 Welcome to Ethical Frontiers in Biotechnology. I'm Christine Mitchell, the Executive Director of the Center for Bioethics. Those of you who have been coming every month will know that our Center Director, Bob Trug, often does this introduction. As some of you may not know, he is both a practicing critical care pediatrician and he is actually on service, saving lives tonight in the ICU at Children's Hospital, so I'm filling in. We are very pleased to see both familiar and new faces tonight when we first talked about this monthly series of opportunities to talk about novel bioscience and biomedical engineering and look at some of the ethical challenges that they raise. We very much wanted to build a new community of people in conversation with one another, bioscientists, biomedical engineers, people in the labs, as well as in our affiliated clinical facilities and the public in conversation with ethicists. And so, in Suhyun, who has coordinated this series, has been very successful at doing that and bringing in some of the people to tell us about the latest biotechnology. Well, thank you, Christine, for that introduction. And I really do welcome you to the Biotechnology and Future of Medicine conference. I think it's going to be an exciting meeting next month. So I would like to take this time to introduce our special guest, Dr. Ching Ping Fu from the University of Michigan. He is Associate Professor of Mechanical Engineering, Associate Professor of Biomedical Engineering, and Associate Professor of Cell and Developmental Biology. He's Associate Director of the Michigan Center for Integrative Research and Critical Care. Received his PhD at MIT in Mechanical Engineering. And it's really exciting to have him here as a speaker because he brings a fresh perspective for stem cell biology as a mechanical engineer. I'll become very clear to you during this presentation today just what is the mechanical engineer's mindset when they approach vexing issues and problems in stem cell-based modeling. You'll see his innovative approach to resolving some of these problems with consistency that he'll note in his portion of the talk. I just flew in from Barcelona from a very impressive bioengineering meeting. And let me tell you, his work is just the talk of the town internationally. He was really being quite rightfully praised as an enormously useful innovator in the space of embryo modeling. And not only that, scientifically speaking, but as an extremely thoughtful, carefully minded research scientist on top of his innovative science. So I'm very pleased to have him here today. He's really in the entire ethical frontier series, our only guest we've flown in from out of state, and it's well worth it to have him here. So as you all know how this works, we're going to first start with some presentation from us, and then we will transition over to a discussion with you, the audience. So expect, you know, just under an hour of presentation, and about half an hour of discussion with you, and we really do invite you to give us your thoughts and your feedback. So as you are presenting, as questions start to arise in your minds, please drop them down so you don't forget, and then engage in discussion and conversation with all of us afterwards. So the topic for today is embryo modeling and embryo cultivation. This is a rapidly moving area in scientific research and in bioethics. This whole new field of embryo modeling is quite, quite remarkable, very rapidly moving, but also serves up some very complex questions about ethics and policy for this area. So we'll get into all these issues. One of the immediate issues that comes up, and it even came up in the way in which I thought, how would I advertise this talk? Like what title do I use? Because there really is a big question mark about what we call these entities. They're so new. Any term you use is going to be suboptimal from some point of view. One idea is to call these things gastroloids, to kind of mimic the figure of speech people have been using for organoids, which are self-assembling, self-organizing, stem-cell-based models of particular organs. Maybe we might use the word gastroloid to represent the stage of development that many of these models represent, which would be gastrulation, but that's really not the best term to use, because the public's going to say, what the heck is a gastroloid? And also the models as you'll see, and a range of developmental timing, which doesn't necessarily always have to do with the gastrulation stage of the developing human embryo. Well, maybe we can just call these synthetic embryos. And in fact, this is the term that I think a lot of the press has jumped on. This is, again, probably not optimal for ethics and public messaging, because synthetic intensive space is somewhat of a bad label to call something. And it, again, may not be all that accurate in terminology for describing this kind of work, because these are made of real cells, real human cells that really do dynamically self-organize in these unique ways. So there's some question about just really how synthetic are these things. This is probably the best term to use, but it's very cumbersome. Stem cell-based models of early human development. We know that if we use this term in talking with the press, they're just going to call these something else. This just takes up too much space in the newspaper column. So I think if you throw this out, they'll just shorten it to embryo models. So with that in mind, I thought, well, we'll just call this embryo modeling for now, TBA, what they're actually going to be called in the future. One of my favorite terms, though, what these have been called in the past were souls, souls, self-organizing embryo-like structures. Now, Jean-Tier, I don't know if George Church came up with this, but I also like the idea of George Church's souls. But this is problematic, just, it rolls off the tongue, but it also makes your heartbeat a little faster. And this work is really not that old in terms of science. 2014 was the first major paper which described this phenomenon. This was from the lab of Ali Briven-Lew, who, by the way, is one of the speakers at our conference coming up. And this time, he'll be talking about chimera research, human animal chimera research, because he does that, too. But from the Briven-Lew lab at Rockefeller University, headed up by Aria Warm Flash, known as the Warm Flash Paper. All these great names, great labels, right? The Warm Flash Paper described this phenomenon, which was really pretty remarkable. All they did was they used micro-patterned culture disks. These are basically culture systems which, on the bottom of the plastic dish, they had these little, stipple, little bumps that make the cells stay in a particular pattern. What they realized, what they found out was if you get circles in a particular diameter and you put human embryonic stem cells on them or human IPS cells, these are stem cells that are derived from skin cells, mimic embryonic stem cells. Whatever cell line they used, in just a matter of a couple of days, it forms this self-organized pattern. Well, what's going on here? You have, if you test the cell types, you have on the outer layer the endoderm, the next layer in mesoderm cell lineage, and the very center endoderm. No, no. Endoderm is on the outside. Endo, meso, ecto. It's just one, it's two-dimensionals, so it's basically like one cell layer thick. But they also have, they describe what seem to be emerging primitive streak-like regions. So these terms will be a little bit more apparent of what we mean by that as we go along in this discussion. But just now, all three germ layers of the developing embryos seem to be represented in this self-organizing structure. And there may be the appearance of a raised primitive streak-like area. So the primitive streak is also going to come into play in our discussion. So that's pretty remarkable, because human embryos, stem cells, and human iPS cells are known to be identical. You're just identical in type. You really can't distinguish one from another. But when you put them in close contact with one another, where they can touch one another, they're forced to remain in this tight circle, give it a little bone growth factor, just a little bit of a nudge forward, and then they emerge in this pattern. So that's really quite remarkable. Now we're getting ever more look-alike models, this time in the mouse. One of this is a real mouse embryo, and the other one is a mouse embryo. Do you know which one's which? Do you know which one's which? The one on the right is the model. Remarkable, right? So at what point does a model become the real thing? Can your model get so good, so complete, that really functionally you made an embryo? So now I'm going to turn it over to Dr. Fu and he'll lead us through some of his work in working on these kinds of entities. Let me fix it out of here. Thanks, Yingsu. Thank you for the kind introduction, and also it's great pleasure for me to be here. So as a scientist, in fact, we over the last few years, I think my group has entered into this exciting emerging area, and people over time realize that the human stem cells in particular, human proponent stem cells, including both human embryonic stem cells and induced proponent stem cells, really take some amazing self-organizing properties and developmental potential. Somehow when you add clayland into suitable three-dimensional culture environment, or maybe even two-dimensional culture environment, and they start to self-organize, as Yingsu mentioned on one fresh paper, and on two-dimensional surfaces, they pattern into a multi-serial structures. Somehow, given signatures consistent or compatible suggesting that there's some embryonic developmental events happening in the culture, so somehow we, in fact, this is a slide summarizing the existing models have been reported from my group as well as from many others, and you can see that on the left-hand side, that's basically the section of images showing the human embryo, and right after implantation, and over time from implantation towards gastrulation, and the next step is narration, and narration is basically the central nerve system, the precursor of the central nerve system, I'm talking about the neural tube, what developed from the actoderm layer after gastrulation. So you can see that, in fact, I think I will just use the mouse, and this is the blastocyst containing the epiblast, and then afterwards there's a cavity formation, this is what we call immunionic cavity, a symmetry breaking containing a bipolar tissue, and afterwards there's gastrulation, let's take a look, that's the gastrulation, you can see the cells start to enter, and beneath the space between the epiblast and the underlying, what we call hyperblast, and soon after, I guess I don't need to provide the details, but afterwards, one of the germ layer, the actoderm, was start to, I would say, pattern and develop into neural tube, you can see the neural tube here now. So more or less over the last, I would say about 10 years now, less than 10 years, and different groups including ours, and we have developed using human proponents themselves, again, both embryonic stem cells and induced proponents stem cells, to develop models that allow us to model different events, different developmental events, and presented on this slide, and early human embryonic development events, and some of the development events, in fact, might not be a single isolated events from this models, in fact, I'll present some models from my group, you will see that it's a continuous developmental process, and showing a very interesting, I would say, morphogenetic dynamics, mimicking a continuous embryo developmental events. Okay, so how we get started, and before I present some of the real experimental data, and I will provide some more information about human embryonic development after the human embryo implied into the maternal uterus, and you can think about this is the human embryo, and this is what we call blastocyst, and containing the prepotent epiblast. The epiblast are the prepotent stem cells, eventually will differentiate, and will form all the cell types in the human fetus, and after implantation, soon after implantation, you can see that somehow the cells, the epiblast cells, they will start to organize themselves, and open a cavity spontaneously, this is what we call proemianic cavity, and soon after, you see some very interesting, what we call symmetry breaking events, where the cells here, next to the invading trophoblast, they start to differentiate into what we call aemianic actiderm. These are the precursor cells, eventually, over time, they will differentiate into aemian, that's the extra embryonic membrane enclosing the developing fetus, and then the cells remaining here, next to the hyperblast, they remain prepotent, but soon after, in fact, you can see that there's another symmetry breaking events, where the cells, the epiblast, as the prospective posterior end, they will continue to develop, and they undergo gasulation, and they will develop into, for example, mesoderm. I should also point out post-implantation, but before the onset of gasulation, there's a very important cell type, is what we call primordial germ cell. These are the precursor cells for sex cells, and somehow, I guess, in mammalian development, including human, monkey, and the mice as well, in fact, primordial germ cells, they will appear, and in fact, post-implantation, but before gasulation, in the embryonic structure. So again, these are the sectional images showing corresponding stages of the human embryo, and you can see the very clear symmetry breaking here. This is the aemianic actiderm, and in closing, what we call the pro-aemianic cavity here, and this is the columnar epiblast, and the reason I pointed out, because this asymmetrical tissue structure, I think as you can see from what I'm going to present, this is almost an embryonic structure feature that's very distinct and very important and to validate, I guess, to confirm the validity of our models. So this has been a very surprising journey for us, and when we started this project, we wasn't thinking about using human proponents themselves to model human development, and more or less we were excited about the existing organoid research and where people using human proponents themselves inject playland into three-dimensional cultures, and they were developing to organ rudiments. So in one of the control experiments in our 3D culture, and where we don't add any exogenous external soluble factors to drive them to differentiate, so we don't have those soluble factors, so in our control experiments, somehow the students start to see some amazing self-organizing events, and somehow the embryonic stem cells, they form a colony, they start to talk to each other, and they start to organize themselves and into, I would say, patterned structures, and bear some significant similarities to developing embryo. So let me go through the videos with you. In fact, the top video here is going to replay, and so it's going to replay soon, and you can see the cartoon here, more or less summarizing what is happening now is placed. You can see originally is a cluster of cells forming a colony, but soon after, you can see it forms a cavity spontaneously without any exogenous external input from us. But soon after, you can see that the cells start to look somewhat different. You can see they become squamous flattened, and you start to see extensive protrusions extending from the basal surfaces. So we did some, we performed quite some molecular characterization to determine, we understand these are the cells differentiating from human proponents stem cells, because the morphology looks very different, and when we stain the cells for proponents markers, these are the markers associated with proponents, and we see these markers are gone. For example, here, a nano, which is a key marker for proponents stem cells. You can see that the marker, the nano is now expressed in the nucleus of the cells. And it took us a while, and at the end, we realized that the differentiating cells, where they are forming the cavity, these are the cells, these are the red cells after differentiation. Then we go back to the cartoon. It turns out the differentiating cells forming the cavity, these are the immune actoderm cells, spontaneously differentiating within the three dimensional colony. But of course, in the first example here, all the cells eventually differentiate, and they form this uniform, what we call squamous immune actoderm cyst, right? Uniform cyst containing all the cells as immune actoderms. But somehow, in the subset of the cell colonies, we start to see additional phenotype. The cell colonies, they were organized, but at the same time, they developed into different structures. Let's take a very careful look. They form a cavity just consistent with the previous video, but soon after, you can see that somehow, only a portion of the cells initiate the cell shape change. They are differentiating, that's this portion, and you can see that more or less the differentiation will propagate and will propagate from the initial site, and then there's symmetry breaking, forming this bipolar, what we call bipolar tissue. And you can see now, very clear, and morphology, distinct morphology difference. And between two compartments of the structure, and when we stem the cells, it turns out the columnar cells, basically this cells, they retain expression for purportancy markers. These are undifferentiated purportant cells. Well, only the differentiated cells, that's basically the cells here, the red cells, they will express markers associated with the immune actoderm as we identified in the first example here. But let's watch the video here, I think it's gonna replay soon. And in fact, if we look more carefully, we see additional phenotypes suggesting continuous progressive development in the same culture and from different subset of cysts. It's the cell colonies, now you see symmetry breaking, the cells here differentiating, right, and forming this bipolar asymmetrical structure. But what's most interesting is happening here, you can start to see that the cells start to almost disseminate, they migrate out from this compartment, this pole of the structure. And again, this is the cartoon summarizing what you observe here, that's symmetry breaking, and soon after the cells here, they start to migrate out, disseminate. In fact, when we stem the cells for markers associated with gastrulation, you can see varturi, snail, and these are canonical markers associated with gastrulation. And you see this gastric, these cells disseminating cells, they will express gastrulation markers suggesting these are gastrulating cells. Going back, in fact, again, I want to call this, like really bring you back to what's really happens in the human embryo, and in fact, I think what we have been able to model using this is very simple three-dimensional culture environment, without any exogenous, I would say external user input, is you can see we are forming the cells for form of cavity, and soon after they break symmetry, and at some point the cells will disseminate, and from one compartment of the cellular structure. So more or less, we will publish the paper, people realize from the early publications, I guess for the first time people realize that indeed, even on the three-dimensional culture environment using human propulsion stem cells, and this propulsion stem cells can allow us to model certain events associated with human development. We're not necessarily modeling the entire human embryo, but at this, the core of the implanting human embryo, I'm talking about the purportant epiplas, I think just using human purportant stem cells that allow us to study their dynamics, the continuous developmental events. So in fact, so the privacy example I talked about, we were using what I called conventional three-dimensional culture environment, you see the cells in a 3D gel, and for more or less, you see the cells in a random fashion, and the cells will start forming initial cell clusters, colonies in a random fashion, that leads to a lot of heterogeneity even from the single, the same experiment. So you can see that it's the real picture here showing a real example, experimental data from a single experiment. We can see how heterogeneous it is from our experiment. Of course, this is what we like to see, and really you can see very clear, distinct asymmetric structure, right? Differential squamous tissue here, and this might be the gas-related cells, and before the cells start to disseminate, migrating off from the epiblast light compartment, but the other tissues here, you can see this is more or less uniformly squamous, the cells should appear to be uniform, where over here you can see some cells might have not differentiated while others have been differentiating, but there's significant heterogeneity, and I would also say that, in fact, conventional three-dimensional culture environment has significant limitation in reproducibility as well. So as Insu mentioned, we have very strong engineering background, we are mechanical engineers, and we know how to build things, or otherwise we can bring additional tools, and in this case, micro-foodic systems, and by using micro-foodic devices, now we, I would say, we have a much more controllable system now, and to generate such embryo-like structures, and I would say stem cell models. So the micro-foodic device is, I can explain very quickly, and so this is the top view of the micro-foodic device, and contain a center channel, this is what we call gel channel, and then the top and bottom channels remain open, and for us to load the cells, and also adding external chemical signals. So the cells, after the cell loading, you can see cell loading, and what we do is, oh, I should mention that, the gel channel is important for us, because after we load the gels, during generation we understand that gel will contract. Because of that, it spontaneously generates what we call concave gel pocket, or form a pocket, gel pocket, concave shape. So because of that, now what we do is, after cell loading, so we just tilt the device for 90 degree, and wait for 10 minutes, allow the stem cells to settle into each pocket, so they will form initial cell cluster, and afterwards we just generally remove the floating cells. So I should point out, in fact, such micro-foodic devices has been very commonly used, and have been developed for many other applications, including, for example, starting, say, cancer cell migration, angiogenesis, and some other important applications, in fact groups like Roger Kent's group as MIT, and they have been, I would say, pioneering this, using such devices for other applications. But for us, after we load the human proponents themselves, that's, you can take a look of the cartoon here now, after the cell loading, and the initial clustering of the cells, the human proponents themselves, they're just amazing. Again, they have amazing self-organizing properties, and developmental potential, and once they start to form commonly, they start to talk to each other, and autonomously you can see that from the cartoon, they organize themselves from a cavity. So you can watch the video here, and this is a video showing that a portion of the microfluidic device were five cell clusters, and synchronized development from these five cell clusters, and obviously you can see the cavity formation, and because in this case, we are not adding any soluble external chemical signals to drive them to differentiate, in fact, this is what people call, you can call them epiblastal-like structures, where the cells remain prepotent, and these are undifferentiated proponent cells. But because this is a microfluidic device, in this sense, now we can add soluble factors, we know are important to drive the human proponent stem cells to differentiate into embryonic ninjas that are associated with progressive development of human embryo. So in this case, now, after the initial cell clustering, now we are adding one soluble factor, it's what we call BMP4, and the reason we are adding BMP4, and you can see that we are adding BMP4 in such a way that only a portion of the cells in the cell cluster will be exposed to BMP4. By doing that, in fact, we can manually, we can control the symmetry-breaking events, and only the cells you can see from the cartoon exposed, directly exposed to the BMP4, this soluble factor treatment will differentiate into aemianic ectoderm that will break symmetry, and now we have complete control because this is BMP4 is added by us, by the students in the lab, so it's not spontaneously differentiating anymore. But because the formation of the BMP4, or sorry, the aemianic ectoderm, the aemianic ectoderm, eventually, I didn't include the data here, eventually the BMP, the aemianic ectoderm, they will continue serving as local signaling center, and they will sending out soluble factors to continuously drive, for example, the epiblast here, compartment here, to drive their continuous development. So they're intrinsic cell-cell interactions, tissue-tissue interactions, guiding the continuous development of such aembronic-like tissues. So let's watch the video here. You can see, again, synchronized development, and there's cavity formation here, and you can see, if you watch very carefully, the cells here, they're exposed directly to BMP4, the soluble factor, they become thinner and thinner. If you watch it again and carefully, so first there's cavity formation, and then you can see the cells, let's watch carefully, the cells here, they become thinner and thinner. They're directly exposed to the soluble factor, they're differentiating, and soon after you can see the cells from the other compartment, and they undergo gasolation-like events, and they will differentiate into the gem layers. So now, in fact, I think the key point, the message is, using engineering tools, now we can control, in a very controllable way, to, I would say, the progressive development of such embryo-like structures, the embryo models. All right, so just another slice, demonstrate the controllability and reproducibility, and to an extent, because this is the micro-footy device, is an engineering tool, and it's very easy to scale up. So indeed, we can now, we can generate a lot of such embryo-like structures, or embryo-like structures quite easily, so you can see, this is just another video showing nine structures commonly as they are synchronized development. I should mention that a key advantage of such system is also, because it's so controllable, and we know where are the cells, it's very compatible with what we call live cell imaging. So we can use microscope, under microscope, we can study the dynamics of embryonic development events. That's hard to study, you can imagine that when human embryo or even animal embryos, right, when they implant into the maternal uterus, they become almost invisible to study, especially the perimplantation development, they are invisible to study. So model systems like this allow us to study dynamics of the development events, and understanding how the cells, they talk to each other, and dictate each other's progressive development. So in fact, as I mentioned that, I think another important data I should present is the primodal germ cell-like cells, their appearance, their emergence in our system. As I mentioned that in human, as well as monkey, as well as some other mammalian models, we understand that primodal germ cells, these are the precursor cells for sex cells, they will appear early after implantation, but before gastrulation. So we decided to look into primodal germ cell-like cells in our model, and it turns out, you can see that TFAP2C, NANOG, and SOC17, and we basically used these markers to identify the primodal germ cells in our system, and the triple positive cells, if the cells show markers, show these triple markers, and these are primodal germ cell-like cells. And indeed, you can see that from the staining images, and we start to see primodal germ cell-like cells in our model. And give us strong indication that indeed, I think our model, I think it's good to model to recapture some key embryonic developmental events involved in human development. Okay, so I think I'll stop here, and more or less this is a presentation about the research activities from my group, and I should mention that there are a few other groups and working on related questions using human proponents themselves, and but here is a presentation about results from my group. Last time that we're switching slideshows. So I'm gonna tell you what I find fascinating about this work from a bioethics point of view. So thank you so much for that wonderful overview of what you're doing. So you may ask, well, what's the benefit of doing this kind of research? What can we gain from this sort of embryo model? And choose, in discussion with Dr. Fu, someone that they really striking potential immediate benefits this research. I'll give you just two. One, women who are pregnant take lots of prescribed drugs, and for 75% of those drugs that women take that are approved by their doctors, there's no data, zero data on the effects on the embryos of these drugs. So if you have a mass scalable, controllable microphoretic system that will give you various aspects of embryo modeling to screen these drugs, that could have an enormous impact on patient safety and the use of these drugs. So that's area number one. Not alone, I think, is the enormous advance. Number two, as Dr. Fu just said, this seems to be able to generate primordial germ cells. There are other teams that are very interested in converting these primordial germ cells all the way down to mature suburban eggs. So if you use IPS cells from living persons, skin cells or blood cells converted into stem cells, loaded them into the channels, you can have pretty much an endless supply of patient-specific, person-specific, primordial germ cells, or further research, and possibly rescue of infertility after the same as true. So I think those are just two good examples of what could come out of this research. But then the question is, how do we know that any type of model really does recapitulate the real thing? How do you validate? How do you know that these models are accurate, especially if they're giving you so-called windows into the black box of human development after implantation when, essentially, women don't know that they're pregnant at that point. So you would have to compare it to your models to culture-natural embryos and fertility clinics. And this is the second part, the second aspect of the title that I gave for today, Embryo Cultivation. So in 2016, there were two groups, Magdalena and Zernicka Getz in the University of Cambridge in the UK, and Ali Brvenlew, at Rockefeller University, published these papers simultaneously in the journal's Nature Cell Biology and Nature Society. And what they did was, they were able to, and you may have heard about this in the news, culture-fertility clinic embryos, which are derived from formalizing eggs, the natural human embryos, as we call it in fact, natural human embryos, culture them in a dish up to the 14-day legal limit, as is the legal limit in the UK, and up to the 14-day recommended limit under guidelines in the US. So the New York group voluntarily stopped, the Cambridge group had to stop otherwise. And what did they do? They were able to cultivate embryos, but with very low efficiency, I was told by Magdalena, she was in the meeting in Barcelona, got about 10% of their embryos to get you right around day 13, or they fixed them on a slide at the end of their experiments, which is still slower efficiency, but it was still remarkable that they got any to go to that point. As prior to Magdalena and Ali's work, the record was I think about seven days. They easily doubled that. Could have possibly gone further, but the culture systems at that point haven't yet been optimized, and further work has to be done. So you may ask, well, what's important about 14-day limit? So I'm gonna let Dr. Fu come up and just explain a little bit more about prolonged culture of embryos, and then we'll get back into what is the 14-day limit and why. I think it's important to talk about why there's still room for improvement, even for prolonged culture of human embryos, but we understand that culture of human embryos in visual by itself still controversial, and they still have some ethical limits, right? 14-day rule is in place for that. But let's take a look of, in fact, I want to go back before I, okay, okay, yes. So you can see, let's go back to the staining images here. In fact, I should point out, you can see that from the images, let's go to day 10, day 11, even though I think as Insou mentioned that, the groups, they have stopped their experiment as day 13, but as that point, even as day 13, as this point, right now the images here, day 10, but you can see that more or less what they have shown here, the human embryo, they still have only been started forming a cavity that's surrounded by the pre-portal epiblots, and this is what we call pro-Aminan cavity. But unfortunately, maybe we still don't know why, and I think possibly because of in visual culture environment, and the human embryo, they are not developing as well as in uterus, because we know that if the human embryo develop in uterus, by day 10, day 11, or I would say towards day 14, in fact, the Aminan act should start to form, and the symmetry breaking should be present. So what I'm trying to say that, in fact, the existing human embryo cultures, and even by day 10, day 11, or even day 13, they haven't studied to observe, doesn't allow us to observe those slightly more advanced developmental events that are still remain unknown for us to study. So let me go back to this. So now, in fact, again, even though we are now able to culture human embryos up to day 14 in a tissue culture, doesn't mean that the human embryo in a culture dish, they were developed as well, compared to human embryo development in a maternal uterus. So there are key developmental landmarks missing from this in visual culture, the human embryo. I'm talking about, for example, the Aminan act during development, symmetry breaking, and the primordial germ cell development as well. In fact, I saw a recent paper from UCLA, and they were working on studying human primordial germ cell development, and I was reading very carefully the numbers, and they culture, they studied, they examined the hundreds of entire human embryos culture up to day 13 in a culture dish. Only one of them, they see one PGC cells, one PGC cells. But, so I think there are room for improvement. And, but of course we understand that it's day 14 for human embryo cultures, and that's reason now people start to rely on other primate models. I'm talking about primate monkey embryo models, and because there's no 14 day rule for primate monkey embryo culture, so now people are pushing and try to improve the culture environment in a culture dish, and to culture the monkey embryos longer and longer. So now, in fact, the two papers, and published quite recently, in fact November 2019, and both papers, and they were able to culture the human, oh sorry, monkey embryos, and beyond early gasulation, and almost towards I would say around day 20, and so people start to see gasulation in this cultured monkey embryos, and so that's another way, I guess for people to leverage the monkey embryos, availability of monkey embryos, the fact that we don't have legal limit for human monkey embryos in a culture dish, and using such primate model systems, hopefully will help us to understand better human development. Right, okay. I'm glad he's black. So we've been talking about the 14 day rule, and some of you just don't know what the heck this is, let me explain it to you. These are the questions that I'm gonna try to address. What is it? How does it apply to embryo modeling research? Let me tell you a funny story about this paper that we published on Star Wars, the A May the 4th, 2016. Funny, not in like, it's hilarious, but it's kind of mildly interesting. So I had written the commentary about how it may be possible in the near future that human embryo modeling research could raise interesting questions of applicability of the 14 day rule, which again, I saw on this one. And that people got accepted in nature and was waiting in the wings. And then I get a call from the editor saying, we just got these two papers accepted, they're coming out on May the 4th, advanced publication on natural embryo cultivation, the two that I mentioned from Magdalena's group, and obviously, they said, can you modify this 14 day rule paper to take into account natural embryo cultivation that got up to the 14 day mark? And I said, well, I didn't know that you got these papers and I didn't know exactly, I don't know, anything about the papers that are forthcoming. They said, well, don't worry, because we have these two other authors who were involved in the ethical review of the New York paper, Amy Wilkerson and Jerry Johnson. So they joined me on this paper. We changed the intro and the conclusion, but kept everything else from my original commentary in the middle, and then we pulled out into a text book to see this publication, the embryo modeling specific language that I had in the original commentary. So this is kind of a strange kind of like hybrid sort of thing that came together at the end. But what we raised was the question of, is it now time to revisit this? And by revisit, we don't mean actually changing it, but just thinking about it again, revisiting that rule. The 14 day rule is a longstanding established limit on how long you can culture research embryos in the lab for study. And this was made famous by the Warnock Committee in the UK which was established after the advent of IVF and mutual fertilization when it was seen realized that not only can you transfer human embryos that were created outside of the womb back into the womb for reproduction, but one might also divert it over for research use, and not only research use but study in culture for some time. So there was a great deal of anxiety, a great deal of public discourse around this. And so Mary Warnock as a philosopher of Cambridge convened a committee and Anne McLaren was also a key member of this committee. Anne McLaren is a goddess of developmental biology and she was the one who helped develop mouse IVF for research purposes. And so there were two key figures, philosophy and science working together with their committee to try to come up with compromises and guidelines which then later became law in the UK. So that's why in the UK you can't buy law go past four or three days whereas in many other countries it's guidelines. Now why is the 14 day limit thought to be important? Does anybody know what people believe happens right around the 14th consecutive day of development time point of human embryology? When you first may see the appearance of primitive street. That's when you first then get north-south axis of the embryo. You don't quite have a central nervous system yet but you have an area that people think will eventually become a central nervous system. But why was this a nice compromise stopping point? Well there are lots of different versions of what makes this a good rule, lots of different perspectives. One is practically very easy. You just mark out two weeks on your lap. The other is don't go past this date. Right, so that's easy. It's easy for regulation because you have to enforce the rule legally. So you have to have some market to know that you're coming up to the line, to the deadline. The primitive street people believed back then was visible. You can see it developing under the microscope and you know that it's about time to wrap it up. But sometimes the primitive street could appear earlier than 14th consecutive day. From a religious point of view, look. I mean you had some people who tried to make this argument and it was persuasive to some. That is that 14 days is the point at which the embryo could no longer twin or fuse together. So that must be the earliest that the soul enters the body because souls cannot be fused together. Souls cannot be divided. So if an embryo in principle could be divided multiple times in a lot, you can actually do embryo splitting. You can create many copies in the lab in early, early stages. Well then the soul can't be there because then God would be just too busy dropping the soul. When you're trying to fight or when two fuse together, where did one of the souls go? You can't have two souls together. So it made some sense from a theological point of view to some people, but it was practically extremely useful. And how useful was it? It was so useful that now we have human embryo research which led to the derivation of human embryonic stem cells, which then led to the development of induced pluripotent stem cells. If you didn't have knowledge about how human ES cells behave, you can make an artificial one. And then out of the field, the rule was so successful in carving out a space to experiment the timeline that we may have outgrown it. Now we're to the point where, well now science can actually get us past this. They didn't imagine back then it would ever be possible to get even past seven days, much less 14, and now we're right there at that limit. Now the 14 day rule is not one rule. It's constantly evolving. So I mentioned the UK Warnock Committee. That's actually the second bullet point. The very first time it ever appeared was in the US. The US Ethics Advisory Board, 1979, who recommended it to the government right after IVF, federal government, guidelines for the funding of IVF research. You know, the guidelines are pretty darn good, but there's a time-honored tradition in the US of getting good guidelines and then not using them and actually not allowing the research funded. So this was just too hot. They didn't want to touch this. But this is the first time they articulated it. 14 days is the important time point. And notice the first version says, 14 days after fertilization. So they're talking about zygotes and the cultivation of natural pneumonia. Why did they pick 14? There are different versions of this. One story that I heard was pretty compelling was if you go too far out above day 14, then you might start to trigger laws at that time that governed over in vitro research and fetuses. They didn't want to trigger that whole complex thing. So they said, we'll stop well before you ever get to that point. Not that we ever think that you can get up to day 14 in a dish. So there may have been some US political legal reasons to drawing the line at 14. While the Warnock Committee, I don't know if they got the idea from the US, but they also landed on 14 after much deliberation. Then you see Canada. Again, first three, they're all fertilizations, zygotes, right? The last one on this slide, NIH embryo panel during the Clinton years. It was another NIH group that came up with funding guidelines for embryo research. Again, beautiful set of guidelines, never used. Too hot. But they actually expand quite a bit. They actually let you go a little bit past day 14 what you wanted to study is gastrulation. So it's actually not a hard limit of what you're kind of interested in. And then they also save space for in the future the possibility, which came four years later, of studying human embryonic stem cells. And you say, these are not embryos. These stem cells. The funniest one will have is when you put them all together. Maybe a single stem cell is not totally boned. It's not a zygote. What if you put them all together in a tight, tight bunch? Then there's the whole composite thing become an embryo. Well, flash forward to stem cell guidelines. 2005, the US National Academy of Sciences. Look, continues to evolve the 14-day rule because now they say, regardless of derivation method, whether they have in mind here, cloning, they also had to do with the transfer. That's not for a while. So it doesn't matter how you derived it, but whatever this thing is, if it can make a baby on an implantation, again, how would you know that? No longer 14 days or formation of the primitive streak because sometimes that becomes earlier than 14 days, which ever occurs first, as they say. What did the International Society for Stem Cell Research say? What do we say now? Well, the latest version that we worked on, 2016 has this language. This is under prohibited. This is, you should not do this or else you will violate international guidelines. Now, what do we mean by this? I'm not entirely sure. George Daley and I wrote this paragraph and at the time we said, this is a very rapidly moving area. Let's kind of be vague, say something, but be a little vague because we know that the science is gonna move forward in unpredictable ways. So what do we prohibit? We said, in vitro culture of any intact human pram implantation embryo or organized embryo like cellular structure with human organismal potential, that was George Daley. I said, well, what is human organismal potential? Well, I don't know. I will let make a baby if you want to. Okay, so that's what I mean. That's how we did it, but I'm really sure. Regardless of derivation methods, so we brought that from NAS, young 14 days of formation of the primitive streak, whichever comes first. So the question is, is the work that Dr. Foo and colleagues doing in violation of this guideline? So it all depends on what we mean by organized embryo like cellular structure with human organismal potential. Definitely didn't want to say souls here. So internationally, this is from the nature of publication that I did with Josephine and Amy. They asked me, Dr. Ken, can you give us a really nice picture of an embryo? I said, nobody, of course I'm tired of seeing pictures of embryos in these embryo papers. Let's come up with a map, a policy map. So the map is coded. Okay, so dark blue is where it is against the law to go past 14 days. So it's Canada, Australia, right? Sweden, UK, Spain. And then the lighter blue is encoded in specific scientific guidelines, China, India, Japan, South Korea, US. But I would actually say that the entire globe, so everything else is like, you know, there's no statement in Africa, countries in Africa or South America. I would actually say that the entire globe is light blue as the international guidelines apply internationally to people doing this kind of research. So there is something out there. I think it's gonna be complex to change the 14 day rule in places where it's encoded in law because then you have to follow the procedures for changing the law into those jurisdictions. So things are moving very quickly. So in December 13th, 2018, Dr. Fu and I, Zernicka Gatz, many other people wrote another commentary in Nature just saying, look, we need to start a debate. We need clarity around this area for embryo modeling. And so that got some of the conversation going again. I'm gonna turn it over now to Dr. Fu and just have him explain how they approach some of their publications because I highlighted their ethic statement for you, my highlighter. And just tell us, what was your thinking here? Obviously, this is the ASCO statement, ASIC statement. We include it into the end of our paper. So we made a statement that we don't believe that, we don't believe that the embryo-like structures, and I have shown you, we don't think that they have human organismal form or potential. And the reason we believe that is because obviously, and those embryo-like structures are generated from human-proposal stem cells, and the structures, they are lacking certain actual embryonic cell types. I'm talking about chovoblasts, I'm also talking about hypoblasts. And those are actual embryonic cell types that will be critical for continuous successful pregnancy and eventually they will develop into yolk sac and placenta. So obviously, those will be needed for successful pregnancy and continuous development of the human fetus. And so that's our argument. And obviously, and all our experiments, they are terminating way before day 14 because in fact, if you saw the time scale in our movies and in fact, we terminated our experiment within about four days. And but I understand that, in fact, the commenting on this human organismal form or potential especially the potential, right? And not to mention so. And that particular term is very hard to experimentally prove or disprove. But given the fact that I think from the scientist's perspective, as this in our work, as long as we can make sure that there are key cell types, I'm talking about, for example, chovoblasts, which is needed for placenta formation, they're not involved, included in our model. And we know that there's no way the cell commonly will be able to develop into a human baby, right? I think that can be accepted. So yeah, and we have been very transparent and all the protocols we used in our research and have been approved by our human stem cell research committee. I'm talking about human propulsion stem cell research oversight committee as the University of Michigan Ann Arbor. And I've been very happy and also to form close, I would say collaborations with people like Insue, I think make sure that the constant class talk between scientists like us and the experts, ask experts and we have constantly communicate and understand. So to make sure that we are not passing the red line. Right, okay. Why is it so important to constantly be in communication with one another, like myself? Well, I don't want you to end up in jail or anybody in this lab. I don't want anybody to inadvertently get in trouble because when people get trained as mechanical engineers or cell biologists, they don't necessarily always get training in bioethics, part of their curriculum. They don't always get training in state law. What does the law say? I look this up. Michigan, state allows human embryo embryonic stem cell research, but has a 14 day limit and bands reproductive cloning. So if you're using IPS cells from living donors, you're making these embryo models or these clones, you know the word clone is from the Greek word clone, which means twig, it comes from horticulture. It's not somatic cell nuclear trans-tree, but the nucleus of a somatic cell is a nucleotide egg and they make that sort of embryo. It's when you take a twig off a geranium, plant that and when it is rice, a whole new plant, then you have a clone. You take a twig and the twig makes a plant. Make skin cells from a person and clump them together and they, under the right bioengineering, create a whole new individual. So some could debate that this is getting us closer to human cloning without using eggs. There's a question of does a 14 day limit even apply here? Massachusetts, let's say here, state law, bands, human reproductive cloning, locates a no baby making cloning technology, creating human embryos for research purposes. So you can't make a human embryo for research purposes, regardless of where you get your funding. And then experimentations on fetuses, but explicitly allows pre-implantation of this, blah, blah, blah. So there's some room for debate depending on how complex these models get of whether something is permitted or not in the state. Things are moving very quickly. So just a few days ago, this report was published in the ISSCR Society Journal stem cell reports. And in fact, I guess just a month ago, we couldn't give this exact same talk because things are really separate, it's just published. So it's me and several members of ISSCR, scientists, development of biologists, et cetera, who just said, look, we need to come forward with clearer guidelines around this area of embryo-like modeling. And so we propose some general statements just to start discussion going because we know the guidelines internationally are currently under the process of being revised. So just a little nudge or a background paper to help the rest of the committee deliberate on these issues. Now, we did set forward some recommendations. Said, look, human models don't integrate all the embryonic and extra embryonic lineages. It should be exempt from mandatory review. So the stem cell committees don't have to review this because they're not complete. I'm going to follow in vitro with this number of cell lines. If you disassemble your embryo model at the time of prune district formation, but you continue to culture the various components, again, should be exempt from mandatory stem cell research ethical review. At no time should you transfer any of these constructs into a uterus of an animal or a human. But then that leaves this question, what if somebody wants to study the complete thing? So my advice to labs like pre-prune foods is for the time being, because a lack of clarity of what exactly of the law, state level or national level apply here, whether 14 day rule applies or not, just to sort of avoid any unnecessary controversy now and to let the field breathe and grow on its own a little bit, try to always leave something out of the model. Don't try to strive to make one model do everything because that's going to trigger all these legal questions. For now, I'm not saying don't ever do it, but for now that might be the wiser thing to go. I don't want anybody in his lab in Michigan to go jail. But the question then remains, what if somebody wants to have all the parts together to see how the whole model runs? That's a very good question. That's the question that right now we are discussing at the international level. So we have international society for stem cell research revising yet again our guidelines, which we think we're probably going to have to revise every five years or so now in case of science. What we said right now is that's the key question. Is the subject to the 14 day rule if you want to try to model the entire embryo? Why or why not? Should someone ever try to do this kind of work? So I invite you now to participate by giving us your comments and your questions. We've just raised lots of issues. I don't think we've really answered anything, but I really invite you now to half an hour. So we have one time to talk and we'll come up here. Oh yeah, so you have mics on the desk in front of you. Make sure that you press the button. You have to keep it pressed because it has to be green for us to hear you. I also wanted to give a plug real quickly. Next month, March 19th, we have Paula or Lara and I organ and present on brain organoids. So you heard a little bit about organoids. We're going to hold the most controversial one out. Brain organoids, you might even bring some to show and tell. And we'll have our conversation here. And then the month after that, first Thursday in April, Roger Cam from MIT who we've mentioned a few times helped to build the microfluidic systems. He'll be talking with us about multi-cellular engineered living systems. And then we have one last one, May. Yeah, it may. And that's public deliberation, precautionary principle. There's sort of a few more to come. All right, so with that, let's go ahead and open it up for questions. Thanks to both of you, super interesting stuff. So I'm curious, you mentioned into some dissatisfaction with how the rules were created but not used. Also there's just the fact that some of the regulations don't seem to be applicable anymore, they're evolving. So in addition to your work, both of you in rethinking the rules, are you also rethinking how these procedures should work? So the process of developing regulations, do you have any insights about how we can do better in moving forward? That's a really, really good question. So I personally think that that should, so let me back up, that international task force to develop revisions to the ICCR guidelines, we're just, I think we're still sort of in the first few months of our work so they're still much more ahead of us than we've already accomplished. My personal view is to exactly do some of that. So if it is on the table right now, we're talking about the 14 day rule for natural embryos and we are talking about whether what are the limits of any embryo modeling work? So we haven't decided anything yet but we're starting that conversation. So A, the topic is on the table for discussion. Then the question is what is the best process by which we do that? Now I think that the Warnock process was, people don't really realize just how political that was. That was extremely contentious. It took about two years and there were like 70 or 90 different societies that commented and gave feedback, medical societies. So I don't know if we wanna wait two years and go through all that. I think in the current political climate in the US it's actually kind of dangerous to offer something like that for renegotiation because you might actually get things yanked away, freedoms yanked away, right? So I don't, that's a really good question. I certainly am gonna bring to the task force the need to deliberate about the deliberation and about the process. I don't really know though what's optimal. It's really tough and the other thing to keep in mind is this is an international society. So we've always held a position that you should always follow local law of course but where the law is silent we offer the guidelines and they're just guidelines or we can enforce them. Is that a different process that they come up with guidelines than law in a pluralistic or democratic society? I think the process could be different for guidelines. So that's the excellent point. I'm gonna bring it up for discussion to talk about process. But I can't even really recommend what we think what we should think is the best process for international society, for that society. Sort of to follow up on that. The 14 day rule was born in the context of in vitro fertilization. So that was aside from the fact that you were uniting gametes natural. Gametes were natural, things like that. And Dr. Fu, I thought your ethics statement was really nice that you included this statement of these constructs don't contain structures that would be required in an actual pregnancy. But I'm wondering when members of the public that are not scientists are part of this debate that distinction of synthetic may really not be appreciated. I just wonder, both of you may be your thoughts on that point that non-scientists, I think, it may not be relevant, that really important point. Okay, well, I'll start off. You're right, you're right. And I think that you'll never make everybody happy. What to say about that? So here's a puzzle I have that I'm wrestling with. How would one know that you don't have that potential unless you try the experiment of uterine transfer? Okay, so I guess we're in some trouble if monkey embryo models make monkeys. I mean, in the time of nuclear transfer, clonings. When Dolly was cloned, people assumed all you gotta do is replace the sheep cells with human cells and you can get a cloned human baby like you got Dolly the sheep. Now we know from years of experience that it's not that simple and the technology is not equally transferable to all these different species. So just because you have mice that are cloned doesn't mean you can have human babies, right? So it's a cloning procedure. But I think that there could be quite a conversation if you start getting mouse pops from these mouse models, if you start getting, especially non-human primate offspring. So how would one know? But back to the messaging issue of like the public even care about these extra embryonic lineages, like does it matter or not? Maybe one way forward is to sort of, you know, again, double down on that red line we seem to have in the current stem-silver parts documented what we're gonna say in the ISACERA. I can be very confident of this. No uterine transfer, right? So the uterus is a new line. Like that's the line. You keep it in the dish, but no uterine transfer. Maybe if we say that, that's still not gonna please the folks who would say for human embryonic stem-silver research. They don't care if it's transferred to the uterus or not, these embryos have complete human organismal potential if they were transferred into the uterus. Why? Because we know from my idea that that happens, that works with reasonable efficiency. So they might still say, oh, likewise here, right? The red line of the uterus does not matter. They still have this potential. I don't know how you would know that without doing the experiment because I think the experiment would be unethical. This here's a mind-blowing question too. Well, what if somebody wants to do that as a way to sort of overcome infertility? So what if somebody wants to create an embryo model and talking with people like Margaret Alaina just a few days ago, I said, would it even be possible to take these embryo models and transfer them, would it do anything? She said, the models are pretty far along. There's well-passed implantation. How would you get the uterus ready for that? So there are all these other issues that maybe you could try to convince people it would never work even if you try, so don't bother. But that's a really good point. Yeah, I mean, people will defend their conclusions that they're really wedded to and committed to it religiously or philosophically. No matter what you try to explain is or is not the case, so I don't know, I mean, like I said, I'll just return to you, you just can't make everybody happy. But maybe try to make as many people happy as possible. I don't have much to add, just as Inge who mentioned that, it's hard to make everyone happy. From scientists' perspective, I think to, as I mentioned, to make sure that we constantly talk, let us constant class talk and input from Essex community and also make sure our research activities in the lab are transparent and to the society, to the administration and to the regulators and to the funding agencies, in fact. And so I think that's also very important and bring, make sure that, for example, for people like me and attending this seminar and give a talk about our research, I think it's, I recognize over time is importance. And as Inge who mentioned that, right, as a mechanical engineer, and sometimes we will, we have been very naive sometimes, very naive, even when we get started working with human components themselves, the thinking was very naive, very naive. We wasn't thinking anything Essex issues. When I first saw the microfluorics and my ass had to fit, doesn't have to look like that. I mean, like mass printing of embryos. And he said, well, it does because you have to, you have to have them all together in the same media. And that's how you get the consistency. Like, baking a dozen cookies at the same time will get you more consistent cookies than trying to do one at a time. So that's how you get the utility for strike screening. That's how you get the actual value socially from this kind of work. But it may strike people in a little off-putting way because what's the immediate trove? It's the embryo generation for commercial use. Sorry to bring that up again, but. I don't know about that. Oh, hold it. Okay, I'll keep my finger there. Let me offer a quick analysis and tell me where I go wrong. Okay, so here it is. First thing is, Dr. Fru, you're a scientist you want to be in touch with in contact and in conversation with the ethicists. Partly because you don't want to go to jail. Sure, yeah. But I can assume also you want to do the right thing. Right, that's right. Okay, all right, now, so focusing for a moment on this ethics thing, doing the right thing. The science is changing and so we ask under these very different circumstances where it's not an IVF, et cetera. Does the 14-day rule still apply? Okay, but to ask that question, surely we have to ask what was the 14-day rule supposed to be doing there? And here we have a big problem. As you said very quickly, you said it was a compromise. And that's the key word I think. On the Lorna Commission, people had different points of view, they finally decided to agree on 14 days but there was no plurality of people who could offer a reason why 14 days is of any significance, whatever. And so you mentioned one of the few coherent rationales that was offered by a member of the committee which is this magical thing about souls, souls not getting together, the supernatural stuff. Now surely that has no place in a secular argument because this is purely theology. So what is the secular rationale? Well there isn't one, it's just that there was an agreement that look, we all disagree, so why don't we say 14 days and each of us will have a mental reservation in our minds, here's my reason for 14 days. Well maybe it's because that's one that would allow a lot of the research we think it should happen without anyone worrying about going to jail. And it's far enough away from the limits of what we can do so we don't have to worry about this for a long time, but that's not a rationale. And so when we ask, well now the science has changed so does it still apply? That's a non-question, what's it? If it was just a compromise, there's no principle whatever. So that means there cannot be an answer to this question, it cannot be. Now what do you do? Well I think the main question, why pick this ball up and rumble this 14 days and you wrote it into the ISZR guidelines. That's a bit of a surprise, but why? Well probably for the same reason as it's been carried along all this time. Well everyone sort of salutes it, no one quite knows what it means, it seems to protect a lot of what we want to do, we don't have to worry about it, but it's not because it tells us ethically what's the right thing. No one agrees on what it says about that at all? Yeah. Okay, it's freaking me out. No, well I'm gonna agree with you. When we ask the question in the commentary, should we revisit the 14 day rule and should we change it? If you're gonna change the rule, you have to kinda know what kind of rule is it to even know what the rules of changing a rule is, whether it's a legal rule or ethical rule or anything like that. And we didn't find any clear answer to the question of what is it, what is the 14 day rule supposed to do? But I think he articulated it great, I mean back then historically it was a compromise, all the other articulations of it that followed the Warnock Committee by the way, it's just repeats basically the same language and what I think happened was, people would say well that looked good, we'll just do that. So I didn't show you the full list, there's Japan, China, they have many, many articulations of it and they just recycle it. So I asked Alta Charo, because she was part of the NIH committee in 2019. So did you know, when I was doing research for the papers, did you know that the first time that showed up was 1978 in the US? And she said oh really? Because on the NIH panel, we thought we were copying the Warnock report. Like they were just recycling it because it was working. So it kind of started as a compromise and then people didn't really know why they were putting it in but they kind of follow it as a good idea because it's popular and it would be weird not to repeat that because then you have to justify why you're not gonna repeat it. So that's why we put it into ISIS or guidelines. He said look, in the first version of 2006 before the embryo modeling stuff happened, right? We said well it would be kind of weird not to mention the 14 day rule so why don't we just throw that in there? Okay so it was kind of this tradition and now we face the question of what is the compromise? What is the compromise now? I think it's back to the original question of back when the first formula of the 14 day rule what's to be gained and what are we trying to avoid? What are the concerns we're trying to sort of try to allay, right? So I think it's a public policy tool to allow practically meritorious science to go forward while at the same time doing something no one's in the study to reassure the public somehow that we're not gonna go down to slippery slope and we're gonna check ourselves. This is all completely just speculative because no one's actually done the study is actually how it functions in society but that's a rationale I think people had in mind. It was kind of like letting, giving us a playing field to do our work and collect data and do meritorious science while at the same time telling people there are boundaries around this football field. There are things that we will not do, we will not cross but people describe it as the line in the sand, right? But I grew up in California and we played on the beach all day and drew all kinds of lines in the sand and we knew that at the end of the day the tides would change and we're gonna lose all these lines. So I think what's happening now is the tides are changing with advancement of science and now we have to say what do we have to gain and what do we have to lose with the changing of the tides and it's gonna have to be compromised again. So back to Matthew's question. I think you're right. I think that's a really nice way to put it. It was what is it? It's a compromise. So when you compromise, both sides have to kind of give up something, right? You kind of have to come to some sort of, some happy medium. So we have to then know, well, who are these sides and what are their interests and kind of work it out? Because we kind of know what the scientific interest is going forward. And then the question is, what are people afraid of? And is the new line gonna be 21 days? Well, then you start getting cardiac cells beating us can imagine a 3D model with a little thing beating in there. That's a little, for some people, over the line, over the comfort zone. So we don't really know where to redraw it. That's the problem. If you're gonna draw the line, where do you redraw it? That would, again, strike a balance. And again, no one's in this study, it's in the sociologically, is it actually make people comfortable? But it's gonna be something like that compromise between two sides, but we haven't identified who the parties are and what their interests are. We knew over two years of Warnock, who the warring sides were and how to get the votes in parliament because they had to pass part of it. Okay, but there's nothing like that in the US and I can't imagine trying something like that right now. Yeah, thank you. Yeah, I think that's it. The it is a compromise. Jean-Tine? Yeah, just briefly hearing these two views, wouldn't then be not more honest to say it's prudential reasoning and not ethical reasoning? Because what I hear you both exchange is a debate about arguments from prudence. Right, right. Yeah, I didn't quite use that term. That's a good term to use in the nature of commentaries. It's a public policy tool. That's what I called it a public policy tool to carve out a space for research while at the same time supposedly allowing people's concerns about science just going way too far. Okay, so and that's why I said this. It was not an ethical rule because I said in the paper, it doesn't seem to make any sense philosophically, secular philosophically, maybe moral theology, I don't know, maybe if you were a Catholic theologian, believe in the soulman, but from a secular ethics point of view, I could not find a good rationale. So it can't be a secular ethics rule. What kind of rule is it? It's not a secular ethics rule. It's a rule for social harmony. It's a rule for, yeah, it's a pragmatic thing. So that's what I try to sort of reframe it as. If that's it, then the rules for that kind of rule change will be pragmatic, kind of, bartering or bargaining, compromising of interests, which has an underlying philosophical basis and kind of, I guess, pluralism kind of basis in the value of that. But it's very different than saying it's an ethical rule because if it's an ethical rule, it just takes one philosopher to come up with the best ethical argument and there you go. You move the line of the argument, it's persuasive. It's not that kind of rule though. Yeah, but the imminent message to the public is moral. Like, this is in all ethics statements because this is the moral way. Well, it was the ethics statement for nature. It's sort of the, I think that's what I would call, it's a little bit, maybe we should think about how to be a bit more honest about it and because moral has like a high price and it sounds great, but it's your case. ISCR says this is not a moral rule. Relax, relax folks, it's not a moral rule. Yeah. You have a question, sir? Good talk. You talked about two technologies kind of moving forward in tandem. The extended embryo culture and the embryo models is the third one, I guess, that ISCR is dealing with which is the chimeras, human embryo, human animal embryo chimeras. So if the synthetic embryo is animal but it's also a chimera with human cells, then what are your, where are the lines in terms of implantation and so on? Cause I think those experiments are probably occurring. Jimmy, is there interest in creating chimera means an entity that has cell populations from more than one zygote and they can be different species zygotes? Okay, so if you have cell populations from iPS cells from human or iPS cells from chimp, is anybody interested in making embryo models that mix pluripotent cells from different species? So I guess the conversation discussion here is hinge on the fact that there's a group from San Diego and working with scientists in China and injecting human embryonic stem cells into monkey embryo and they have been capturing those monkey embryo up to, I would say, day 30. And I guess the claim is the fact that they are hoping that such human monkey chimera system in the long term future will allow to generate for the possibility generating human organs and those organs will be useful for organ transplantation and using monkey as a host. So I guess that's really the conversation here. But I think, I don't think there's a legal guideline right now available for such research. I guess a lot of research activities are moving towards different directions or so fast and sometimes the legal guidelines or they're trying to catch up. Yeah, and the ISSR has a lot of work ahead of us. So I'm the chair, co-chairperson of the committee on guidelines for organoids and chimeras. And so your question is the hot button question right now. What are the limits of any around using monkey host embryos and transferring human pluripotent stem cells into them and culturing them in a dish? Because as you saw in one of Dr. Fu's slides, there's no 14-day rule for a primate embryo cultivation. There's no limit. Okay, so if you can transfer a whole bunch of human stem cells into that, is that a way to sort of bypass a human 14-day rule? So I'll tell you what happens in the UK. In the UK, everything's regulated by the human fertilization embryology authority. And they have a definition of an admixt embryo. So I had to look all this stuff up, folks, for our ISSR guidelines. So you really dig into the weeds of some of these regulations. So in the HFPA, what they say is an admixt embryo is defined as one that is a mixed embryo of species where the human contribution predominates. And then my question is what does that mean? Is that percentage of cells, or you might think of it as maybe it's not the majority percentage of cells, but it's all in the central nervous system. So it's really the central nervous system that makes something human-like, moral, and that's what we're doing. So it's not clear, because I don't think anyone's done an experiment to propose that to the HFPA, but what they said is it's reviewable and, in principle, allowable. They said, if you're going to mix embryos, if it's human predominance there present, then HFPA will impose a 14-day rule on that entity. Because that's their answer. And then the question is, well, can you give us more clarity? I don't think anyone's actually tried that work. So there's some precedent in the UK for something on paper about that. Is that permissible or not? So we have to discuss what will the international society say, because all we have is this one vague language in the HFPA in the UK, and there are people who may go to China to do this work or may do it in other places outside of the UK, outside of that jurisdiction. So we have to contemplate this. I'll tell you right now, it doesn't look like from the data that we are aware of, unpublished data, that you're gonna get more than just like 3% human contribution, because that's really hard to make the human stem cells last there, but maybe, who knows, they might get better. So they're kind of far away from any definition of human preponderance, predominance. But that is the issue that's keeping me up right now with our guidance. I can also follow on this conversation. In fact, I think for example, the stem cell models we have been generating using human stem cells, human preponderance stem cells on a cartridge dish, and this human embryo models might be useful for starting questions like Camara, right? Human monkey, Camarison, and without involving in-type monkey embryos, which is, I think this particular measure is actually very challenging to convince people this is a viable, this is a useful way to conduct research. I should also comment the fact that I think people are talking about, do we, you mentioned that, what if scientists have been able to generate a synthetic embryo model that somehow have all the embryonic and actual embryonic images? And so at that point, likely, I think those models will be considered equivalent to human embryo, and at that point, whether 14-day rule should be applied. But going back, in fact, I want to step back. I think scientists like us, many scientists working in this area, and we're hoping building models to ask fundamental questions about human development, advancing understanding about human development. We know much better mouse development, chicken development, animal development, than human development. But a lot of important fundamental questions about human development, they don't necessarily require us to establish complete human embryo models to study them. So which means that in fact, scientists, we can come up with different models and they are lacking certain cell types, they are incomplete models, and still allow us to probe those fundamental questions without the possibility bypassing the limit. I think that's going to be a viable, a really promising, I guess, approach to push this field forward. Yeah, just to give you a little preview of what's coming in the next two presentations for the series, so brain organoids and multicellular engineered living systems. So you're going to see in those talks, I didn't want to give it out too much in this talk, but I think there's a general guiding principle, what's the minimal level of complexity you need to answer your very important research question, and just leave it at that. Don't add any scientifically unnecessary completeness to your model, because that'll just raise further ambiguities morally. So same thing for the brain organoid models, like what are you trying to answer? So getting back to the human cells and to the monkey embryos, people might say, well, why are they doing that? There's been great difficulty getting human cells to survive in a pig or sheep embryo to get trans-foundable human organs. Some of you have heard about that kind of work, you want to get trans-foundable human organs by knocking out the ability of the animal to make the organ and then rescuing that ability with human cells, and then you get 100% human organ. They can't get the cells to survive in those animal systems because evolutionarily, a pig and a sheep are just too far away. So they said, we need to learn more about how to make the human cells more competitive in a different host environment. So that's what they're using the monkey embryos. So to answer that question, you don't have to gestate it, you have to see them in culture and see what stage of pluripotency, because the different stages of pluripotency you put into the animal host, what stage of the host do you do to get the cell survival to happen, to then make an incremental step to then maybe going further and further out away from human beings with your species, then to get to the large animals. So people confuse this, they think, oh, they're gonna grow human organs and monkeys, that's terrible, because you're gonna kill the monkeys. No, there's no gestation, it's just to study cell-cell competition of the species. And then later, for all the people who eat bacon and lamb chops and stuff, they're gonna use those animals to grow organs for transplantation. So that's why I thought it was interesting to ask the question, if that's all you're trying to do in the system is to use the monkey embryo to see how the cells can survive in that foreign system, would it be informative if you put chimp IPS cells, because that's even closer demand than the recess monkey IPS cells that they're using, or the host embryos that they're using for the other experiments, in a very controlled system to see what it takes to get the human cells to predominate over the monkey or the chimp IPS cells in that controlled environment. So you have to always ask why are they doing this? What's the minimum required to answer that question or to interrogate that question? And let's at least start with that as a general principle just because you just really don't need to go down the road of all the other things that get stirred up when they say, oh, the steam is putting human cells into my gamutus, well, what do you think? People are gonna then jump to what's gonna happen to the animal after it's born, well, no, it's not gonna be born, right? So that's gonna be the principle that's gonna come up in the next two talks, because it's very non-organized, kind of sugared these questions of how far are you gonna go? That's kind of answerable by, well, what is it that you want to study? Same thing with the engineer systems, these weird ambiguous semi-living systems that they're making over in Cambridge, okay? Yeah, I don't know if you want to say. So I just want to thank you both, Dr. Poulouse is enormously informative and interesting and we very much appreciate your coming to talk to us about it. For those of you who are accustomed to coming to this seminar on the first Thursdays, remember that the brain-organized one is out of sequence a little, so it's on March 19th. And please join me in thanking them both for that. Thank you. Thank you.