Hi, everybody. I would like to welcome you to the Ethics and Research and Biotechnology Consortium series. This is a monthly series that brings together ethicists and researchers to talk about ethics issues at the cutting edge of both biomedical research and biotechnology. My name is Zinsu Hian. I'm the host for this series. I'm a faculty member and the director of research ethics at Harvard Medical Schools Center for Bioethics, and I'm also a professor of bioethics at Case Western Reserve University School of Medicine. So I'd like to just go over a few of the ground rules for submitting questions and how you can be involved as an audience member. These are meant to be interactive sessions where we first present material to you, and then there's plenty of time for discussion and interaction after the formal presentation. So just a reminder, this is being recorded and live streamed via Facebook. The event video will be posted on the Center for Bioethics Facebook page and on YouTube pages if you want to go back and take a look at this again, or if you know other people who might be interested in this topic. You can submit questions at any time using the Q&A feature. Try not to use the chat function because we actually might miss your question if you put it into the group chat. It's much better to put it into the Q&A feature. And at the end of the formal presentation, we'll be selecting key questions from the Q&A. We probably won't be able to get to all of them, but we'll get to as many as we can. And both Madeline Lancaster, our speaker today and me, will try to answer as much as we can. If there are any technical issues along the way, just use the chat function for that and send a message to the panelists and staff who might be able to help you. And then of course, we've got upcoming events in this series that you can find at the Bioethics at HMS website. It's right at the bottom there. So with that, let me go ahead and introduce the speaker for today. We have Dr. Madeline Lancaster. She's a group leader in the Cell Biology Division in the Medical Research Council Laboratory of Molecular Biology, which is part of the Cambridge Biomedical Campus in Cambridge, UK. We're very lucky to have her speak to us about brain organoids. In fact, she pioneered the first human brain organoid research paper back in 2013, kickstarting this very dynamic and very interesting field. So let me turn it over now to Dr. Lancaster and she can get started with just saying a brief hello and starting with her presentation. Take it away. Yeah, so thank you. And it's really a pleasure to be able to be here. Let me just get my shared screen going. There we go. Good. So yeah, it's really great to be invited to talk about brain organoids, how we're using them to advance COVID-19 research and also the ethical questions surrounding brain organoids, especially as it pertains to things like consciousness. It's, I think it's quite nice to be here at the LMB actually because I don't know if everybody knows but this is where Francis Crick got his start. And in fact, we have a chalkboard just outside my office that he signed. And I'm reminded of that every time we get on the topic of consciousness just because then of course that became a main interest of his later in his life. And so I'd like to think that he might be happy that somebody like me is working now at the LMB. And so yeah, so I'll just jump right into it. I want to first start by talking about brain organoids and kind of what they are and particularly sort of what the brain organoids are that my lab is using because they're a little bit different from lab to lab. And so before I get into brain organoids as well I wanna first just talk about some of the really big questions actually that my lab is interested in and why we need brain organoids for these big questions. So one of the biggest questions really in neuroscience is what is it about our brains that makes us special that gives us our special cognitive capability and really makes us who we are. And so this is a question that has been of interest to philosophers and scientists alike for hundreds of years. And we still don't have the answer of course but we're getting there, we're getting some hints now. And one thing that we now know is that for example that we know now that there isn't a particular part of the brain that makes us human. So for some time it was thought that maybe there's specific brain regions that don't exist in other animals and that's just simply not true. So even regions of the brain that are important for speech for example, Broca's area and Wernicke's area there are the equivalent regions in other apes for example. And so the basic architecture, this basic architecture of having a cerebral cortex, a thalamus, a cerebellum, brainstem all of these structures are present in all other mammals. So there's not just, there's not some brain region that makes us human. So if it's not that, could it be something a little bit more microscopic? So if we look inside the brain and we start looking at its architecture, we also see that there's no human specific sort of architecture, we all mammals, we all have this gray matter made up of the neuronal cell bodies around the outside and then the white matter made up of all of their connections on the inside. And that's true of mice as well and other mammals. And if you look a little bit more closely and you start really looking at the neurons, you see a particular architecture where you have these beautiful neurons that are organized in a sort of layered fashion. And this layering is the same as well. So across mammals, again, we have these six primary layers in the cortex, they're a little bit more complex in the sense that there's sort of more neurons in each of these layers in primates and in humans. But there's no, suddenly a new layer that exists in human, for example. So qualitatively, there doesn't really seem to be something different about the human brain. So what I mean is there's not some feature that exists that does not exist in other animals. But quantitatively, that's where we really see the differences. And so what I'm referring to here really is brain size and neuron number. Our brains are very big just in general. Even if you wanna see a brain that starts to reach the size of ours, you need to start looking in really big animals, like elephants and whales. But we're not nearly as big as elephants and whales and yet we have a really huge brain. So the best way to think about this is to think about how big is our brain for our body size. And that's what this so-called encephalization quotient is communicating. And so without necessarily getting into it in too much detail, what this number is telling you is that our brains are around seven times larger than they would be expected to be for our body size. Whereas gorillas, for example, gorillas have quite large bodies. So although their brains are quite large, they are not that much enlarged compared to their body size as ours is. And on the contrary, mice, for example, actually have a smaller brain than you would expect for a body size that they have. And of course, brain size is related to cellular makeup and the neurons being the key computational units of the brain, we tend to focus on those. And so our brains have around 80 to 100 billion neurons. So it's often reported to be 86 billion simply because there's a very nice publication from Susanna Herculano-Halzell where they actually counted the number of neurons. But this varies from individual to individual. So it's really more likely a range of 80 to 100. And instead in other apes, we have about a third, the number of neurons. And also brain size is about a third of the size of humans. Now, just to put this number in context, because it's very difficult, I think for our minds to really understand these kinds of huge numbers. So to put that into context, this 80 to 100 billion neurons, if you were to spread this out over the nine months of gestation and calculate how many neurons would need to be made per hour over nine months of gestation, you get 12 to 15 million neurons every single hour of that nine months of gestation in order to reach that huge number. So you're getting the equivalent of basically, so a mouse cortex, an entire mouse cerebral cortex, adult mouse cerebral cortex has four million neurons.  So it's really a huge number. And I want to sort of drive that point home because I'll be coming back to it quite a bit, I think. And actually, I will come to it now if my slide will change. Just to briefly take a little foray into consciousness and just kind of open this up a little bit. And then I'm sure we'll come back to it later. But on this topic of sort of the quantitative difference in humans and the human brain compared with other animals. We obviously don't have a good definition of consciousness yet. So, and I won't even claim to have any idea about this myself. But one thing I think most of us can agree on is that consciousness is some sort of emergent property of a highly complex brain. And it's related to cognitive capacity. So the higher the cognitive capacity, the higher the degree of consciousness. So we may never really have a good definition of consciousness. And so I'm sort of skeptical about whether it's even worth necessarily trying to define or to find consciousness in different organisms or in organoids for that matter. But at least we know it's related to cognitive capacity. And we can think about cognitive capacity and we can kind of measure that. And so if we sort of put a number of animals on a scale here, we think that cognitive capacity, that there are degrees of that cognitive capacity and humans are sort of at one end of the spectrum and worms and flies would be over here. And then if you now look at the number of neurons that matches really well with what we sort of understand to be cognitive capacity. And actually I wanna point out mice and birds here. So for a long time, we know that birds can do some kind of complex things like learning songs and such, but they weren't really thought to be conscious or to have more complex abilities. And recently it's been shown that they can make the same kinds of complex decisions that even primates can make. And actually if you look at their brains, they have, and this is just for a songbird, corvids, which are very, very smart. So crows and magpies have around four times this number. And so birds actually are very smart. And in fact, you see that they actually have more neurons than mice do. So yeah, so I think the number of neurons really, it's this quantitative aspect that relates also to the degree of cognitive capabilities. And it's a bit like computers, right? You can, if you have a really, really simple computer, basically just a calculator, it can only do a few simple calculations. But as you increase the computing capacity, the RAM storage, the processing unit, you get more and more complex abilities. And really I probably should actually put this, even this gigantic supercomputer is probably still way down here on the spectrum if you wanted to draw a comparison to animals. We are still way ahead of even these guys. So, and again, it's just wrapping your mind around that huge number of neurons. I think that it's really many magnitudes of difference that we're talking about. So now I'm taking a shift back to what we do in my lab. So the reason that we are interested in these questions in what makes our brains unique is not just because it's very interesting, but also because it has a lot of implications for human neurological conditions. And actually there are a huge number of people in the world who are suffering from mental illnesses and other neurological conditions. So one in four people will actually be diagnosed with a mental health condition every single year. These are UK numbers, but they're actually highly similar in the United States as well. And despite that high prevalence, mental health and neurological research doesn't receive nearly as much funding as other areas like cancer. And one of the reasons for that is because it's a bit of a chicken and egg issue because these conditions have been very difficult to treat. They receive even less money because the success rate is so bad. So in the area of neuroscience, the attrition rate for new drugs is higher than any other area. So basically it's very high failure rate for developing new drugs to treat these conditions. And that's because of something that some people have called the clinical trial cliff, which is where quite a lot of drugs have successfully made it through preclinical trials and have cured various neurological conditions in mice, for example. So spinal cord injury, for example, there have been drugs developed that have been able to make mice walk again after spinal cord injury. But when they take them to the clinic, unfortunately they fail every time. And the reason is either because they lack efficacy so they just don't work in humans, or because of toxicity, they cause toxic side effects.  And so when you use these animal models to develop drugs, they're not applicable to the problems in humans. And so that suggests we need a human model that we can use to treat this huge number of suffering individuals, really. And so that's where brain organoids or cerebral organoids come in. So these are, this is a method that I developed when I was a postdoc in your clinical lab in Vienna in Austria. And so this method has been around for a little while. We've improved it and made some modifications. And what I'm showing you in this schematic on the left is kind of a schematic of the current method that we use in my lab. So we use a bit of bioengineering. We can make organoids from cells that can be made from any individual, really. You just need a sample that contains live cells and we can reprogram them and make these pluripotent stem cells, which are basically stem cells that can give rise to any cell in the body.  So what we end up with are these tissues that have these sort of little outgrowths, what we call lobes. And each one of these lobes is essentially equivalent to the cerebral cortex here that you see in a human embryo. So this is a section through developing human embryo. This is sort of a schematic of what that looks like at that stage, around that stage. You can see the cerebral cortex is really large in humans. It really expands outward. And so each one of these little sort of buds here or lobes is equivalent to that. And if you section through it, you can also hopefully see the similarity that I'm talking about. So you can see each one of these lobes has this fluid-filled space here. And I forgot to mention, so we've filled these with a blue dye, so you can actually see that fluid-filled space there. And when you compare with a mouse fetal cortex, you see the same organization where you have the fluid-filled ventricle, just like here. And in our organoids, we see the same sort of architecture as well with these stem cells on the inside and neurons around the outside. So the difference here, of course, is that we have several of these lobes. And obviously, in an actual embryo, you would just have two cortical hemispheres. So we tend to look at each of these lobes in isolation and study that as a sort of little cerebral cortical unit. I have a question for you at this point. I think it's fascinating that cerebral organoids in the human recapitulate the fetal stages of brain development. So it kind of starts from very early development onwards. I know that you're aware of a paper published about a year ago by Alison Motri's team at UC San Diego, where they very controversially claim that they observed electrical signaling in their more mature brain organoid models that seem to be very similar to what one might expect in premature infant brains. So I'm wondering if you have thoughts about, is that the best way to think about that data? What are your thoughts on that kind of paper? Yeah. So yeah, so we're also doing more and more looking at the electrophysiology. So what they were looking at was the electrophysiology, the neuronal activity, and we're also doing that in our lab, looking at our organoids in that way. But I think there's a couple of things. So one, we know in science that when you compare data that's collected in a different way, you know, there are issues with, that basically there are issues with comparing data that's collected in different ways. So the data from preterm babies is EEG data. And so what that is is an electrode on the skull. And what it's really measuring is the combined activity of millions of neurons. Really millions. There's a reason you can't do an EEG on a mouse, because a mouse doesn't have enough neurons for one of those electrodes to be able to record the activity. So, and then just the way the recording is done is different. And instead in the organoids, they're recording using what's called a multi electrode array, which it's also electrodes, but these are electrodes that are placed right next to the neurons and are receiving information from individual neurons. So the difference is recording from huge populations of neurons in a human brain and recording from a few neurons in an organoid and then trying to draw similarities there. So that's one major caveat with that study is comparing different data collected in very different ways. I just don't think it's comparable. The only way really to do it would be to implant electrodes into baby's brains, but nobody's gonna do that. So let's just leave it to now. I think probably it'd be more relevant to compare with mouse data, for example, which is collected in the same sort of way. And in that sense, I do think that the electrical activity that they're recording is quite intriguing. It's quite mature in a sense. So it is, you do see quite mature activities, but as I think I'll come to as well, just seeing neurons become mature is not enough to say that you have a human brain there. Oh, that's great. I really appreciate your perspective on that, because I think the temptation is to leave too far forward in your conclusions based on limited data, or as you suggested, maybe questionable data comparisons, and then to stir up a lot of controversy either in the bioethics field or among regulators over brain organoids. It's very important to have ethics and analysis grounded in the actual science and good methodology. So thank you for that. So I'm going to interrupt me further. Go ahead and proceed. Wow. Okay, please do interrupt me, though, if you want to. And so I think there's definitely places. So I mentioned that I would also, I just wanted to also mention a little bit sort of how the organoids that we're working with kind of fit in the grander scheme of different organoids that are out there and other systems that are out there as well. So we're not the first to be growing neurons in a dish. And actually people have been growing human neural stem cells in a 2D culture like this in a dish for over probably a couple of decades now. And we can learn a lot from those neurons. And actually those neurons can become very mature. You can see quite mature activity there. Neurons connecting with each other. And we can use that to learn something about human neurons. But the issue is that the neurons are sort of disorganized. They're not exhibiting the kind of architecture that you would actually see in a brain. Our brains have a particular architecture. It's not just 100 billion neurons randomly connected with each other. They have to be connected in a particular way. And so while this is a great system to look at individual neurons and how human neurons mature, it's not so good for looking at networks and more complex kinds of behaviors. So the field has been moving towards more increasingly complex systems. And these cerebral organoids that we work with are much more complex. They exhibited a variety of different brain regions often. I have to say that our newer protocols when I just showed you in the last slide is actually focused more just on the forebrain. So mainly the cerebral cortex. But we can make organoids that contain a variety of different brain regions. But the issue is that you start to sort of lose control a little bit over what brain regions are made where. So you get kind of a disorganized blob of tissue where you have multiple cortexes, for example. And then maybe one retinal region and then maybe a thalamic region, for example. And of course in vivo, in the actual brain, you would really just have two cerebral hemispheres organized in the right way with a thalamus and two eyes and one cerebellum. And we might have five cerebellums in one of these things. So in that way, they're quite disorganized. But we can look at each individual brain region and actually get a lot of information. So one of the analogies that I draw is sort of, it's a bit like if you had an airplane and remember those things, remember airplanes? It's a bit like if we had an airplane, we took all the pieces of the airplane off and kind of put them back together in a very jumbled fashion. So we'd have one wing on top of the fuselage, you know, a propeller at the back, the wheels on the top, the engine sitting in the middle somewhere. And that plane would never fly. So it's not going to be functional. Just like an organoid is not going to be able to think. But we can study each of those components in isolation and understand something about them. So we can understand something about the wing, about the propeller, oh, it turns, you know, that kind of thing. And so the same is true here in the organoids. We sort of focusing on those regions individually to learn something. So yeah, so this sort of just shows you some of that complexity. This is a movie through a cleared organoid where we're imaging each, what you're seeing here is the individual cell bodies. And then we've traced out these ventricles and you can actually see how they're interconnected with each other.  Now, more recently, we've also been interested in looking at neuronal maturation and activity. And so that's what I was also mentioning. But one of the issues that we've faced with organoids is that when cultured in three dimensions like that, they become big. Now, when I say big, I mean probably the size of a very small pea. So they're still pretty small by most, you know, general public sort of standards. For a biologist like myself, where we're always used to looking at everything under a microscope, they're really big. But like I say, they're pretty small. But still, they're too big to get, to basically to survive and grow without vasculature. So they have no blood vessels. And because they have no blood vessels, they have no blood supply or oxygen being supplied to the inside. And what that means is you get a lot of cell death. So in order to overcome that issue, we've now started doing something called air liquid interface slice culture. So we take our organoids, so my mouse there it is. We take these organoids and before they become too big that they start to die, we cut thick sections. We just, we use a blade to cut them into these thick sections. And then we put them on this filter where you have the nutrient bath underneath and the air above and they get the oxygen and the nutrients that way. And what that leads to is a really nice maturation of neurons. So this is a stain for neurons. And so in red, you're actually looking at all the axons. So the processes that neurons extend through the white matter to connect with other neurons. And in green are some individual cells that we've just specifically labeled. And what you can see is these individual cells that are sending these long processes over to the other side of the organoid. So we get these really long range projections. And in our brains, we know that these long range projections are very, very important for transmitting information from one hemisphere to the other, for example. And so we can see, again, I can see these really long range projections not only within the organoid, so projecting from one side to the other, but also leaving the organoid. And actually if we provide a target tissue, so what I'm showing you here is actually an x-plant of a mouse spinal cord from an embryonic mouse. So we basically dissected out the spinal cord of the mouse and we've left a bit of muscle attached to the spinal cord. So a bit of the back muscle attached. And so that's what this is. And we've just placed it next to one of these air liquid interface cultures. And what you're seeing here are human axons. So this thing in magenta here is staining specifically just human cells. And you're seeing these nice long axons projecting into the mouse tissue. And the reason we put mouse spinal cord there is because one of the long range projections that's so important in humans is what's called the corticospinal tract. These are motor neurons from the cortex that actually project down into the spinal cord. And those are the neurons that control movement.  And so these are the neurons that are actually triggering those movements. So what this allows us to do then by having this mouse tissue and the muscle attached is to see if these projections are functional, to see if they're actually sending out information. So what we've done here is we've actually used an electrode to stimulate only the human tissue here and then watch the mouse tissue. So this is the mouse muscle tissue when we stimulate. And so this is just a trace of the contractions that you're going to see. And this is while we're giving a series of pulses of stimulation. And what you'll see if it will play is the muscle tissue contracting once we trigger those stimulations. This is just a trace of that. So each little, if I can find my mouse, each little line up here is showing you one of the stimulations we've given. You can see these contractions in response. So that tells us that these neurons in these organoids can be functional and actually trigger muscle contractions. Now, more recently we've started looking at other important functions within the forebrain. One of the regions of the forebrain that doesn't really get very much attention is what's called the coriplexus. And this is a tissue inside the brain. It's very deep inside the brain. And it actually generates the cerebrospinal fluid. So cerebrospinal fluid is this really important nutrient rich fluid that surrounds and bathes the brain.  But it's much more than that. It's providing nutrients, certain vitamins that aren't provided by the blood. And it's also helping to clean out the brain from toxic byproducts of all of this high metabolism that's going on in our brains and also clear out aggregates, things like A beta, the protein that actually aggregates in Alzheimer's disease. So the way I kind of think about cerebrospinal fluid is it's a bit like the water and plumbing system of your house. It's providing much needed liquid and it's also helping clear out all of the waste. And if something goes wrong there, it's very bad news. So imagine if all of the toilets in your house clogged and you couldn't get a plumber in. Very bad news. So we were very interested then in seeing whether our organoids could also shed some light on this tissue and also help us learn something more about the cerebrospinal fluid because there really is very little known actually in humans. Like I say, it just hasn't really gotten much attention. It's not the most sexy part of the brain. When you talk about plumbing, it doesn't sound that exciting, but it's so important. And so to do this, a post-doc in my lab, Lara Pelegrini, she developed a method that modifies our existing brain-organized protocol and generates these beautiful corid plexus organoids. So they look just like the actual corid plexus that you see in the actual human brain. And they make these really large fluid-filled sacs, fluid that looks a lot like cerebrospinal fluid. So we actually extracted the fluid from within those and did something called mass spec. So to look at the different kinds of proteins that are being secreted there. And we found that there was a really huge overlap between the fluid that we're seeing in these organoids and actual cerebrospinal fluid from actual human beings. And so that suggests that these organoids are making a CSF-like fluid. And part of generating cerebrospinal fluid is also part of the role of the corid plexus is also acting as a barrier. So because the CSF has free access to the brain, it can flow in and out between glia and neurons. It like the rest of the brain, it needs to be protected by a very selective barrier. So our brains are sealed off from the rest of the body. Drugs, toxic compounds, pathogens are not able to enter the brain, not even our immune cells. So T cells and B cells and such are not able to enter the brain. And that's because of this highly protective barrier. And the CSF has to be protected by the same kind of barrier because otherwise anything that could get in the CSF can get into the brain. And so indeed, when we tested the barrier surrounding these tissues, we found a really selective barrier that was able to prevent entry of even small molecules like dopamine. So we know that dopamine doesn't enter our brains, but it would allow L-dopa, which is a precursor of dopamine, it did allow that to cross. And that's exactly what you also see in vivo in the actual brain. And in fact, L-dopa is given to patients who need dopamine therapy treatment. Right. So just now looking at sort of what we're using these different organoids to model. So one of the first things that brain organoids were used for and are being used for is to model human neurological conditions. So the idea is you can take cells from an individual with a condition, you can reprogram them to induce polypone stem cells, make organoids, and then compare them to the control and see how they're different. So we did this in our first paper with cells from a patient with microcephaly. So many of you may have heard of microcephaly because of the Zika virus epidemic, but this was a genetic form of microcephaly. So this patient had a mutation. And we were able to generate organoids and compare to control. And what we found is that the organoids were overall smaller than the patient, than the healthy control. And we could actually then use the organoids to understand why. And what we found is that the neural stem cells, within these tissues that are gonna give rise to neurons were becoming depleted too soon. And they weren't being maintained so that they could make more neurons. And I mentioned Zika virus. And in fact, indeed, this has been one of the real sort of big success stories, I'd say, of brain organoids because they've been used by a number of labs to look at how Zika virus causes microcephaly and organoids have shown that the reason is because this virus infects the neural stem cells and similarly depletes those neural stem cells. And so now they're even being used to develop drug therapies to try to treat this condition. Madeline, this is fascinating because you're now able to model human neurological disorders using an original donor sample so you can model either the development of a disorder or even use healthy controls to compare these disorders up against. I'm wondering if there's any concern in the near future maybe about what we might call a developmental incidental finding because in the course of genetic sample research or even a brain scan research there's this whole problem that arises that the investigator may see something incidental to what they were looking for that may have health related consequences and may even be actionable by the patient themselves. And then the question is, do they need to return these findings to the clinician and to the patient? I can imagine that there could be cases here where it wouldn't be evident from a genetic analysis of the person but maybe even if it's the health control that you would have to see manifestations of something irregular during the developmental phases of your model. Do you think we're just still too far away from that becoming an issue? The issue of developmental incidental findings and what obligations people like you may have in the course of your research? Yeah, I don't know. I think that's a really interesting question. That's definitely a new one that I've not actually got before. And I think it's a really, I do think it's an important question to start thinking about. I think it is probably still early, but and usually, you know, like for the, for example, for these human neurological conditions that we've been modeling, these are, you know, we know the patient has a condition and so we are therefore using the organized to try to understand the mechanism, you know, leading to that condition.  And I'd say probably not so much for developmental conditions because those will be obvious in the patients. And so you'll already know that when you get the sounds. But maybe for something, maybe for, for example, for neurodegenerative conditions, you know, later on set conditions. Right. People are beginning to use organize to model Alzheimer's, for example, and have been able to see, you know, in, in so neurodegeneration actually is one area where brain organoids have a lot of potential because mouse models of neurodegeneration are there's issues with them. So you tend to have to, you have to introduce a lot of mutations before you start seeing neurodegeneration, much more than what you see in patients. So we know that in human beings, if they have a mutation in pre-Centralin-1, for example, they will generate familial Alzheimer's disease. But in a mouse, if you introduce the same mutation, you don't see, you don't see the neurodegenerative effects. You have to add on top of it other mutations and other mutations, and you finally start to see something. So clearly something different going on there. Now in 2D cultures of human neurons, people have also started looking at this and they start to see some features. But the classic pathology that defines these conditions, which is that the tangles and the plaques, you never see both of those together in any of the models so far. But organoids and neurons in 3D, for some reason the three-dimensional conformation, they have been able to see the pathology you see in patients. So now that we know we can see that in organoids, the question becomes, yeah, if I go and make organoids from a control individual who's maybe 30 years old, let's say young, and I see that, do I need to go and inform that person that they have an increased risk, an increased likelihood of developing neurodegenerative disease? Yeah, I don't know. I'd say take a page from genetics and follow their example there. Thank you so much, yeah. So we'll see what the future holds, but as these models get more and more accurate and informative clinically, I think there really is a possibility, as you suggested, for these control groups. Thank you. Thanks. Okay. Right, so let's move to COVID now. So one of the reasons why I told you all about the Corid Plexus organoids is also because it leads into this COVID work that we got into. So this, we started becoming interested in COVID and in particular in the virus causing COVID, the SARS-CoV-2, because of the increasing reports of neurological manifestations. So this is a poster that was put together by an Emory. And just, I think it's a nice kind of just visual representation of the variety of different kinds of neurological manifestations. See, and this was back in April already. And of course, since then, we know of even more neurological symptoms. And some of these symptoms are probably not necessarily directly a result of a neurological problem, but rather a side effect. So for example, stroke is probably more likely an issue of problems with vasculature. But since April, we've also learned about patients who are exhibiting, for example, psychosis, depression and anxiety, confusion, this so-called brain fog. And now of course, long COVID, these people who, we didn't even realize that they had these kinds of symptoms because they didn't end up in hospital, we're learning now there's a huge number of people that actually have long-term complications of COVID-19, including neurological complications. So we became interested in trying to understand where or why some of these neurological complications might be arising and whether it's because whether this virus might actually infect the brain. So we figured organoids would be a good model to look at. So the first thing we did was just to take our organoids and to look at the individual cells within the organoids by doing something called single cell RNA-seq. So what we're doing here is we're taking our organoids, we're breaking them apart into single cells and then we're sequencing each one of those cells to identify what those cells are based on the proteins that they're making. And so by doing that, we can define different cells and say what they are. And we can also say what are they making? And one of the things we wanna know whether they're making is this ACE2, which is the main receptor for SARS-CoV-2 and this gene called Tempris2, which is a co-entry factor. So these are necessary for entry of this virus into cells. And so if a cell expresses these factors, it's likely that it could be susceptible to this virus. Then what we found is that these factors seem to be present in certain cells of the organoids, but not all the cells. So we see it highest in all of the choride plexus, that's what the CHP stands for, in the choride plexus cells of our organoids, but not in the neural cells of our organoids. So that's just maybe it could infect choride plexus. And in fact, actually that matches very nicely within vivo data. So this is data taken from a database called the Allen Brain Atlas, where we're looking at ACE2 levels across different brain regions. These are just ordered by the regions expressing the highest levels of ACE2 and choride plexus here is expressing the highest levels. So this suggests that maybe the choride plexus, this CSF producing part of the brain might be susceptible to this virus. So to test that, we started out by using what's called a pseudovirus. So this is basically a safe virus. So not the actual SARS-CoV-2, but another safe virus, what's called a lentivirus. And then all we do is put on the SARS-CoV-2 spike protein. So you might have heard about the spike protein because it's the protein that all these new vaccines are targeting. So that's the protein of the virus that actually then binds to those entry factors, that ACE2 and that Tempest 2. And that's what actually then triggers viral entry. So when we put spike protein on a pseudovirus like this, we can look at what cell types this spike protein is able to infect. And indeed what we can see is infection of cells of the choride plexus of these choride plexus organoids. And the way we know that is because the virus that we put in there is also carrying with it a green fluorescent protein. So we can just see that those cells must have been infected with virus. Now interestingly, if we do the same thing on cortical tissues of our organoids, we don't see any infection at all. But we do see infection if we use, instead of the spike protein pseudovirus, we instead use a lentivirus that carries an entry protein that can that can bind to any cell type. So it's not selective at all. And that then does infect. So we know then that this is a positive control. So basically what we're seeing here is that we know the system works and our virus is working here. It's just that the spike protein is not recognizing cells of the cortex. Now that could be because maybe for whatever reason, the way that we've designed those experiments, maybe the pseudovirus is not really replicating what the live SARS-CoV-2 is doing. So to test that, we teamed up with a group of virologists here at the LNB who were starting to work with live SARS-CoV-2 and actually put live SARS-CoV-2 on some of our organoids. And for this experiment, we actually took organoids that had a mixed identity. So these are organoids that have a choroid plexus regions along with cortical regions. This is very nice because we have the tissues together and we can put the virus on and see where does the virus preferentially infect. And so here we're staining for the spike protein of the virus. And you can see that it clearly infects only the choroid plexus. So the choroid plexus here is the white cells because of this marker HDR2C, which specifically marks choroid plexus and does not infect the neural tissue here. HUCD is a neuronal marker. And then in blue is all the cell types, all the different cells. And even if we leave the virus on for a long time, we still don't see any specific infection of neurons. So it seems to only infect choroid plexus cells. And also we found that when we infect choroid plexus organs, we can see a productive infection, meaning that there's replication of the virus. So the virus actually goes up over time. So that suggests choroid plexus is not only susceptible, but also represents a productive cell type that's able to replicate more and more of this virus. We also did a variety of other experiments where we put the virus only on cortical tissues where they don't have any of this choroid plexus. We thought, well, maybe the choroid plexus is more susceptible and it's kind of acting like a sink, like it's sucking up all the virus and none of the virus can infect the neurons. But when we do that, we still don't see any specific infection of cortical tissues. The only times that we could see any infection of cortical tissues is if we increase the amount of virus at least 10 times and left it on for at least two days, then we could see one or two neurons being infected, but it was very minimal. I mean, it's hard to even show a picture of that. So we really don't think that neurons are very, or at least neurons and organoids are very susceptible to this virus. Now, what does the virus do to the choroid plexus? Well, I talked about how important the barrier is that the choroid plexus is protecting the brain and the CSF. And it turns out that this virus, when it infects the choroid plexus, we see a breakdown of the barrier. So this marker here, this Clodin-5, is marking basically outlining the connections between cells. And the cells have to make these very, very tight contacts between them. It's almost like glue so that they don't let anything pass. But when we have this virus, you can see these connections are broken down and it becomes leaky. In fact, we can actually see fluid leak out of the organoids. So we can actually measure the fluid inside the organoids. We can see it's dropping in these guys, and then it's actually diluting the protein that's outside of the organoids. So basically what we found is that choroid plexus cells here that are generating the cerebral spinal fluid of the brain are susceptible to this virus and that it leads to a breakdown of the barrier here. And that's important because not only because then that would allow virus in, but actually I think probably more concerning than that would be actually the entry of other things that are not supposed to be getting into the brain. Things like immune cells and inflammatory cytokines that would lead to a very broad sort of neuro inflammation. Like I say, the brain is normally supposed to be highly protected from the immune system and from toxic compounds in the blood. When you have a breakdown of this barrier, you start getting T cell activated T cells and macrophages going into the brain and start attacking neurons themselves. And I think that's probably more concerning than the virus entering necessarily. Cause as we've shown, we don't think the virus really has too much tropism for at least cortical neurons. And rather, I think that the idea that neuro inflammation might be a major factor here also fits with the kinds of phenotypes that we see in patients because this kind of brain fog and confusion and the actual inflammation that's actually also seen in the brain of some of these patients fits very well with that. I also should state that the blood vessels here of the chloride plexus.  But in the chloride plexus, blood vessels are really leaky and they're leaky on purpose because the chloride plexus has to take stuff from the blood and make CSF. But that also means that the virus if it gets into the blood would have very easy access to these chloride plexus cells. So probably not necessarily so common in the more mildly affected patients, but in patients where they have varemia, so where they actually have virus getting into the blood, then this may be more of a concern. Okay, so I will just switch gears now and go back to this kind of big question about what is so special about our brains and how we're trying to use brain organs to understand some of that. And this will kind of bring us full circle back to some of those questions about, what makes us human and consciousness and such. So we thought that organoids could give us a unique insight into this because it's impossible actually to study the brains really of our closest living relatives, the other apes. They're highly endangered species. They are protected. We don't do experiments on chimpanzees anymore.  There's no examples of that. So we don't even really know, for example, what a gorilla brain looks like in vivo, during development. So there's still a lot of questions about what's really human specific. So organoids give us a unique window into this because we can make organoids from any cell type, because we can reprogram them. So we've been getting cells from different apes, from zoos. So these are basically samples that are taken because the animal needs to undergo some sort of veterinary practice or has to have a blood test or something. And then they have leftover blood. They would have just thrown it away. They give it to us and we are able to reprogram those cells and generate pluripotent stem cells and make organoids. And so the animal isn't harmed at all and we're able to learn something here. And so we're making organoids from different species. I'm gonna focus on the ape for this part and then comparing them, looking at how they look different. So these are just some of the examples. We have a number of different cell lines from different individuals. But what we find is that chimpanzee and gorilla organoids, first of all, they look beautiful. We're able to make beautiful organoids just like we can with human cells. But interestingly, and you can see, sorry, you can see these nice lobes just like we see with the human, these nice cortical lobes. But interestingly, the overall size of these organoids tends to be a little bit smaller than the human. And it's not a huge difference and I wouldn't expect it to be a huge difference because after all, chimpanzees and gorillas actually do have quite large brains.  So what we decided to do was to look at earlier stages and try to find out when this size difference first appears. And it turns out that it actually appears really, really early when the neural stem cells, so they haven't even started making neurons. The neural stem cells here are just expanding. They're just multiplying and increasing their numbers. And what we find is that in the human organoids, they seem to be expanding in a sort of a different way. They look different, these tissues look different. They have bigger spaces on the inside, these fluid filled lumens, the spaces on the inside are bigger. And this is just quantified here. You can see these are bigger than in the other apes. And so it turns out that the reason that those lobes are different shape is because the cells are a different shape. It turns out that while these cells are expanding in humans, they have more sort of like a column shape, what's called columnar epithelial shape. And you can see here, they're on their contacts here on the inside look pretty similar in width to their outside. But in the ape, they're really thin on these inside regions. And you can see that even better when we do a stain for what's called zoe one. So this is a marker that goes around the outside of the cell just at this side here. And then we can look at them on face. So we can actually look at them all sitting next to each other. And we can see that in the ape, these connections between cells are smaller. So the cells are much more thinned out. And then what we see is that in both humans and apes, the cell shape changes over time, but in humans, the cell shape change is slower than in other apes. So without getting into too many of the details, basically what we've identified is a new sort of cell stage here during development, something we're calling transitioning neuroepithelial cells. So these are the stem cells that are gonna give rise to all the different cell types of the brain. And in non-human apes, we see a change in their cell shape before we see that change in cell shape in humans. And simply because of a change in cell shape and a delay in that change in cell shape, humans then have slightly increased numbers of those neural stem cells, those progenitors that are gonna give rise to all the neurons. And because they have an increased number of those founder stem cells, then once they switch to making neurons, you just have more to work with, so you make more neurons. And so that will lead to a general increase in all of the different neuron types. And in fact, that's exactly what you see in the adult brains when you compare them. The fact is that human brains are around three times larger and all neuron types are about three times increased compared with other apes. And so basically it's a matter of timing. By delaying this transition in humans, we see a slight increase in the number of progenitors that leads to a pretty dramatic effect than later on in terms of number of neurons. And this whole process is really delayed when you compare with something like rodents where the whole thing happens in a matter of hours. And in humans and apes, it takes over a week. So it's all about timing here. So with that, I'll just thank some of the people in the lab that did the work that I showed you. So I mentioned Laura, she did the work on the chloride plexus organoids and the SARS-CoV-2 work. Sylvia did the work on evolution. And Stefano worked on the air liquid interface work that I showed you. Thanks. That was wonderful. Thank you so much. So clear and you covered a lot of ground. Let me just, as for gathering our questions for the Q and A portion, let me just start off by asking the following question. It was fascinating work, beautiful work you did with the plexus and the infection routes with COVID. Do you think this could lead to any interventions or strategies and what would that look like? Yeah. So I don't, I think it's probably too early to say interventions or strategies, but I think what it does suggest is that we really need to do more investigations of patients themselves. In particular, I'd like to see some investigation of CSF samples. It's not common necessarily to take CSF samples, but I think that we should start looking into that, particularly given this increasing rate of neurological conditions, because we need to understand whether, is there really, well, first of all, is viral presence in the CSF a common finding or not? We still don't really know. There's a few papers that say, yes, we see it, but a lot of papers that say, no, we don't see it. And so I think we just need more information there. And secondly, even in the absence of virus, are you getting an abnormal inflammation there? Are you getting things like T cells and macrophages and inflammatory cytokines entering the brain when they shouldn't be? And that would then hopefully give us some insight into whether there is a breakdown there in vivo, in the actual patients and whether we should start considering things like immune modulation, so dampening down the immune response using things like steroids. It may be that some of the treatments that are already being used for other parts of the disorder, other lung inflammation that you're seeing will also help with the neuro inflammation, but we need to understand that because you don't wanna actually hurt the patient. So maybe you need to have inflammation. Maybe the virus is still present and needs to be killed off by your immune system. So I think we also, we just need to understand a little bit more from actual patients. Thank you. So I'm gonna turn it over to my graduate student, Christina, who's gonna be moderating the questions that have come in. So take it away, Christina. What's our first question? Well, thank you, first of all, what an amazing presentation and the applications of the research that you've pioneered are tremendous. So it's delightful to have you here. Along the lines of COVID and the work that, you know, the potential application there, there are two questions. One is whether or not you know whether Pfizer or Moderna or any of the other companies that have been doing work in the area of vaccine have relied on any of the organoid information or research that you have. And what ethical issues might you see going forward specifically with respect to COVID-19 research? Yeah. So I don't know of any, I don't know for certainly not from the immune, sorry, the vaccine development perspective. I don't think organoids are necessarily gonna give you much information when it comes to that, but more for probably for the treatment of patients who already have COVID or who get COVID despite having a vaccine maybe. There I think then organoids might give us some insight. I don't personally know about, I'm not interacting for example with any pharmaceutical companies. I hope that they are reading the papers that we are putting out there. I do hope that that at least is happening and that they may be able to use some of these organoid systems as well. We are making these methods widely available and accessible. There will even be a kit that people can buy that companies can buy for making brain organoids and for making choride plexus organoids in particular. So we're trying to make it as easy as possible for these companies and things to come in and be able to use these models definitely. From an ethics perspective, ethics surrounding COVID-19 and organoids, I don't know, I guess, I mean, I think that when it comes to patient modeling and that's basically what we're talking about sort of modeling a disease here, right? I think that we obviously have an ethical duty to provide the findings as quickly as possible. So we're trying to do that. And obviously using things like bioarchive and putting, so we put this out on bioarchive as soon as we could and then publishing it as quickly as possible and open access. And then when I think about modeling patient conditions, usually I think this comes more into play when you're actually taking cells from patients. So in our case, we're just, we're using an established human embryonic stem cell line that's from an individual that doesn't exist. So there is no sort of, but if you take patients, if you take cell lines from a living patient, I think that is also something we should be talking about more probably in particularly in the brain-organized field about telling patients or asking patients if they're comfortable with us making brain organoids from their cell lines. Because a lot of times we just do these patient consent forms that are really broad and don't really specify what it is we're going to be doing with them. And I guess they write them on purpose like that, but it's a little bit, I think it's better to tell the patients and have them decide whether they're comfortable with it. So that leads to an interest. And many people will agree with you on that one. And it leads to an interesting question, I think, about asking for donor permission to use biomaterials for brain-organized research. If the future prospect of such research is entirely unknown at this stage, so how can you create the parameters and set them forth so that informed consent is accurate and not encroached going forward? Yeah, and that's the problem with a lot of these kinds of things is, you know, a lot of times the patient consent that we have on IPS cell lines, for example, that we have in the lab, some of these IPS cell lines have been around for, you know, well, as long as IPS cells have been around. So, you know, over 10 years sometimes. And 10 years ago, brain-organized didn't exist. So when patient consent was obtained, this wasn't even on anybody's radar, why would you even include it? So I think there's also a question of whether should we try to go back to some of those patients and ask them if they're comfortable with this? In the future, I think we should start telling patients what we may do with these cell lines. And it might be that repositories that are establishing these database, these sets of cells could have in their patient consent, you know, are you comfortable with? And then you could have a number of different things. Human embryo-like tissues as well, for example. I think a lot of people might not necessarily want to have their cells used for that, but maybe they'd be fine with kidney organs. So you could kind of, you could have a checklist of am I fine with this or not? And people could check it off. And then in the data, in the repository, you know, I would go to the repository and I would see that, well, this cell line can't be used for brain-organized. So I won't get that one, I'll get a different one. We have some other questions about moral status. And specifically if we were to be able to implement brain-organoids into animal models and the vascular system would allow that to thrive. One, how would you be able to know if the cognition capacity and potentially consciousness is expanded? And so where would then that entity lie on the spectrum of moral status? I'm happy to take this on, but I'm curious about your thoughts about, you know, transfer of brain-organized into animal models. I don't think that's an area that your team works on, but there have been others who have done such a work. Yeah, so we're not doing anything with that. We're not doing any sort of transplantations. Yeah, the reason we're not doing it is not necessarily because I feel that it's ethically not okay. I actually think it's, for example, putting organoids in mice, I think from a, maybe I can get into why that is, but I don't think that that's actually really too much of a concern. But the reason we're not doing it is just because I'd like to have a model that can be fully in vitro because it's just easier to do things with. You know, we're not gonna be able to do drug screening on mice with brain-organoids implanted in them. So to be able to have organoids that just stay in vitro so we can do lots of different perturbations and really study them carefully that way, I think it's better for the kinds of questions we have. But I mean, in terms of why I don't think that's a problem, the reason I don't think it's a problem is for mice in particular. And for example, I might think it's more of a problem if you were to transplant brain-organoids at a very, very early stage of a pig embryo, for example, or a cow embryo or something like that. And that's just because of size. I kept driving home this point throughout my talk on purpose because I think size is really, really important here.  I actually calculated out so that you understand also what we're talking about when we're talking about size. So I calculated out how many of our organoids we'd have to grow in order to reach 100 billion neurons. It would be 25 to 55,000 organoids. So we'd have to somehow get 55,000 organoids all growing in one giant blob in order to get enough neurons to make human-sized neurons. Or I also calculated out if we were to grow them on dishes, on a tissue culture dish, you would need a tissue culture dish the size of the floor plan of my house. So you would need a hundred square meter tissue culture dish to fit that many neurons. So it's really a human brain. You can do it, I believe in you. No, but let me put a little bit of background on that question, right? So there have been teams at at least one team that's transplanted human brain organoids into rodent models. Couple of things to keep in mind here. One is that when you do an experiment like that, you have to justify why you're using that species, host species. And if somebody wants to jump right away to not human primate, or as Marilyn said, you know, pig or a much larger animal, the question for the research oversight people would be, what is your research question? And why do you have to use that particular animal model? I can't think of a very good reason right now, scientifically, why you would jump to that kind of host and not use a rodent. If you're talking about a much smaller animal, then you've run into the major limitations of physical space because what you not only have to do is squeeze the brain organ right into the skull of the small animal, but you also have to excise out part of the brain tissue to do the replacement. Now, there's only so much you could take out before you kill the animal or greatly harm the animal to the degree that the animal research committee would not allow that kind of research. So there are these other constraints that are kind of, I think, militating against the concern that people may have about these radical models, you know, using human brain organoids. One more point to raise on this point, and that is if we did get evidence that the human brain organoids somehow contributed to anything of the animal's experience or anything of its abilities, it's a rescued function. The clinical implications of that are astonishing. And I think we greatly outweigh any concerns people may have about, gee, is the mouse having a bad experience or is this oriented to question its reality, right? The clinical implications of having transplantable human brain organoid to recover function is enormous for stroke victims is enormous for human health. I said, we also have to kind of keep in mind that other narrative that would emerge from that kind of experiment. But that was a good question. Thank you. That's a really good point, too. I didn't actually consider that one, of course. If you could actually get an organoid to contribute and integrate and recover, let's say an animal with spinal cord injury, I mean, that would have huge implications. And as I try to also remind a lot of people who bring up the consciousness issue as well and such, you know, there are a lot of truly conscious human beings out there who are suffering from conditions that have no good treatments. And organoids are one of these new models that can finally, I hope, provide some new treatments. It's already showing promise in the Zika virus. Like I said, there's already small molecules being developed. And I think it's only a matter of time, probably in the next, definitely in the next decade, we're gonna see new drugs being developed with the help of organoids. So there are a lot of questions in the chat about consciousness and we'll move a little bit in a different direction. One question that's interesting is a hypothetical. Adult humans who have had significant parts of their brains removed can still be conscious with the plane analogy in mind, as well as the evidence of functional nerve tracks, is it possible some arrangement of an organoid could become conscious through a randomly fortunate connection, even if not all brain regions are present or if they're arranged poorly? So that's a very interesting idea. I'd like to just quickly stress that or highlight the facts that in those patients that this person is referring to, those are of course human beings who before having lost those parts of their brain had the part and it was organized and they were interacting with their environment. And that is so key here that actually, first of all, developing the brain needs to have that kind of interaction with the environment, both input and output. So it's got to have receiving some sort of sensory input and having some sort of interaction with that input. So you have to have a closed circuit to make a functioning neural circuit. You've got to be able to not only see the thing, but touch it, move it around. We know from animal studies that if you prevent an animal from being able to interact with something, they functionally can't see it, even though their eyes work just fine. So there's that. So you've got to have it developing in a body, interacting with the world. Then, yes, okay, then if you start taking away that kind of, you know, those inputs and outputs, for example, or you start taking away parts of the brain, there might be some remnant of consciousness, but to develop in the first place, it has to have a body, it has to interact with the world. That's a terrific answer. I think that's also a really good reason why you have to have ethics and science together, because a lot of philosophers wouldn't think about that angle in those constraints. Very good, thank you. Sure. And let's talk a little bit about access and justice. We have a question here. As far as exponential applications of organized research applications, how affordable accessible is organized research to other areas around the world, especially low and middle income countries? And to that, I might add a question about different public or scientific attitudes towards this sort of research in the UK or the US or elsewhere. So in terms of cost, I mean, we have, so we, there's a kit we can buy, you know, that actually we use in my lab now that's developed based on our method. And it's very convenient, but it's also more expensive. That's also why it's, you know, that tends to be right. When a company comes along and makes something really nice and convenient, they're gonna charge you more for it. But you don't have to use that kit. You can also make your own homemade media using all the basil. We publish the recipe for it. You can buy all the components and make it yourself. And we calculate it out. When you do it, your home, when you make it homemade, I think we calculated that to grow an organoid from start to sort of the stage when you might wanna do some different analyses was about a dollar. So it's not actually that expensive to make an organoid. We don't usually make just one. So we usually make, you know, like 25 or 50 or something at a time so that we can compare across lots of different organoids and stuff. But still 25 or 50 at a time, $25, $50, that's really not too bad, I think. So I do think it's affordable. What becomes more expensive is actually the stem cells maintaining the stem cells, but that's just stem cells. And we can't do much about that. The organoids have to be made from stem cells. I don't know, Insu, do you have other questions that may? There was another question you asked at the end. Maybe Insu can also comment on that. Right, about like attitudes about organoid research, UK versus US, for example, take two, two locales. Do you see differences? Yes, I definitely see differences. So I mean, I'm American. I did my undergrad and my graduate work in the US in California and I did my postdoc in Austria. And then of course, now I have my lab here. So I've seen, yeah, I've been in three different countries and experienced different attitudes. And I can tell you that I've seen quite different attitudes across the three. So for example, in Austria, they're highly conservative when it comes to stem cell research. I think organoids are sort of viewed in the same kind of way as stem cell research generally. They're kind of lumped in together with that, even though they come with their own sort of ethical things. But it sort of starts from the stem cells. And in Austria, they're very conservative when it comes to stem cell research. So we had to be sort of careful just with how we communicated our work and kind of emphasizing the fact that a lot of it, we mainly use IPS cells, so induced pluripotent stem cells and not so much embryonic stem cells. And instead in the UK, it's sort of the opposite. And then sort of the United States is kind of in the middle, I'd say. So in the UK, actually the attitudes towards stem cell research and also sort of by extension organoid research is more progressive, I guess. So they're really not too concerned. Here, for example, you can use public funding to generate new embryonic stem cell lines from blastocysts. You can work with human embryos and do experiments on human embryos up until day 14, obviously the day 14. And I think the UK was one of the first countries to allow genetic engineering of human embryos for research purposes. Whereas in the United States, that work is not yet allowed. And I think it's, or at least not with public funding. So I think that they are more, I guess, progressive on that front, but where they are more conservative in the UK is actually animal research. So they're much more conservative when it comes to limiting animal research. It took me a very long time to get a license to do any work with animals. And actually in the end, we just don't do any work with animals in the end. And I have to say, I think I like that. I'm kind of, I myself am more concerned about the consciousness of a mouse that I might work with than I am about the brain organoids that I have in the dish. Let me ask a follow-up question. I thought it was really fascinating what you're seeing in the developmental differences between the great ape, organoids, and human. I'm wondering if you were to do the similar kind of analysis of other non-human primates that are actually used in biomedical research, neurological research. And if you were to uncover some key differences there, wouldn't that be a way for those who are actually pretty hesitant about non-human primate research invasive for neurological studies to say, aren't these really as informative models as we thought they would be for human conditions? I can imagine some sort of, you know, route to being a little bit more skeptical about the scientific usefulness and justification for using non-human primates for neurological studies because the assumption seems to be, well, they're the closest thing we have to man that we can actually use, you know, practically and legally. But it might be that actually, maybe that's not that warranted. There are some key differences there. And maybe brain organoids might be kind of a way to either be an alternative to using non-human primates themselves for this kind of work. Yeah, yeah. So we're only now really getting into other, so like macaque, for example, which is used for, and also the two different macaque species that people use for non-human primate research. And we have cell lines for those and we've started making organoids. And what, so, you know, we also make mass organoids too. And what you see is that the non-human primate, so that the monkey organoids basically are sort of somewhere in between in terms of the many sort of readouts that we use between rodents and humans. So what that means is that, now it depends on the feature. So we're generally looking at these kinds of early events that I talked about. And we see that in mice, so for example, in mouse organoids, this transition that I talked about, I mean, we can't even catch it. It's so fast. It happens in a matter of hours. And in the non-human apes and humans, so basically in the apes, this transition takes about a week. And in the macaque, for example, it's sort of somewhere in between. It looks like it takes a couple of days. So meaning that they are closer to humans. And so this is just one feature we've looked at. But if this is true sort of across the board, then organoids may tell us that, well, they may confirm that they're closer to humans. So meaning there are probably some things that we will still wanna do in macaques. But if there are things that you can do in an organoid, then absolutely, I mean, we should do it in an organoid. But unfortunately, I think we just don't really know everything that organoids can do yet. So, and that's also part of what we're doing in my lab, just trying to characterize everything that they can model. I have one last question and I don't come from a scientific background, but as you see these lobes that are growing in you and how much control are you being able to get in directing what type of cell, what type of lobe, what type of area? Yeah, that's a very good question. That's a very, very good question. I mean, that's one that my scientific colleagues always ask me, so very good one. The original, when we originally developed this protocol, we really had no control. I mean, every lobe developed to be something different. It was very messy, really. But since then, I mean, that was, well, seven years ago now, we've steadily been improving the method that we use. And others too, I mean, I want to emphasize, this is a big field, I'm one player in this very big field now that's been reaching all across the globe really. And so others too have been modifying the protocol and using different factors to help control it a little bit better. And it's a little bit of a sort of give and take. So you kind of, if you push it too far, so if you try to control it too much, you start overriding the intrinsic developmental programs and then you start losing some of the actual development and then you're not able to watch processes that would happen normally in vivo because you've completely overrided them. But I think we're reaching a balance now where we're able to kind of gently help guide it. And I can tell you that all of those lobes that I showed, they are all cortex. We know because we can look at later stages and we can use different markers and see that we know for sure we're getting cortex. But, and we're able to still maintain the intrinsic sort of developmental programs there in that way. Great, cognizant of the time, I just wanted to point out that there are many people who would love to hear your ideas of what consciousness might look like or how it might need to be redefined. So if you do decide to broach that topic and I'm not gonna pin you to it now, you started saying that we weren't gonna go down that road but many people would love to hear your thoughts on that down the road. Yeah, why don't you just take us down the home stretch on this? Yeah, sure, I mean, I'm happy to.  And I think that's a good way to start from. So consciousness we may not be able to define but I can at least, I think there's, we have enough information from neuroscience to tell us, regardless of how we define it, what are some of the prerequisites to get that? And one is size, I kept going on and on about size. I think it's about size. Now you could have, let's say a human brain organoid that has the same number of neurons as a fish, which would still be a huge number of neurons and way more than what we currently have. But then, you know, maximally it would, in what I would say is probably have the consciousness of a fish, which is probably not as advanced as ours. But even then, even if you've got this large number of neurons, it's also got to be organized. So we don't have a hundred billion neurons just like randomly thrown in there and connected randomly. It has to be organized in a particular way. You've got to have the two cerebral hemispheres, the thalamus, I talked about that, right? All mammalian brains have that structure. So if we don't have that structure, if we have seven cerebral cortices and no thalamus and I don't know a random cerebellar tissue growing on there, that's not gonna have the right architecture and organization. And finally, I think you've got to have, you've got to have it develop with the interaction with the environment. You've got to have sensory input and output. And it's got to be in a way that's rich, not just, you know, I poke it and it randomly twitches because, you know, even sea slugs can do that. You poke a sea slug and it'll like pull back. That's not consciousness. And so it's got to be a very rich interaction with its environment. And really, I mean, you get that when you're in a body. So the fact that it's completely bodyless is very small in terms of the number of neurons and is not organized tells me that we are very far away from human consciousness, whatever that is. But I think we're very far away from it. If you wanted to get that, getting all of those things in combination, if you could do that, then yeah, I would say, yeah, you're gonna have a conscious brain organized. But do you think it would be possible to not go that far but try to model pain, for example? So connect nerves to brain organoid and try to see if there's any kind of way of setting pain pathways. Would that be possible? I think it might be possible. I mean, you can certainly, for example, we hooked it up to mouse spinal cord. And if you went the other way and sort of hooked it up to mouse skin, for example, and had the peripheral nervous system there with the pain sensors, because of course there's no pain sensors in the brain.  But if you had it hooked up to the peripheral nervous system with pain sensors, yeah, I think you probably could. Now, what do you mean by pain? That's another point because what we mean by pain, I think it's not just the actual, what's called in neuroscience, a noxious stimulus, which is just a stimulus that's bad, but it's the emotional aspect of it that this much more complex aspect of pain. And I don't think that would be present in organoids. Well, that's terrific. I think I want to wrap it up there. I appreciated your response to the consciousness issue because that looms very large over this field for non-scientists. So thank you so much for your thoughts on that. So I'm going to conclude now our session. I want to alert everybody to the fact that on December 11th, we have our last consortium for the semester, which is on Right to Try Laws and their impact on research and patient access. And for that, we have George Daley, who's the Dean of the Harvard Medical School alongside Allison Bateman-House from NYU. So that's December 11th. And then we have four very exciting talks next semester as well, so be on the lookout for those. I would like to thank our guest, Dr. Madeline Lancaster, for joining us all the way in the UK. I want to also thank my graduate student Christina Larson for filling the questions for Ashley Trotman and Angela Alberti for all the logistics for this program. And on behalf of the Center for Bioethics at Harvard Medical School, I want to thank you, the audience for joining us. I hope you have a great weekend. Thank you, goodbye. Thank you.


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