat pancreas, must be functioning. Right, in mouse environment. However, interestingly, as I mentioned, you know, in this case, we have to use mouse as a mouse surrogate mother. The size was chimera size of the chimera 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-sized 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. That we tried to generate mouse pancreas in rats. So, we did exactly the reverse experiment. We generated Pdx1 knockout rat and we injected normal wild-type mouse iPS cells. And as you can see, as expected, we could see a rat-sized rat-to-mouse chimeras and we were able to find mouse pancreas. This huge chimera. But this time, rat-sized mouse pancreas was too obviously too large, too big to transplant back to mouse. So, this is the, the size of the mouse pancreas generated in rat. This is a normal mouse pancreas. This is 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 islet cell transplantation for type 1 diabetes patients. Islet is a, you know, cluster of cells in the pancreas, composed of, you know, beta cells or other cell types that produce various hormones, and most importantly, insulin produced by beta cells. So, people isolate islets and transplant to the diabetic patients. So, we sort of mimic that, you know, therapy. So, we prepared islets from, uh, this mouse pancreas generated in rats and transplanted 100 islets only 100 per mouse to the drug-induced mice to see if whether they are rejected or not rejected, or if they are able to, you know, normalize or treat diabetes. So, we, these are the two mouse-to-rat chimeras, and these are the pancreas. So, we prepared it from these pancreas and transplanted to the diabetic mice. And this shows the blood glucose levels. And all of them, because of the streptozotocin-induced diabetic mice, they have very high blood glucose levels initially. But after transplantation of 100 islets, within 60 days, the blood glucose levels were normalized. So, 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 were very high, indicating that those transplanted 100 islets are responsible for this cure for the diabetes. And most importantly, there were some rat cells remaining in the graft. But they are completely removed because they are immunocompetent recipients. And also, we didn't use any long-term immunosuppressant, only for the first five days, just to avoid acute, you know, rejection. But after that, no immunosuppression necessary, indicating that, you know, these are truly self-islets and no need for immunosuppression. So, this is indeed a proof of concept data for the ultimate goal, 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 organ. And also, if it's a self-autologous islets, we only need a very small number of islets to treat diabetes. 100 mouse islets, less than two percent of the islets that a wild-type mouse has. So, these two things we were able to learn.

Now, the question is, you know, obviously, what about 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, you know, become good enough, large enough to provide human organs. So, we generated, by transgenic and also somatic cell nuclear transfer technology, we generated pancreatic pigs, pigs that cannot make a pancreas. And we injected exogenous orange color labeled transgenic pig stem cells. We used a blastocyst, so kind of ES-like cells, to prove that blastocyst complementation also works in large animals like pigs. And indeed, it worked. And some of them grew into adulthood, and, you know, they're entirely normal for, you know, pancreatic function, including blood glucose levels. So, at this point, this was like 2013 that we published this data. We are able to inject human iPS cells to the a-pancreatic, pancreatogenesis-disabled embryos. However, at that time, implantation of human-animal chimera embryos was prohibited by the guidelines in Japan. So, although we have all the, you know, proof of concept data and also the materials to inject, like a-pancreatic pigs, we were not able to test or perform experiments. So, that's why I moved to Stanford University because here it is, it was possible, it is possible. And so, we continue, we try to continue our study here in the US. However, after I moved to Stanford, set restrictions on chimera research. So, they stopped funding these research. So, it was really disappointing.

Now, Insung has 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-gastrulation stage, so pre-implantation embryos. And they really called out, for example, non-human primate blastocysts 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 Hiro would like to do. They have, 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 certain committee that has formed. I want to contrast this with England. In the UK, there's what's called the HFEA, the Human Fertilisation and Embryology Authority, which gives all licenses for human embryo research. And interestingly enough, they have a category there called "admixed 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 "predominates." It's not really clear what they mean by predominance. Is this 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 to try to deal with this type of research and put it under review under the 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, Hiro?

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 going to be an overlap between biological humanization and what might call moral humanization. And so, what we have here in the photo is, you know, what maybe one sense of morally significant humanization would be appearance. There have been actually some bioethicists that I'm aware of who have raised the 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 chimeric modifications to all three germ layers, mesoderm, ectoderm, endoderm, and only limit that chimerization 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, and, you know, others have quite an active imagination. So, that might be one concern. What about a 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? Yes, sperm and eggs. Again, I think the idea there is that the concern that there could be an inadvertent fertilization event between a chimeric animal and non-chimeric animal, you end up with a human-animal hybrid. Again, nobody wants that. And there are strict guidelines right now about not breeding chimeric animals that could have germ cell formation. We are going to 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 want to leave you with one thought before I turn it back to Hiro here. 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. A 100% human, that newborn brain is not going to 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, um, 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 want to just leave it there and I want to return now back to the science. And I'll turn it back to Hiro. Take it away.

Okay, thanks. So, I was not able to get NIH funding, but luckily I had funding from CIRM, 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 a strict Pdx1 knockout. It worked. And then we also started to make human-sheep chimera embryos. In this photo, we are injecting a TdTomato, which is the red color labeling, TdTomato-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, we, it took some time, but we started to do this kind of human making human-animal, human-sheep kind of. And 24 hours later, in culture, we were able to see human iPS cells still there and, you know, well mixed with these sheep cells. Looks like. Then, our collaborator, Professor Pablo Ross and his team, is injecting these chimeric human-sheep chimeric embryos into the uterus of a whole foster sheep. This is the uterus, this is a sheep, surrogate sheep mother. And, oops, he is injecting these kind of embryos into the uterus. And then three weeks later, we recovered all these embryos. And in this case, I think nine out of 20 embryo, sheep chimeric embryos showed more than one human cell in 100,000 sheep cells. Even so, this is a very small number compared with the rat-mouse chimeras. So, somehow, the human-sheep, and also some other group has shown that human-pig chimera is far more difficult to make compared with rat-mouse chimeras that we have shown. So, at this point, our real challenge is to overcome this interspecies compatibility. We call it xenogeneic barrier. And human iPS has minimally contributed to human-sheep chimeras. And there must be some kind of barrier to prevent efficient chimera formation. So, we continue to, we go back to rodent studies and analyze carefully what happens to chimera embryos after transfer. So, we generated rat-to-mouse chimera and followed their fate after transfer. So, this is the chimera of each embryo after transplantation. So, at E9.5, we see, you know, many embryos with high rat chimerism. But at around E11.5, all these embryos are gone. By E14.5, only a very low chimera embryos are surviving. This is in contrast to the mouse-to-mouse chimeras, where even at E15.5, you know, more than 25% of chimera, most embryos have averaged 25%. And we also tested the embryonic survival rate. And in the case of rat-to-mouse, rat-to-mouse, the survival rate goes down very quickly after it already turned. This is in contrast to the mouse-to-mouse chimeras, more than almost 80% of them survive even at 15.5. So, clearly, interspecies chimeras have a problem, particularly with a high degree of chimerism. 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 chimeras between mouse and prairie vole, or called goldman. They are one of the most distant species within rodents from the mouse. So, rat is about diverged about 25 million years ago, whereas mouse from prairie vole, they diverged by 44 million years. So, we generated iPS cells from all mice, two independent lines, and then we injected them to mouse blastocysts and see how they behave, how they make chimeras. So, two independent cell lines using two individual independent experiments. In both cases, we do see chimeras at earlier stage, like E10.5. But E13.5, the number of chimeras 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 chimeras. So, chimera generation with between mouse and vole was possible, but much less efficient. So, these are the mouse-to-vole chimeras. So, a little weird-looking chimeras, but they could survive. So, it appears that this xenogeneic barrier is probably a result of evolutionary distance. Distance. So, mouse-rat is right here. Chicken-quail, these are chimeras that have been reported. And also, we tested this mouse-prairie vole, which is around here. So, we are able to make chimeras relatively easily if it's less than maybe 50 million years of divergence. But now we have to make chimeras between human-pig, human-sheep, human-mouse. This is about more than, you know, 90, 90 million years of divergence. So, this is a little difficult to overcome.

So, to further understand what happens if we use, you know, higher, you know, and, you know, evolutionary more, up on top, closer to a human being. But obviously, we cannot make human-monkey chimeras. So, we decided to test what happens if we try to make non-human primate, non-human primate chimeras. So, mouse that diverged about 24 million years ago. Human and chimpanzee are much closer, like only six million years of divergence between these two species. Very close to human. So, is the same true for rhesus macaque and pigtail macaque? They are, you know, very close in terms of evolutionary distance. So, we cannot use human, but we can use chimpanzee iPS cells. iPS cell technology is so convenient. We can make iPS cells from very various species quite easily. So, we obtained some blood from the chimpanzee and then we established also other big sub-is chimpanzee RK cells. So, we use these chimpanzee iPS cells and also iPS cells from these different subspecies of macaque. We try to see how they behave when they try to make chimeras among these non-human primate species. The experiment is extremely difficult, obviously. We can generate iPS cells from and we can label these cells with TdTomato. And we transfected BCL2. This is the anti-apoptotic gene. And we know from our rodent studies that BCL2 expression helps to increase the chimera or survival of the injected iPS cells. Then we inject 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 is very difficult, and it's not easy to maintain them in culture for more than 48 hours. So, we analyzed in this preliminary experiment, we analyzed the 48 hours after the initiation of culture. And we have, you know, different experiments. But here we try to see how they behave. They mean human-human or human-sheep, you know, culture. And then they developed into muscle cells. And these human-human, human-sheep were both, you know, differentiated well and cooperated functionally. Right. So, human and chimp, although there are some difference in culture conditions, but they behave very in a very similar manner. So, we're again, we're very close. However, if we co-culture mouse-mouse, a mouse-human, unlike mouse-mouse, mouse-human, they do not integrate very well. As you can see, they grow independently. They do not, they tend not to mix together. We also analyzed the, you know, chimera, 48 hours between these NHP and HP chimeras. As you can see, it's much better than mouse-rat chimeras. So, in some cases, more than 90% of them showed chimeras, particularly with the help of BCL2. So, they do well, addressing this very short subculture period. But we have to, of course, see observe much longer. But there are some technical difficulties. And hopefully, we'll be 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 xenogeneic barrier.

Meanwhile, you know, it almost took 10 years to revise the guideline, but finally, the 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, you know, by this time, like two years ago, we know that, you know, human-animal chimera is not easy to make. So, they, I think they realize that it's not necessary to worry too much about the humanization of the pig or sheep, any animals when we make human-animal chimeras. So, now I got formal approval to do a human-animal chimera research last year. And we started to the injection of human iPS cells into pigs and rat and mouse 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 knockout, meaning all the pancreas and vessels are lucky. So, we should be able to, if it works, make a human pancreas, also the blood and vessels are from iPS human-derived. And we're studying 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 xenogeneic barrier between human and pig.

So, in order to, you know, over the years, I realized that, as Insung mentioned, many people concern about the generation of animal with a human brain cells or germ 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 iPS cell lines that cannot at all contribute to human forebrain or germ cells. So, just to test this idea, we knocked out OTX2 and PLP14. These genes are important for the generation of brain and also germ cells. 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 high manners. So, essentially, if we use this, similar to this double knockout iPS line, then we do not need to worry about generation of, you know, some ambiguous animals, pigs with human brain or human germ cells. So, this is one approach to minimize, to reduce the people's ethical and social concerns. So, we are planning to use this kind of iPS cells for our future research, human-animal chimeras.

I think Insung has a comment on this. Sure. Thank you. I want 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, you know, go to the website and enter their, 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 in 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, you know, how representative that is of actual individuals. And a lot of the comments have to do very broadly with things about, you know, you shouldn't be playing God, or this is, this stem cell 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, you know, 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, some of my colleagues at the Hastings Center. They 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 besides the pig-organ chimeras. So, so I think those are those are pretty legitimate concerns there about this paper. Now, the authors did respond. They didn't respond by saying that they acknowledged these limitations in the paper itself. That's what they claimed. And that 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. You know, the authors of this study claimed that the public attitudes had greater acceptance in Japan for this type of work. But I think this calls for a need for more 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 revising them. 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 U.S. and out of the U.S., we had to pretty much proceed as 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, where 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, you're going to make them better. Right? But the reality is, if anything, modified animals have a radical, you know, 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-human 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.

Okay, all right. So, to wrap up my talk, as a conclusion too, so xenogeneic system iPS cells contribute to chimeras much less efficiently. So, there's a general barrier. And so, what, what is the mechanism? There could be a number of reasons, but mostly dependent on the evolutionary divergence or evolutionary distance between two species. This, this could be differences in ligand-receptor interactions. They may have lower affinity, for example. This could be, there could be several 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, you know, interspecies chimeras. So, in conclusion, I think we need to understand and manipulate better the barrier. 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 xenogeneic barrier that exists between human and animals. I, I think it's also important to, you know, keep a 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 the possibility of infection or tumor development and so on. So, that's, you know, all about what I want to say. And these are the names of the people who contributed to this project, and also the funding agencies. So, I stop here and I get, you know, get back to Insung.

Okay, great. Thank you so much. So, we've had some questions come in. And so, while I'm, I'm going through those, let me start with a question that's come in by Bob Truog. 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? How, what are your thoughts on that? And, um, are there advantages or disadvantages as compared to the strategy that you're pursuing?

Yeah, that's a very good question. I think, actually, I was trained as an immunologist, and maybe a dropout. Immediately, I still think that no immune system is very precise. And it's not just the Major Histocompatibility Complex, MHC, or HLA that determines the, you know, self-non-self. So, I think their 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 patients have to have, you know, immunosuppression for a lifetime, I guess. So, it could be, could provide a good bridging therapy until the donor becomes available. But eventually, I think it's, it's not an ideal situation. It's okay, it's it's needed, but it's it's not an absolute, you know, 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 xenogeneic barrier. But once generated, this organ is autologous. So, it should provide 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 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 would make it a little bit more foreign to the 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, uh, organs generated in pigs should have some pig cells, you know, sort of coexisting, contaminating. But as I showed you in the rat-mouse experiments, you know, we are transplanting these into the immunocompetent recipients. It may, we may need to do some immunosuppression initially. But, you know, since the immunocompetent, eventually they will eliminate all the pig cells, you know, so eventually they will, the patient 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. Let me just get these to you 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, um, how do you actually get all the cell types you need in, let's say, you know, a pancreas or 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 look at Pdx1, all the pancreatic cells are deficient. So, we are able to replace it with the iPS cell service. But of course, you know, vessels, hematopoietic cells, or fibroblasts, there are many stereotypes. 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, all are deficient. So, you know, at least, making, you know, similar combination knockouts, we should be able to replace most of it. May not be all. But as I said, eventually, it, once transplanted, that, you know, human cells will somehow replace, eliminate pig cells and replace by his own. That's my optimistic. We also recently just published. We found another approach to do organogenesis in xenogeneic systems. So, this is a little different from blastocyst 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, and 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, uh, 20 people die waiting for, uh, 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 our food. This is an almost negligible number of pigs. And I think, you know, from the, uh, medical viewpoint, I think, uh, there's this is this approach is, I think, uh, rational. I mean, we can, you know, uh, argue against the kind of, you know, criticism.

Yeah, yeah. I must say, from my own point of view, there's an important distinction between the 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, um, they're actually going to be treated as medical products. Um, you, I would, I would think that the, um, housing and the care and even the, the euthanasia of these animals would be more along the lines of like a non-survival surgery. And, um, and pretty, pretty, I wouldn't, it's not free-range, but a pretty, uh, more comfortable environment than, uh, 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, um, 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. 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, uh, unsafe work environments for staff and for, for the animals themselves. So I don't know if that's going to change anybody's minds, 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, um, for organ retrieval, um, and, you know, maybe that might matter for some people thinking about those differences.

Okay, um, someone asked, did you ever, did you inject mouse ES cells into monkey embryos? What type of, what other type of cells did you put into monkey embryos? And did the mouse, did mouse ES cells, does that work, or did you just stick to, um, yeah, 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, but, uh, because we, the reason why we performed that is we know that mouse ES cells are truly, uh, naive or pluripotent stem cells. 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. But we didn't, you know, do, we didn't spend so much time on that because now we're working on human iPS cells.

And somebody else asked, um, even if this technology is feasible, would you be able to grow an organ soon 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, uh, for those with, uh, uh, for example, heart or kidneys, we have, you know, artificial organs to support their life for a while. So depending on the organs, but we, we think we can, if it works, we should be able to prepare organs within 10 months or so.

You know, I, I think where some of the attendees are really quite fascinated and 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, transplantation waiting list, and there's no, you know, money involved. But if you're able actually able to grow organs in the kind of like a 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 fairly, you know, get organs into people who maybe on, on the current waiting list are ranked very high, but maybe because they don't, they can't, you know, have access to researchers who will can use their iPS 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, uh, this, this future, and have you thought about any of that?

Well, that's an interesting question. Uh, you know, first of all, I should, as I mentioned, you know, there's a black market. You know, actually, 10% of transplantations performed worldwide, uh, are using, uh, you know, those, uh, organs from the black market. It's clearly, you know, for the rich people, I would say. And even that, even even though, even after transplantation, they have to have, uh, immunosuppressants throughout their life. It's a lifelong, you know, immunosuppression, which is very costly too. So it's a very expensive, uh, 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 because, you know, we, you know, sacrificed 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, uh, you know, artificial, you know, organs and immunosuppressants. So, yeah. So once, uh, then, so that means, uh, probably initially, we have to think about how we triage the patients. But once the system is established, it should provide a relatively inexpensive way. So, uh, 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, uh, relatively short time, not worrying about the, you know, price cost involved.

Right. So, uh, so your work focuses on the transfer of human cells and trying to develop, try to trying to fill in the the missing organ of interest. But obviously, you also have to work equally hard, maybe with another group, and developing these organogenesis disabled animals. And I've noticed that, um, in your slides, that you, you've had pancreas disabled, and you've got various other organs, but I didn't see heart disabled. Is that a particularly challenging one? You know, because your other slides show that the, in, 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, uh, 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 U.S., uh, cardiology is very well advanced. Many good cardiac cardiologists, cardiac surgeons here. However, the basic science of heart development is not that advanced, I would say, compared with, you know, organs like a pancreas, kidneys, liver. Uh, I think it's a little more, you know, complex, the development of a heart. So we may have to knock out, uh, probably because heart is a very important fundamental organ, you know, it starts development, starts early, and involves involves several different transcription factors. And we still need to know much more about the development of heart in molecular terms. So that's why, although I'm very, very much interested in the generation of heart using this approach, but we still need some more, uh, 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 side effects or unpredictable, unpredictable effects in the process that you're pursuing?

Well, that's, uh, that's what, you know, I'm constantly thinking about. I mostly, uh, I don't concern much about, you know, human-like pigs. But, as in the COVID-19, you know, I worry about, you know, uh, zoonosis, some, you know, viral new virus coming out of this type of, you know, human-animal chimeras. So that's one thing I worry about. But this is, you know, I think Joe's group's contribution, uh, now we can screen whole genome pigs and look for any potential, you know, uh, viral-like, you know, uh, sequences and we can knock it out. So, you know, we may have some, uh, you know, uh, potential initially. But once this system starts to work, I focus my all attention to the safety of this type of therapy. And technically, I think it's possible.

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. I, you know, it makes me rich, hopefully, but that's not the only purpose. You know, it 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, unnecessary capitalistic involvement in this kind of treatment. So I'm, I'm hoping that, you know, I don't, it doesn't need to be migrated, 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, uh, yeah, that's my idea.

You said that recently the Japanese government changed their 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, kind of any cultural differences, anything like that?

Well, over the years, almost 10 years, you know, my work, our research has been, you know, published and also, you know, the media, newspapers, TVs, they also, you know, uh, talk about or broadcasting or talk about our research. So people realize that, you know, initially it was a kind of, uh, shocking, uh, resource for them, but now they realize what's going on. I try to, you know, maintain the transparency of our research also and keep telling them what we are trying to do. So I, I think I've got better understanding of the people about what we are doing. And also, uh, the government has realized, uh, that, you know, human-animal chimera is not like making a monster, and a high contribution of human cells is not so, you know, easy. So those are the things, the government has changed their attitude also. People have also got a little better understanding of what we are trying to do.

Yeah. So you've been at it for quite a while now. Uh, what do you think is that the fact or factors that that have really contributed to to the slower pace of what you're hoping to do? Is it funding issues? Is it, are there scientific technical barriers? What are kind of like the biggest reasons why, you know, the work, 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, uh, bad disclaimer. So, and this 100% disclaimer, uh, is, uh, possible. They do not want to change. Their currency 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 chimeras and almost no contribution to human cells, I think then they thought, you know, it's okay, it's safe, uh, to change the guidelines. So they're, they're not interested in, uh, introducing new treatments or new technologies. They just want to be conservative, not to be blamed. Yeah, but it's a general attitude of Japanese, you know, bureaucrats.

So since coming to the United States and and working under CIRM funding, um, do you find that CIRM funding, this is the California Institute for Regenerative Medicine. It's a state bond money that's devoted to stem cell and regenerative medicine, just for the audience to know. So do you guys, CIRM funding, do you find that that is a pretty good substitute for NIH funding, or is there anything you kind of, you know, wish, uh, any reason that you might want to wish for the moratorium at the NIH to be over and that you can apply for NIH funding?

I think, you know, from my experience, CIRM 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, 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, uh, trials or just, how can I say, I cannot use this money to do anything else. So I cannot do, uh, any innovative or just challenging experiments, uh, if I solely depend on NIH grant, whereas CIRM is more flexible. I hope, and they take more challenging proposals. So that's something I like.

Right. Yeah. 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? Um, I'm curious about why, you know, sometimes in your graphics, you show both sheep and pigs, and we, and we actually do talk at the ICCR 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 our experience, this xenogenic barrier, uh, is, you know, also, uh, they show variation organ to organ. So, in other words, mouse-rodent chimeras, when rodent cells are injected into mouse processes, or even between us, say, in mouse and rodent, somehow, interestingly, if we make chimeras, for example, the blood cells tend to be mouse dominant, whereas lungs or some other, you know, organs, blood cells tend to become dominant. So there's some, you know, organs, probably some organ-specific cytokines or 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 gives makes better, uh, human. Okay. So if possible, I'd like to try many different species. But, you know, sheep and pigs are the probably the only reasonable animals to try because it requires a certain level of embryogenesis technology, and those technologies are not available besides big and cheap at a reasonable level. So that's, that's why. So I think it's, uh, 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, um, why do you have to?

It's an interesting question. Now, we work together, and we have the same ultimate goal, that is to make organs, human organs, in a 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 in 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, um, do you ever have conversations with your lab members and your postdocs about career development? I mean, I, I would think that some people may be a little concerned in your lab about, you know, if if there is ever sort of a bad press around this kind of research or or public public disapproval, that, um, their career trajectories could suffer a bit. Do you ever talk to them about, you know, these other concerns about, you know, um, they're starting off in their science career, they're part of your lab, and this is for some people very controversial research. Just curious about how that mentoring goes.

Well, I don't think, no, they have that worry about it much about it. And, and I think they understand that, you know, this is like a, what do you call, a moonshot experiment, you know, very challenging experiment. Yeah. I don't think any postdoc, 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, 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 human organs in pigs yet, but, you know, on the way, just like Apollo 11 project, we have, you know, internet or GPS, many, you know, by-products from this, you know, long shot, big, you know, project. So I think they're enjoying it, rather enjoying it. So we, including myself, do not worry about those, those things. We may be too optimistic, but yeah, they are publishing good papers and getting good, you know, uh, 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 in the manuscript phase. Sometimes I get asked to do bioethics reviews. I wonder, do you ever get bioethics reviews, or what does some of the reviewers typically like focus on? I'm curious about that.

Oh, yeah, that's an interesting question. So maybe seven, eight years ago, even our first Cell paper, I think editors, uh, some are skeptical about what we're doing. And to tell you the truth, I wasn't trained as a developmental biologist. So I think I made, you know, some, uh, warnings or technologies, uh, for the developmental biologists. I was so naive. So they collected some of our usable language terminologies. I mean, I mean, the reviewers, the editors, they were somewhat skeptical. And some editors, uh, you know, worry about the, uh, you know, virus issues, or this cannot be accepted socially or ethically, that kind of, you know, uh, reviews or comments I used to get. But these days, you know, making chimeras 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. That's a good thing.

Okay, so I think you have time for one more question. And this comes again from Bob True. And this is a really nice segue to, by the way, for the audience, our next presentation next month. But, um, 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, 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 is a little different from, you know, Western, maybe U.S. or European countries. So we still do not believe in brain death, but now it's, it's okay, getting better. There's still a 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 is becoming an issue for the medical economy viewpoint. So I think there's a big demand to generate, you know, transplantable kidneys, for example. And now the same is true. We have somehow, Japan has technology to make good artificial kidneys, these artificial hearts. So the many number of patients on artificial heart is increasing dramatically, and this is very, very expensive, much more expensive than hemodialysis. And 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, as I thank you for sharing your time and your expertise with us. For the audience, I want to also announce that we have another session on March 26, Friday, with Nina Sestan from the Yale School of Medicine. 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 and goodbye.