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Dr. Pașca on Huberman Lab: How Assembloids Cure Autism

Assembloids are lab-grown brain circuits from stem cells in a dish; Pașca uses them to map what goes wrong in profound autism, epilepsy, and schizophrenia.

Andrew HubermanhostDr. Sergiu Pașcaguest
Aug 18, 20252h 23mWatch on YouTube ↗

EVERY SPOKEN WORD

  1. 0:00 – 2:08

    Sergiu Pașca

    1. AH

      Welcome to the Huberman Lab podcast, where we discuss science and science-based tools for everyday life. I'm Andrew Huberman, and I'm a professor of neurobiology and ophthalmology at Stanford School of Medicine. My guest today is Dr. Sergiu-Pasca. Dr. Sergiu-Pasca is a professor of psychiatry and behavioral sciences, and the director of the Stanford Brain Organogenesis Program. During today's episode, we discuss autism, schizophrenia, and human brain development generally, both brain development during pregnancy, as well as during childhood, and leading all the way up to our third decade of life. During today's discussion, you will get the most up-to-date information about autism and its treatments. You'll learn why the prevalence of autism is rising, the role that genes play in autism, and the novel treatments that Dr. Pasca is developing to treat what is called profound autism, which are the most severe cases of autism. Dr. Pasca is one of a small handful of researchers that pioneered the discovery and development of what are called organoids and assembloids, which are essentially human brain circuits derived from stem cells that form in a dish so that one can study them directly. And while that might sound artificial, today he explains why those organoids and assembloids are immensely powerful for understanding exactly what is wrong in psychiatric illnesses like profound autism, schizophrenia, and other psychiatric challenges, and for developing cures. So today, you're going to learn a lot about human brain development and about stem cells, which is going to be important for anyone interested in how the brain wires up, how to treat various diseases of the brain, but also for anyone who is considering stem cell therapies. As you'll soon learn, Sergiu is an extraordinary scientist, but also an extraordinary teacher. By the end of today's episode, you'll have the latest information on stem cells, organoids, autism, and what is being done to cure autism and other psychiatric conditions. Before we begin, I'd like to emphasize that this podcast is separate from my teaching and research roles at Stanford. It is, however, part of my desire and effort to bring zero-cost-to-consumer information about science and science-related tools to the general public. In keeping with that theme, today's episode does include sponsors. And now for my discussion with Dr. Sergiu-Pasca.

  2. 2:08 – 7:16

    Autism Spectrum Disorder, Incidence, Genetics

    1. AH

      Dr. Sergiu-Pasca, welcome.

    2. SP

      Thank you. It's great to be here.

    3. AH

      We're old friends.

    4. SP

      Mm-hmm.

    5. AH

      Shared a laboratory space years ago. We'll get back to that a little later. In the meantime, these days, there's a ton of interest and I think misunderstanding about autism. As soon as the topic of autism comes up, immediately some people will say, "Why are we trying to cure this thing? I know autistic, uh, children and adults that are delightful people that lead functional lives."

    6. SP

      Yeah.

    7. AH

      "They might be a little bit different, or a lot different than other people, but why are we trying to, quote-unquote, 'cure' autism?" And then other people will say, "Well, there are people with autism who need constant care, who will never live independently." Tell us about autism, what this spectrum really is, and then we'll talk about what your laboratory is doing to try and literally find cures for the most debilitating forms of autism.

    8. SP

      Well, autism is a complex condition. It's a spectrum, as you said. Uh, in a way, you could say autism and neurodevelopmental disorders. It's behaviorally defined. There's no biomarker. So, in a way, it's a condition that is defined exclusively by observing behavior.

    9. AH

      Mm-hmm.

    10. SP

      Which is actually the case for most psychiatric disorders. Um, but it's essentially diagnosed by the presence and absence of certain behaviors in a certain period of time or up to a certain age. And, of course, what triggered, I think, a lot of discussions in recent years is because the number, uh, or the prevalence of autism has increased, right? So now it's close to almost 3% of the general population, which, of course, it's a big number.

    11. AH

      3%?

    12. SP

      Almost 3%, yes.

    13. AH

      Wow.

    14. SP

      So it has increased even since I was in medical school. When I was in medical school, actually, it was considered a rare disease.

    15. AH

      Mm-hmm.

    16. SP

      The reason why I actually studied autism, because it was a very rare disease and we had very few resources, so we thought studying a rarer disease would be easier.

    17. AH

      Mm-hmm.

    18. SP

      But now we also know so much more about this condition. So, we do know, for instance, that there is a strong genetic component to it, uh, which for a while, obviously, we- we- we didn't. In fact, uh, in early days, the psychoanalytic perspective dominated, especially in the '50s and '60s. So, it was thought that it was resulting from having very cold parents, in particular a cold mother.

    19. AH

      Emotionally cold?

    20. SP

      Yeah, emotionally cold.

    21. AH

      Mm-hmm.

    22. SP

      It was the so-called refrigerated, refrigerator mother hypothesis, uh, of autism. And then in the '70s, some of the first biological studies were done, primarily in twins, that showed something quite remarkable, that if you have twins that are identical, genetically identical, and one has autism, then the probability that the other one has autism is very, very high.

    23. AH

      Even with different mothers?

    24. SP

      Sure, yes.

    25. AH

      Mm-hmm, mm-hmm.

    26. SP

      But generally, we think that there is a strong heritable component to autism. So, that was, like, in the late '70s. And really just in the last 10, 15 years, we've learned, actually, that there are genes associated with, uh, autism, and with, certainly with very specific forms of autism. And so, that's what we would call generally profound autism today, the conditions that are severe, uh, that are causing an impairment. Uh, they are very often associated with other conditions, such as intellectual disability, so low IQ, epilepsy. So, because it is a spectrum, of course it creates a lot of confusion. Uh, and certainly there's no doubt that, uh, there are individuals that have autistic traits that are fully functional in the general population. But the reality is also that there are, uh, kids that have autism who are very impaired and will require, actually, lifelong care of sorts. You know, another way of, like, thinking is, about autism is that autism is not one disease. And I think, you know, no psychiatrist or even biologist who's studying autism would ever consider that this is one single disease.The way I look at it, um, sometimes is, like think about the fever of the 19th century in medicine, right? So, you see this very often in movies, right? They will say, "Oh, he has a f- fever, high fever. He's going to die from high fever." Well, that fever could have been a viral infection, a bacterial infection, could have been cancer, metastatic cancer, right? It could have been an autoimmune disease. The treatments are very different, but in that time, that's all we knew. It was, we were observing that behavior, in which case raising of the temperature, but we didn't know the biology. Today, we will use very different treatments for those conditions, and some of them for- of course, we don't even treat, right? We just observe. So, I think i- uh, in autism research, as it is the case for many psychiatric conditions, they are defined behaviorally, but there is a disconnect with the biology. Very often, we don't have good biological mar- we don't have biological markers by definition, and so that disconnect, I think, creates a lot of confusion.

  3. 7:16 – 9:35

    Is Autism More Common in Males?

    1. SP

    2. AH

      I have a couple of questions. First of all, is the prevalence of autism higher in males? I've been told yes. Um, w- if it's 3% overall, is it, um, eh, what i- what's the distribution for males versus females?

    3. SP

      Yeah. The ratio varies, also based on severity-

    4. AH

      Mm-hmm.

    5. SP

      ... but generally, it's been one to four, so more, more males than, uh, females.

    6. AH

      And, uh, we just recently had our colleague Neerav Shah on the podcast, who basically said the difference between a biological male and female comes down to this SRY gene, not even-

    7. SP

      Yeah.

    8. AH

      ... necessarily on the Y chromosome.

    9. SP

      Mm-hmm.

    10. AH

      If, if a baby has the SRY gene, you're going to get a fully functional male.

    11. SP

      Yeah.

    12. AH

      Um, if not, you're, you're essentially dealing with a female. So presumably, something about the SRY gene-

    13. SP

      Hmm.

    14. AH

      ... is conferring a vulnerability to autism.

    15. SP

      Yeah.

    16. AH

      I think it's fascinating.

    17. SP

      Well, there are a lot of discussions, of course, like what causes this difference? And you know, some discussions are just in terms of diagnosis, that perhaps some of the girls are not getting diagnosed properly-

    18. AH

      Hmm.

    19. SP

      ... that they're ... We, we do know that some of them are very good at sort of what we call, like masking the symptoms, or sort of like, you know, learning the skills, uh, social skills, and sort of like covering for that diagnosis. But what we do know for sure is that there are differences in how the male and the female brain, especially around birth, can actually take up injury.

    20. AH

      Hmm.

    21. SP

      So, think for instance about premature birth. You know, one of the best predictors for a premature baby in terms of outcomes, it's actually to be a female. Just in general, females, preemies will do much better for whatever reasons, you know, uh, you know, the way the nervous system is built, their resilience. We know that the maturation stage is also different-

    22. AH

      Hmm.

    23. SP

      ... right? Uh, for the male and the female. You know, think about like, acquisition of certain milestones that happen much faster in girls. They generally tend to speak a few months earlier, to walk a few months earlier. So, just the nervous system, uh, is maturing a different, uh, uh, at a different pace and can take injury differently.

    24. AH

      Hmm.

    25. SP

      So, it could be that, that, that, uh, is certainly the cause, but at the same time, and as we were talking, since autism is not one single disease, it is very hard to point out to one specific factor that is behind it.

  4. 9:35 – 11:56

    Sponsors: David & Helix Sleep

    1. SP

    2. AH

      I'd like to take a quick break and acknowledge one of our sponsors, David. David makes a protein bar unlike any other. It has 28 grams of protein, only 150 calories, and zero grams of sugar. That's right, 28 grams of protein, and 75% of its calories come from protein. This is 50% higher than the next closest protein bar. David protein bars also taste amazing. Even the texture is amazing. My favorite bar is the chocolate chip cookie dough, but then again, I also like the new chocolate peanut butter flavor and the chocolate brownie flavor. Basically, I like all the flavors a lot. They're all incredibly delicious. In fact, the toughest challenge is knowing which ones to eat on which days and how many times per day. I limit myself to two per day, but I absolutely love them. With David, I'm able to get 28 grams of protein in the calories of a snack, which makes it easy to hit my protein goals of one gram of protein per pound of body weight per day, and it allows me to do so without ingesting too many calories. I'll eat a David protein bar most afternoons as a snack, and I always keep one with me when I'm out of the house or traveling. They're incredibly delicious, and given that they have 28 grams of protein, they're really satisfying for having just 150 calories. If you'd like to try David, you can go to davidprotein.com/huberman. Again, that's davidprotein.com/huberman. Today's episode is also brought to us by Helix Sleep. Helix Sleep makes mattresses and pillows that are customized to your unique sleep needs. Now, I've spoken many times before on this and other podcasts about the fact that getting a great night's sleep is the foundation of mental health, physical health, and performance. Now, the mattress you sleep on makes a huge difference in the quality of sleep that you get each night. How soft it is or how firm it is all play into your comfort and need to be tailored to your unique sleep needs. If you go to the Helix website, you can take a brief two-minute quiz, and it will ask you questions such as, "Do you sleep on your back, your side, or your stomach? Do you tend to run hot or cold during the night?" Things of that sort. Maybe you know the answers to those questions, maybe you don't. Either way, Helix will match you to the ideal mattress for you. For me, that turned out to be the Dusk mattress. I started sleeping on a Dusk mattress about three and a half years ago, and it's been far and away the best sleep that I've ever had. If you'd like to try Helix Sleep, you can go to helixsleep.com/huberman, take that two-minute sleep quiz, and Helix will match you to a mattress that's customized to you. Right now, Helix is giving up to 27% off all mattress orders. Again, that's helixsleep.com/huberman to get up to 27%

  5. 11:56 – 18:48

    Eye Contact in Babies, Fever; Proposed Causes of Autism; Genes

    1. AH

      off. You mentioned that autism is diagnosed by behavioral measures or the lack of, uh, behavioral symptomatology, what we call positive and negative symptoms-

    2. SP

      Mm-hmm.

    3. AH

      ... which can be confusing language, because people think positive means good. No, positive is the presence, negative is the absence.

    4. SP

      Yeah.

    5. AH

      Um, I haven't looked at this literature in a while, but, uh, the last time I did, it seemed that babies or young children failing to, uh...... focus their own gaze on the eyes of other people is one of the major diagnostic criteria. Uh, it seems they look at the face, uh, they'll, they'll, um, more holistically or they'll zoom in just on the nose, but they're not really, um, making as much eye contact.

    6. SP

      Yeah.

    7. AH

      Is that still a, uh, diagnostic criteria?

    8. SP

      It's not part of the diagnostic criteria. Um-

    9. AH

      Hmm, interesting.

    10. SP

      It's, uh, but it is one of the features that has been observed.

    11. AH

      Mm-hmm.

    12. SP

      Uh, of course, it also has to do with just in general, like joint attention is one of the earlier. So, you know, uh, if you just tell a child like, "Oh, look here," right? So, if they kind of like have that attention, if they engage in that attention, uh, it's one of the features that is associated with autism. It's not certainly diagnostic. It's not, uh, pathognomonic, so to speak. So, it's not specific to the disease in any way.

    13. AH

      Mm-hmm.

    14. SP

      Uh, but there's certainly many deficits and some of them can actually be compensated later.

    15. AH

      Interesting. There were some other things I've heard over the years. For instance, that when children with autism have a fever that their symptoms improve.

    16. SP

      Yeah.

    17. AH

      Is that still the case?

    18. SP

      Yeah. So, those are mostly anecdotal reports.

    19. AH

      Mm-hmm.

    20. SP

      Um, of patients who would have a very high fever and then, for instance, they were non-verbal. So, many patients with autism, uh, or individuals with autism will have, you know, will be non-verbal. They have very few words or if they, you know, they're, they're not able to communicate. And so, there are a few reports of parents saying that when they spike a very high fever, they'll start talking in sentences, like very briefly or, like engage. And in fact, I mean, that is known. Uh, you know, kids in general, when they have a high fever, they tend to be more talkative. It activates somehow the nervous system. There've been a lot of hypotheses about this. Um, some of them, uh, having to do with how the noradrenergic system is activating during fever. Others saying that there are some of the cytokines, the immune molecules that are present during fever that are somehow getting into the brain, activating the nervous system. And others as simple as, oh, ion channels, right? The ion channels will open, uh, more when the temperature rises, so something about the circuits functioning differently during that. But it's, it's mostly anecdotic, uh, at this point. It's, and it's certainly, again, probably not present in all individuals with autism. Also, because autism is, again, not one single disease. So, we would not expect it to be present in all.

    21. AH

      A few years ago, there was a lot of excitement about the idea that autism might somehow be related, perhaps even caused by deficits in the microbiome. Um, there were some mouse experiments of doing-

    22. SP

      Yeah.

    23. AH

      ... uh, fecal transplants from what we call wild type or healthy mice into mice that were, uh, re- had some symptoms that resemble autism, and there were improvements observed, um, to the point where I think there were some human clinical trials using fecal transplants.

    24. SP

      Sure.

    25. AH

      Um, whatever became of that?

    26. SP

      Well, I think, again, almost everything has been associated or thought to be causal. But generally, demonstrating this is very, very difficult. So, you know, we cannot deny that perhaps improving the microbo- biome will improve the, you know, the quality of life of some of these individuals. But whether it's really causal, there's no, um, clear evidence for it. Think about it. Just to give you another example, think about sleep. Many patients, uh, will report, especially the ones that are profoundly impaired, will have severe sleep dis- disturbances. I mean, 70%, 80% of them. You know, they can have nights where they sleep very little, right? And do that for, like, a week. So, just imagine even just improving the quality of sleep for those patients can do miracle. I mean, all, all of us, right? If we don't sleep for three, four days, our social skills, you know, we become socially impaired. So, I think, of course, correcting, uh, a lot of these issues. So, for instance, many patients are picky eaters. You know, they don't like certain textures. So, they will, they will never eat, for instance, veggies, all right? So, that creates, in the early days, for instance, we thought that, uh, you know, there are dietary disturbances that really at the core. Of course, it remains to be seen whether just simply correcting those is gonna be just improving or certainly reversing, um, some, some of these forms. But again, most of the evidence points out towards a very strong genetic component, uh, behind it. And in fact, we now have hundreds of genes that we know when, uh, when they are mutated, they are strongly associated with specific forms of autism.

    27. AH

      I'm curious, uh, what sorts of, um, proteins those genes are upstream of. And I ask because, uh, David Ginty at Harvard, um, he did these really beautiful experiments where he induced mutations just in the periphery, so outside the brain-

    28. SP

      Yeah. Yeah.

    29. AH

      ... of these mouse models for autism and saw a lot of the same symptomatology-

    30. SP

      Yeah.

  6. 18:48 – 21:37

    Genetic or Idiopathic Autism Diagnoses, Timothy Syndrome

    1. AH

      um, with a child that's diagnosed with profound autism, what is the treatment? Uh, l- let's set aside the, uh, the potential for epilepsy, which hopefully they would treat as well, or other things that might be secondary. Um, but what is the typical treatment? Are they doing... And let's assume infinite resources, which of course nobody has.

    2. SP

      Yeah.

    3. AH

      Most people don't have. But if one had infinite resources, what would be done? Would it be behavioral training? Would it be something to control the activation state of the brain? I mean, as far as I know, there's no single treatment for autism.

    4. SP

      No. There's no single treatment for autism. Again, in the context of this not being one single disease. What we can say today is that if, um, you know, a family walks into the clinic with a diagnosis of autism or perhaps, like, they receive it into the clinic, there's still like a 20% probability that they will leave the clinic with a genetic diagnosis, meaning that it will be pointed out to them that this gene is mutated in your child. And it may be sometimes a mutation that was present in one of the parents and got transmitted, or maybe it was present in both and somehow, you know, the child got two copies that were, uh, modified now. Or many of the genes are actually mutated de novo, meaning that the mutation was not present in either parent, but something went wrong during development, perhaps early in the sperm cell, in the egg cell, or perhaps in early stages of development, and a new mutation was acquired. But that is also the g- we- we acquire a lot of mutations, all of us. They ha- we have a lot of new mutations, right? About like 80 new mutations. 30 of them are protein truncating. So certainly, the challenge very often is to, even when you see a- a gene that is mutated, to know whether that gene is truly causing the disease. So very often, the way we know is that we find many patients that have a similar presentation clinically. Let's say maybe they'll have syndactyly. So they're webbing of the finger, and they have autism, and let's say epilepsy. And they all have a mutation in one single channel, let's say in a calcium channel. So that would be Timothy syndrome, a genetic form of autism, where the mutation is very clear. Actually, there is one single letter in the genome that is changed and causes a relatively similar presentation in all of these patients. So about 20% of the patients will get a genetic diagnosis. Now sadly, that doesn't do that much today because we don't really have specific therapies for those forms. I think the hope is that perhaps we will have individual treatments, whether they're going to be genetic or otherwise. So being part of that community is generally useful. And then the rest of the patients will essentially fit into this larger category of idiopathic, meaning that we don't really know the precise l- uh, cause.

  7. 21:37 – 26:46

    Rise in Autism Diagnoses

    1. SP

    2. AH

      I want to talk about Timothy syndrome, and I also want to talk about genetic approaches for fixing genes.

    3. SP

      Yeah.

    4. AH

      So-called gene therapy. Uh, before we do that, would you be willing to just speculate on why you think there's this fairly dramatic increase in the incidents of- of autism? Um, people will always say, "Well, maybe it's better detection, better diagnosis." So I'd like your thoughts on that. And if there are increases that can't be explained with that, I- I'm- I just would like your thoughts. I- I realize-

    5. SP

      Sure.

    6. AH

      ... we're not talking, um, formal biostatistics here.

    7. SP

      No.

    8. AH

      I just, in your experience, you're an MD, you think about autism a lot, uh, you're working on potential cures for autism and other neurologic conditions, how do you think about this increased prevalence issue?

    9. SP

      Yeah. Well, the- certainly the increase is still puzzling, right? So I think on one hand, there's no doubt that the changes in diagnostic criteria, which have happened over time, I mean, we had to just refine what autism really is. That changed, uh, you know, to some extent the prevalence. We've also seen, you know, a diagnostic migration, so to speak. So some children, for instance, you know, 30 years ago would have been diagnosed with intellectual disability, and today they fit the criteria for autism. And about a third of individuals with autism also have intellectual disability, so there is also great overlap between the conditions. So there's been a move sometimes between the diagnosis over time. Of course, there are all kind of discussions about, you know, availability of services and to what extent that is also contributing, uh, right? But, uh, you know, we don't really un- you know, we don't truly understand all the reasons behind, like this, uh, this increase. There's- there's no doubt. We can't explain... We know that it's highly heritable based on genetic studies.

    10. AH

      Mm-hmm.

    11. SP

      So we know the heritability is very high, one of the highest for psychiatric disorders that we know of. Uh, but of course we can... We don't have the genes for every single form, so it is likely that some of them are very rare, right? So essentially just think of it as like, you know, they're individually rare form, but collectively common. So it will take a while until we sort of like map all of them and then, of course, there are environmental factors that we do know historically can contribute to this. So there are various exposures to environmental factors like in early days. Uh, thalidomide, for instance, was one of them that we know increases, uh, the risk, uh- uh- uh, for autism. So of course those are contributing, but...

    12. AH

      Thalidomide was a drug given to pregnant mothers to try and prevent miscarriage, right?

    13. SP

      Exactly.

    14. AH

      It's no longer prescribed.

    15. SP

      It's no longer prescribed.

    16. AH

      Because it caused major, uh, birth defects.

    17. SP

      Defects. Exactly, yeah.

    18. AH

      Okay.

    19. SP

      So there- there certainly, you know, i- i- it's quite complex because first of all, the definition of the condition is- is quite difficult, right? And I- I think that is in general like the challenge with psychiatric disorders, right? Um-And, and perhaps one of the reasons we've made such slow progress in understanding these conditions, because of course, the power of modern medicine is in molecular biology. You know, we kind of deploy this remarkable force of, and understanding. And in order to do that, you need two things. You need, first of all, to, um, have a very clear definition of what that disease is, generally, biologically, right? Think about, like, myocardial infarction, you know, very clearly defined in terms of, like, what it actually means. You need to have biomarkers, like the patient walks in, you take blood, you can immediately tell. Yes, in 20 minutes you can tell that they have a myocardial infarction based on a biomarker. And then the other one, which is certainly very important, which and, to a large extent, is sort of like, (laughs) you know, is the source of all the work that we've done, is the unbearable inaccessibility of the human brain, so to speak. And to a large extent, the human brain is inaccessible for most of its development. And so if you look, actually, across branches of medicine, you can see that there's a very strong correlation between how accessible an organ is and how many cures or therapies we actually have. Think even just in cancer, right? Think about in cancers, uh, you know, which used to be, of course, uh, an incurable disease, right? Uh, a century ago. Think about, like, uh, leukemias in children. They were, like, 90% lethal in the '50s and the '60s. Today, they are maybe 10% lethal, and that is because blood from these patients, right, it's very easy to collect. We've been bringing it to the lab, studying it, like what goes wrong, and then deploying molecular, uh, uh, biology to develop therapeutics. With the brain, sadly, you know, there's no way of doing it. And so largely to, uh, you know, what we've been trying to do is to, like, find a way of shortcutting that process. But I do believe that the major challenges that we're facing in understanding brain disorders, whether they're neurological or psychiatric, are on one hand, you know, the in- inaccessibility of the organ of interest, the brain.

    20. AH

      Mm-hmm.

    21. SP

      And on the other hand, our challenges are very often defining some of these conditions with biological markers, because they are much more complex.

    22. AH

      The degree

  8. 26:46 – 31:34

    Cause, Correlation & Neurological Disease; Schizophrenia, Do Vaccines Cause Autism?

    1. AH

      to which correlation has been leveraged to try and understand neurologic diseases is kind of staggering. Um, I'll just share a couple, and I would love your reflections. I remember when I was an undergraduate and in graduate school, there was this prominent theory that, uh, a mother who contracted influenza, the flu, um, toward the end of her second trimester-

    2. SP

      Yeah.

    3. AH

      ... had a much higher probability of having a schizophrenic child.

    4. SP

      Mm-hmm.

    5. AH

      And there was so much said of that, and then now we barely hear anything about it at all. Although I think schizophrenia is more prominent at the, uh, toward the poles where you have harsher winters as opposed to around the equator. But someone needs to check me those, uh, on that, because those statistics might have melted away with more careful analysis.

    6. SP

      Yeah.

    7. AH

      I don't know. The other thing is that you'll nowadays hear a growing interest in, uh, populations for which a given disease is very rare. So one of the things that's circulating out there now, uh, that's related to the vaccine debate.

    8. SP

      Mm-hmm.

    9. AH

      And by the way, I'm just gonna... I'll, I'll myself go on record. I don't think there's any solid evidence that vaccines cause autism.

    10. SP

      A- and there's not.

    11. AH

      Uh, right.

    12. SP

      Epidemiologically, there's no evidence.

    13. AH

      There, there's not. I mean, there's this open question as to whether or not vaccines of all kinds can increase inflammation, and there might be things downstream of inflammation. But for the record, there... Right now, there are no published papers that have not been retracted (laughs) that-

    14. SP

      Right.

    15. AH

      ... uh, that support the vaccine-autism link. Uh, I think those papers are being reinvestigated under the new administration, but let's leave that aside for now. People will say, um, "Well, you have groups like, uh, Amish populations, um, where the incidence of autism is significantly lower." Turns out, it does exist. I looked at these data. But it's significantly lower. And then people say, "Well, it's the absence of food dyes. It's the absence of, uh, vaccines perhaps," et cetera. But then as a genetic disease, you could say, "Well, there's also, uh, there's a tendency for people in the Amish community to reproduce with other people in the Amish community."

    16. SP

      Sure.

    17. AH

      So it's a more restricted genetic pool.

    18. SP

      Yeah.

    19. AH

      And so that could explain it as well. And I, I raise this, uh, not to, um, create any additional arguments. There are enough out there, uh, between people.

    20. SP

      Yeah.

    21. AH

      But just because I think the correlative nature of all this is what kind of raises the opportunity for anything that's observed, like a fever, they get better.

    22. SP

      Sure.

    23. AH

      Uh, but as you said, healthy kids without profound autism also talk more when they have a fever.

    24. SP

      Sure.

    25. AH

      And so there's, there's been so much made of autism and the various conditions that could create it.

    26. SP

      Yeah.

    27. AH

      And I think it's been very confusing for the general public, even as a, as a, you know, trained scientist, it's been very confusing for me. I feel like every six months or so, every year, we have a new, uh, pet hypothesis.

    28. SP

      Yeah.

    29. AH

      And, um, but nothing's really, except for these genetic data, nothing really is rock solid.

    30. SP

      Right. And then of course, it's... The, the other issue is also that these conditions are disorders of the human brain, right? So if you think about it, right, even talking about schizophrenia, right? Hallucinations, right? Or, or phenomena that are very difficult to study, and of course, we don't know this. We know that schizophrenia is present in almost every population that we know of, even isolated population at 1%, right? And again, it's a little bit easier because it's done in adults, right? I think in children it's much more difficult. And in fact, many of the genes that were early on identified for, uh, autism were identified in these populations. In the Amish populations, for instance. There is a very classic example of a gene that is associated with severe epilepsy and autism that was identified there for the first time. It's present, uh, in other places as well. So, uh, yeah, I, I think, of course, the, the, the complexity of the problem is that you also wanna make sure that you don't just associate something. You also wanna reverse it in a way, right? So you would wanna do the other experiment where you change it and then it goes away. But you can never do that in the human brain. We can't just turn things on and off to see whether they are truly causal. And then of course, human brain developments also takes an incredibly long period of time.

  9. 31:34 – 41:05

    Global Increase in Autism; Gene Therapy, CRISPR, Follistatin

    1. AH

      all kidding aside, before we get into the incredible experiments that you're doing and the direction that you're taking to tackle these really hard diseases, I have to ask two questions. First, is the incidence of autism also increasing outside of the United States, or is this something unique to the United States and Northern Europe? Um, I don't know why we always pair those two.

    2. SP

      Y- y- yes.

    3. AH

      Or, I should just be fair, to the United States and Australia, or whatever. Um, or is there something going on in the United States in particular that autism is increasing faster here?

    4. SP

      No, this... Yeah. No, this, the, you know, the, so like the prevalence for autism, you know, has been actually reported to be higher in other countries even before this. Uh, some of the early reports many years ago showed that in Korea, for instance, uh, you know, the inci- the, the prevalence was very high.

    5. AH

      Mm-hmm.

    6. SP

      Uh, now that the studies are done, uh, also like in Scandinavian countries, it shows that it's probably around the same, um, you know, kinda like rate, one in 30 to one in 40, so somewhere between. Um-

    7. AH

      Okay, so it can't be whatever is, uh, attached to whatever United States-specific, um, conditions. It, I mean, uh, you know-

    8. SP

      Yeah, no, very likely.

    9. AH

      Well, because you hear these arguments-

    10. SP

      Of course.

    11. AH

      "Oh, you know, it's the glyphosates in the, in the-"

    12. SP

      Yeah.

    13. AH

      ... uh, the crops in the United States." And while I don't favor that argument, I, I do think we need to be cautious about what's in the food supply. But-

    14. SP

      Absolutely.

    15. AH

      ... um, those same people often will, uh, leverage the argument that, "Well, in Europe they're not using these things." Well, if the incidence of autism is the same and rising-

    16. SP

      Yes.

    17. AH

      ... that, that sort of does away with the-

    18. SP

      Right.

    19. AH

      ... at least the clean logic of that.

    20. SP

      And perhaps another argument which is very important to, you know, bring is that we find the same mutations, right? I mean, the same mutations, if we're talking let's say a mutation in a specific calcium channel, you know, you'll find it in a patient in Denmark, right, as well as, like, one in Africa or in, let's say, Australia. So, I think some of these genetic mutations are certainly the same. Yeah.

    21. AH

      Could we briefly talk about gene therapy and CRISPR? Just briefly.

    22. SP

      Sure.

    23. AH

      Because I think in the context of a discussion about these n-neurologic diseases for which currently there aren't perfect cures, or even cures in many cases-

    24. SP

      Yeah.

    25. AH

      ... uh, gene therapy does hold some promise.

    26. SP

      Yeah, absolutely.

    27. AH

      Um, in simple terms, uh, that I and everyone else, uh, can understand, could you just explain what CRISPR allows physicians potentially to do?

    28. SP

      Yeah.

    29. AH

      Uh, in other words, can genes be fixed in adulthood? Do they have to be fixed in the embryo? Um, just give your thoughts generally about, about CRISPR and gene therapy. Because I think most people have heard of it-

    30. SP

      Yeah.

  10. 41:05 – 43:41

    Sponsors: AG1 & BetterHelp

    1. AH

      I'd like to take a quick break and acknowledge our sponsor, AG1. AG1 is a vitamin mineral probiotic drink that also includes prebiotics and adaptogens. As many of you know, I've been taking AG1 for more than 13 years now. I discovered it way back in 2012, long before I ever had a podcast, and I've been drinking it every day since. For the past 13 years, AG1 has been the same original flavor. They've updated the formulation, but the flavor has always remained the same. And now for the first time, AG1 is available in three new flavors: berry, citrus, and tropical. All the flavors include the highest quality ingredients in exactly the right doses to together provide support for your gut microbiome, support for your immune health, and support for better energy and more. So now, you can find the flavor of AG1 that you like the most. While I've always loved the AG1 original flavor, especially when I mix it with water and a little bit of lemon or lime juice, that's how I've been doing it for basically 13 years, now I really enjoy the new berry flavor in particular. It tastes great, and I don't have to add any lemon or lime juice. I just mix it up with water. If you'd like to try AG1 and these new flavors, you can go to drinkag1.com/huberman to claim a special offer. Right now, AG1 is giving away an AG1 welcome kit that includes five free travel packs and a free bottle of vitamin D3K2. Again, go to drinkag1.com/huberman to claim this special welcome kit of five free travel packs and a free bottle of vitamin D3K2.Today's episode is also brought to us by BetterHelp. BetterHelp offers professional therapy with a licensed therapist, carried out entirely online. I personally have been doing therapy for well over 35 years. I find it to be an extremely important component to overall health. In fact, I consider doing regular therapy just as important as getting regular exercise, including cardiovascular exercise and resistance training, which of course I also do every week. There are essentially three things that make up great therapy. First of all, it provides the opportunity to have a really good rapport with somebody that you can really trust and talk to about essentially any issue that you want. Second of all, it can provide support in the form of emotional support or directed guidance, or of course, both. And third, expert therapy should provide you useful insights, insights that can help you improve in your work life, your relationships, and in your relationship with yourself. With BetterHelp, they make it very easy for you to find an expert therapist who you resonate with and that can provide those three benefits that come from expert therapy. Interestingly, in a recent survey, 72% of BetterHelp members reported a reduction in negative symptoms as a result of their BetterHelp therapy sessions. If you'd like to try BetterHelp, you can go to betterhelp.com/huberman to get 10% off your first month. Again, that's betterhelp.com/huberman.

  11. 43:41 – 52:03

    Stem Cells, Ethics, Yamanaka Factors, Human Stem Cell Models

    1. AH

      Let's talk about stem cells, organoids, and assembloids, and you'll explain what those are. Um, but let's wade into this through the, uh, the way it happened chronologically.

    2. SP

      Sure.

    3. AH

      Um, most people have heard of stem cells, cells that could become other things. Uh, when I was a postdoc, any laboratory that worked on human stem cells worked on human embryonic stem cells.

    4. SP

      Mm-hmm.

    5. AH

      Literally cells that were collected-

    6. SP

      Mm-hmm.

    7. AH

      ... from aborted fetuses. This was... And given for, um, medical study. There was an incredible discovery, which you'll tell us about, which basically made that technology obsolete and also allowed, um, scientists to bypass a lot of the ethical considerations.

    8. SP

      Mm-hmm.

    9. AH

      Serious ethical considerations.

    10. SP

      Mm-hmm.

    11. AH

      Regardless of where you sit on that debate. I mean, you're using the, the, the tissue from a human embryo to, to study things. You could say some people will support that, some people won't, but then a new technology comes along-

    12. SP

      Yeah.

    13. AH

      ... and basically makes that technology obsolete, allowing you and others to do the work on stem cells and assembloids and so forth without having to take cells from, uh, human embryos, which is spectacular. So could you please tell us about that discovery of the, uh, stem cell technology that really changed the entire game-

    14. SP

      Mm-hmm.

    15. AH

      ... and did away with this ethical-

    16. SP

      Yeah.

    17. AH

      ... um, serious ethical battle. Let's call it what it was.

    18. SP

      Sure. Let's start first with stem cells and what they are because I think it's also important, uh, to define them. So stem cells are cells that have two properties. First of all, they, in principle, can become other cells. And if they are of the most potent type, they will be totipotent, so they can make everything. If they're pluripotent, they can make almost everything. And then of course, there are, you know, lower levels of potency for the cells. So we all carry stem cells in us, right? Not in the brain or fewer in the brain for sure, but you know, in the liver and in other organs, like in the gut as we renew the gut, uh, you know, every few weeks, that is done primarily through the stem cells. But those are restricted. They can't make everything. They can make mostly that specialized cell type for which they have been so, like, primed. Now, the earliest, earliest of stem cells, like those pluripotent, they are very important. Those are present at early stages of development of the embryo. Um, and of course, that happens post-conception. So the challenge has been that you have to remove them from a fertilized, uh, egg. And if conception, if life starts at conception, then of course you are interfering. So I think a lot of the ethical debates have started because of that. But, you know, in early days, even if you were to do that, you wouldn't be able to keep those cells. It turns out that the cells are very difficult to maintain. And this brings us, actually, to the second property of the cells, which is that in principle, they can be maintained forever. If you provide the right conditions, they will divide and stay the same forever. Those are the two properties. So, uh, you know, you can keep them forever. You can freeze them down, put them in a, you know, liquid nitrogen, bring them out anytime and they'll start exactly where they left. And then with the right guidance, they can become other cell types. So only around, you know, 1998, that it was, that when we could actually maintain some of the cells in a dish. So somebody figured out a soup of chemicals that you can add and these cells will survive because after that point, it was not possible. So that triggered, of course, the promise of this field, that now we'd be able to take those cells and derive various organs, right? Perhaps transplant them, replace organs. Uh, of course, that ended up being much more complicated. And of course, there were all these ethical debates related to the source of those cells and what does it actually mean to use this embryonic stem cells? And yet we've learned a lot about those cells in early days, what are the properties of those cells? And then almost 20 years ago, uh, Shinya Yamanaka, who was a scientist in Japan and at UCSF, came up with an absolutely brilliant idea. You know, we were... Always thought that the development, the development of the human or of any, it's, it's a one-way street. Once you go down development, you never come back. So once you start making, you know, a stem cell that is more restricted and then at the end you make, let's say, a liver cell, you can never go back and become that pluripotent stem cell again. And that generally is thought to be useful to protect us from, like, cancer or like any other, so we don't have, you know, parts of our hands, like de- differentiating into something else. And he thought that maybe you could do that, not in a natural way, in an artificial way. And that, of course, would be very useful.So, what he did is he went and he looked at the genes that are expressed in pluripotent stem cells at very, very high levels. So, very, very high levels. And almost as gene therapy, 'cause we were talking about gene therapy, he took, like, kind of like the top couple of dozens of these genes, and then started adding them inside skin cells. So, he took skin cells, initially from mice and then from human, and then started adding them one by one, two by two, three by three, four by four, five by five, six by six, to see whether any of those cells, once they have this combination of genes that are expressed in pluripotent stem cells, would somehow get confused and think that they're actually a pluripotent stem cell, and then go back in time and actually become a pluripotent stem cells. And he showed indeed that a combination of four is enough. Of course, you can have six. And that ended up being what we today call the Yamanaka factor. Uh, in a, in a way it was like, it was almost like alchemy, right, where you sort of like, you know, transform something into something else, right? You make, uh, out of this metal, you make gold. It was pretty much like that. It was kind of like the essence of alchemy. And it turns out that that discovery was so profound, because suddenly you could take a skin cell from anybody and put those genetic factors in, turn those cells into pluripotent stem cells that we'd later on learn they're almost identical to those embryonic stem cells. And now have those cells from any of us and use them for various purposes, perhaps for, let's say, making blood cells in the future or perhaps to, you know, model something outside of the body. And I was finishing my clinical training around that time, and I remember even seeing that paper. And of course, in my naivete at that time, I thought, "Wow, this is it. This is going to be, you know, the entry point for studying human neuroscience." I was doing experiments at that time studying actually the cortex and recording from animals electrical activity of those neurons, and always, like, thought, it's like s- I saw this disconnect between what I was seeing in the clinic, which were these patients with severe profound autism, and then recordings from the brain and thinking, "We're never going to be able to do that. How are we going to understand this complex disorder of the brain if we cannot even listen to the activity of those cells live?" And then suddenly, like, seeing that discovery, uh, you know, again, naive at that time, thought, "Well, that could be perhaps the way in which we could make neurons from any patient." And so very soon after I came to Stanford, which I guess where we met, uh, with sort of like this idea in mind that we will be able to make neurons from these patients and rebuild maybe some of the cells or some of the circuits of the brain outside of the body without doing any harm, 'cause we're not doing a biopsy of the brain or anything invasive, just essentially creating a replica of some of those cells outside of the body, and then finally study them at will in a dish and do all kind of experiments where you remove things and add things, and perhaps one day even develop therapeutics. And here we are, 16 years later, uh, since that process really started, took a long time, but now for the first time, we've gotten such a good understanding of some of these conditions. A- and one of them, uh, in particular, that actually a therapeutic is inside, and we're preparing for the first clinical trial, that is really arising exclusively through studies done with these human stem cell models, without actually using any animal models, just essentially creating, recreating cells and circuits outside of the brain of those patients.

  12. 52:03 – 59:30

    Umbilical Stem Cells; Stem Cell Injections & Dangers, Autistic Kids

    1. SP

    2. AH

      It's amazing because it allows you to study human cells, which has im- immense benefit. Um, they're essentially limitless in number.

    3. SP

      Yes.

    4. AH

      Uh, because all you need is one fibroblast-

    5. SP

      Yeah.

    6. AH

      ... one, one skin cell-

    7. SP

      Yeah.

    8. AH

      ... or, or some cell that you can provide these Yamanaka factors, uh, to, and essentially grow other cells. Um, and we'll talk about what those cells that you create are capable of becoming, not just cells, but circuits-

    9. SP

      Yes.

    10. AH

      ... i- in a few moments. But, um, I know it's going to be in the back of people's minds, and it's certainly in the back of my mind, uh, this idea that when one has a baby, that you should, uh, keep the umbilical cord because the umbilical cord-

    11. SP

      Yeah.

    12. AH

      ... uh, contains stem cells. Uh, usually, I think the umbilical cord is discarded.

    13. SP

      Mm-hmm.

    14. AH

      Maybe some people keep it. I don't know. Um, what is the current thinking on stem cells that reside in the umbilical cord? People pay a lot of money to freeze those-

    15. SP

      Sure.

    16. AH

      ... and most people don't have a, a minus 80, uh, freezer-

    17. SP

      Right.

    18. AH

      ... around, so they pay f- uh, to do that. What, what is the potential for umbilical stem cells in the future? Is it-

    19. SP

      Yeah.

    20. AH

      ... something that, um, parents, I don't want to say should invest in, but if they have the disposable income, uh, that they would be wise to do that?

    21. SP

      So, those cells, uh, that are collected from the umbilical cord are stem cells, but they're already quite restricted in what they can make.

    22. AH

      Mm-hmm.

    23. SP

      So, their applications are also restricted mostly to blood disorders. So, I think it's, it's important to keep in mind that they're not sort of like a universal, uh, you know, solution to anything that would ever involve pluripotent stem cells in the future or stem cell therapies in the future. So, again, I think it's important to know wha- that while they have certain applications and there have been quite clear cases where the availability of those cells were useful in a blood disorder in that child later on, um, they're certainly not, you know, they don't have these universal uses as maybe sometimes they're being advertised.

    24. AH

      When we hear about people typically leaving the US, uh, to get, quote unquote, "stem cell injections"-

    25. SP

      Yes.

    26. AH

      ... where are those stem cells coming from? Are they coming from those patients? And I should mention that there was a clinic down in Florida, um, that was offering stem cell injections into the eye for people with, um-

    27. SP

      Yeah.

    28. AH

      ... macular degeneration, and that clinic was shut down, and all stem cell injections in the United States, to my knowledge, all were shut down because those patients, uh, not only did it, uh, fail to rescue their vision, it actually made them go blind very quickly.

    29. SP

      Right.

    30. AH

      Uh, so the FDA shut down, uh, commercial stem cell injections. I think there's still places where they do a kind of workaround.

  13. 59:30 – 1:12:22

    Organoids, Modeling Brain Development, Intrinsic Development Timer

    1. SP

    2. AH

      Mm-hmm. So, let's talk about the other approach, uh, which is the one that you are, uh, you've been embarking on. I'll never forget when we were post-docs. Folks, we were post-docs in the same room.

    3. SP

      (laughs)

    4. AH

      It was D222.

    5. SP

      Yes.

    6. AH

      Uh, we had a lot of pride in that room.

    7. SP

      (laughs)

    8. AH

      We had benches, uh, on opposite sides of the room, and we sort of, uh, took over that room as an empty room. This is, you probably couldn't do this anymore, but it was like, "There's an empty room. Let's bring some microscopes in there." And we just started doing experiments there. And I'll never forget, um, when you started building organoids. You started building nervous systems i- in a dish and how excited you were.

    9. SP

      (laughs)

    10. AH

      And, uh, and it's been remarkable to see your, your arc, uh, to, from that. Um, and it's, uh, not lost on me that you were working extremely hard then-

    11. SP

      (laughs)

    12. AH

      ... and have continued to, to become, what, really one of the luminaries of this field. Um, tell us what organoids are. Tell us why they're useful, and what they're telling us already about how the brain develops and their therapeutic potential.

    13. SP

      Yeah. So, let's start from the beginning. So, around like, you know, 15, 16 years ago, we were able, for the first time, to get some of the cells that are now known as induced pluripotent stem cells.

    14. AH

      These are the Yamanaka.

    15. SP

      Yes, or iPS cells, iPS. So, induced because they've been induced to become pluripotent in an artificial way. But again, they stay like that, so you can share them with anybody else, like, afterwards. So, we got some of those first cells in those early days. And now the question was, how do we make neurons? And what you do is, you really kinda, like, leverage the...... everything that is known in developmental biology, right? So we already know that there are certain molecules that are very important for making neurons. So all you do is you put those cells in a dish, right, in a plastic dish, in a Petri dish, and then you start, almost like when you cook, you start adding various molecules on top, and you see what happens. And we knew that it's actually quite easy to make neurons. That was already known. There'd been a lot of experiments done the decade before that showed that even if you just remove some of the factors that maintain those cells pluripotent, those pluripotent stem cell will start now to differentiate, and they like to become neural cells. Hmm, by default. Almost by default. So it's actually not that difficult to make neurons. So in those early days, you know, you would take those cells, plate them nicely, those pluripotent stem cells, in a dish, and then remove some of these factors. And then within a few days, you will see that they'll change shape, and within a few weeks, some of them will really look like neurons. And, uh, when you look at them you can even sort of, like, look at proteins that only neurons will have. You can actually get an electrode inside a cell and listen to the electrical activity. So it was very exciting as, uh, maybe you remember in those days. Uh, I mean, you know, this, uh, burst in curiosity is always sort of like, uh, you know, the ATP of the li- the, you know, the life in the lab, so to speak. It is. Mm-hmm. Right? I mean, you just, like, kinda like wanna wake up, right, and wanna go see what happened to those cells. And it was clear in those days that, you know, we would be able to make those cells, but w- would we actually see any abnormalities in those cells, I think was, like, the question. You know, how would you know if you derived cells from a patient with autism? How would you know that you found anything abnormal? I think that was like, uh, the question. Wh- you know, we didn't even know what would be abnormal in the brain. And so that's when we decided, actually, to focus on something that would be relatively predictable, and that was this mutation in a calcium channel, which was discovered just a few years before in very few patients that had essentially one single letter in their entire genome changed in a gene that makes a protein known as a calcium channel, sits in excitable cells, meaning cardiac cells and brain cells. And every time a cell receives electrical input, this protein opens up and lets calcium go inside the cell. And that's very important because it couples electrical activity of the network with chemical activity inside the cells. And what we knew about that mutation at that point, and that's pretty much all we knew in those early days, is that it probably allows the channel to stay open slightly longer, just a little bit longer, so more calcium would go inside the cells. Of course, there would be no way to know because you can't get a neuron or a cardiac cell from those patients to actually test it. So what we did is, essentially, we m- made, uh, we recruited some of these patients. We flew them to Stanford. Uh, then we got a tiny skin biopsy, made these iPS cells. This takes months. This takes already, like, four or five months. And then we took those cells in a dish, started to deriving neurons, and after about five, six, seven weeks, then we put them under a microscope, and we started looking at the calcium. You can measure calcium inside cells through a microscope and just literally look at it. And I'll never forget that day, um, you know, when we did that experiment. Was looking down the microscope, and we essentially stimulated the neurons, and you could just see how control cells will go voom. Calcium goes inside the cells, and then it goes out. And then in patients that had Timothy Syndrome, so in Timothy Syndrome-derived neurons, you could see how the calcium will go voom, and then it will stay longer. It takes longer to go out. So it's just, like, the first defect that we saw in patient-derived neurons that were actually not coming from a biopsy. They were not coming. So that was incredibly exciting, as you can imagine. But it was still relatively simplistic, just a few neurons at a bottom of a dish. And of course, for me, what was particularly frustrating was that we couldn't go very far in development. So think about the cerebral cortex, the outer layer of the brain that presumably makes us human, right? It has multiple layers, a large diversity of neurons. You know, it takes 27 weeks to make all those cells in the cortex, 27 weeks to make all those neurons, and we're not even talking about glial cells. They're supporting cells that are coming much later for several years afterwards. But just making those cells takes about 27 weeks. And it turns out, something that we discovered in a, uh, through experiments done in a dish, is that the timing of the development of those cells, it's actually recapitulated in a dish as well. So if you keep the cells in a dish, they'll actually essentially develop at the same pace. They're not, like, much faster. And it's very difficult to keep neurons in a dish for 27 weeks to get all the neurons. Essentially, they peel off, you know, every time you start to move them to another plate. Then at one point, they just die. And so then we thought, how about, like, never letting them just sit down on a surface? How about just essentially aggregating them as bowls of cells and then letting those float? And in those early days, there was this, uh, amazing, uh, scientist from Japan, Yoshiki Sasai, who started doing, uh, really beautiful experiments, where he was already moving some of these studies that he was doing of development in 3D cultures, where he showed you can make an optic cup, a part of the eye. And so it was clear. It was in the air, this revolution of actually moving cells from 2D flat cultures to 3D self-organizing, and that actually unleashed amazing, uh, new properties of the cells. So essentially all we did in those days is, I ordered from Germany these plates that were counterintuitively coated so the cells never stick, right? I mean, every time we keep cells in a dish, you want them to stick. That's the major problem. So they were actually coated so the cells will never stick, and then there were, like, this bowls of cells. They were floating there, and of course, I, I remember, uh, talking in the lab and everybody was like, "Oh, they're not gonna survive. It's gonna be a couple of weeks, then they're gonna..."... and then, uh, a week passed, and two week passed, and then they kept growing and growing, and of course the enthusiasm of every g- every day to see are they still alive, right? And then we discovered that we can keep them for months. Uh, and these three-dimensional cultures, uh, are now known as organoids, which is perhaps not the most fortunate name because it suggests that it's organ-like, and of course they're not an entire organ, right? So they're not a representation of the entire brain. But that's sort of like the term that we refer, these days, to anything that is sort of like three-dimensional and organizing in some way. And so we started keeping these cultures, and then at one point actually we discovered that we can pretty much keep them indefinitely. My lab maintain the longest cultures that have ever been reported, like literally going for years, for two, three years in a dish. And at one point, uh, in those early days when actually I was running out of funds in the lab, and I came one day in lab meeting, uh, uh, really, uh, you know, determined to... for us to actually, like, cut costs. So I told everybody, "Go into your incubators, 'cause we're spending so much money in feeding the cells, and everybody throws out 20% of your cultures." And then people started saying, "So should I throw the ones that are, like, 500 days old?" And somebody else like, "The ones that are 800 days old?" and I said, "What? You guys are keeping them for such a..." "Yeah, they're just keep growing there in the incubator." So then we actually did the first study, and then we had a series of three studies done over the years of, like, trying to ask how far do they go in development? So if you have a clump of human neurons that you've made from pluripotent stem cells and you keep feeding them in a dish, how far do they go in development? Do they move much faster? Do they move much slower? Are they stuck at one point in development? And it turns out that they actually keep track of development beautifully, to such an extent that, for instance, we discover when they reach nine months of keeping them in a dish, so about the time of birth, they literally switch to a postnatal signature. Really? On their own. In a dish? In a dish. So, you know, uh, there's this classic example in developmental neurobiology. There is this, uh, uh, there's this, uh, protein that usually changes around the time of birth. It's, uh, an NMDA receptor, so maybe some people know about NMDA receptors. Uh, binding glutamate. They're very important. But they change a lot during development. They're made out of different units and the units change, and it was very well known that during early development, so prenatal, before birth, you primarily have 2B subunits, and then after birth, they're primarily 2A. So if you look in brain development, you just see how essentially 2B goes up and then it goes down, and 2A goes up, and when you look, they meet around birth. So very often people thought that it's birth itself that triggers that switch, that canonical... It's called a canonical switch 'cause, uh, we all thought that it was, like, so classic. And then you take an organoid that you maintain in the dish for 600 days, and of course we're not inducing birth. We're not changing media. We're not doing anything special. Yeah, no hormones from mom. No hormones changes- Yeah. ... like, you know, we keep exactly the same media, which is certainly a very simplistic, uh, uh, you know, kind of like soup of chemicals, but we don't change it. And then you just look at these two subunits and you see how, like, 2B goes down and 2A goes up, and they pretty much meet that nine months of keeping them in a dish. That's amazing. So that tells us that there's some sort of intrinsic clock. Once you started development, the cells measure really, really well the time of development. That does not mean that all aspects of development are gonna now be recapitulated in a dish. But it tells us that there is this incredible ability of cells, especially in the nervous system, because of course those cells will keep for the rest of our lives. Now, we're not... never gonna renew neurons. It's gonna be different for liver cells or gut cell. But for neurons probably in particular, they'll need to keep track of time really, really well. So that was, like, the first discovery that we've sort of, like, made, which is still stunning today. We still don't know the mechanism. We're still working really hard on figuring out exactly how the cells are keeping track of time, because as you can imagine, if we understand what that molecular machinery is... We used to call it a clock. We now call it a timer. We think it's more of a timer than an actual clock. Uh, but understanding what the molecular biology of that is will allow us actually to play with that clock, right? So if you want to make neurons that are, you know... oh, a 70 years old neuron from a patient with Parkinson, you know, I don't have to wait 70 years in a dish. Could I make it in, like, a few weeks? Or perhaps could I take an aging neuron and somehow, you know, rejuvenate it by playing with that, with that timer? But just to make it clear, we still don't know that... you know, we have some clues about, like, what it may be, but I think... it's still early days. And I think that was, like, one of the first things that these cultures allowed us, uh, uh, to do. Uh, just watch development, human brain development, outside of the human body in a dish, and actually witness that some fundamental aspects of brain development are actually recapitulated even outside of the uterus and, of course, of the brain.

  14. 1:12:22 – 1:21:22

    Assembloids, Brain Cell Migration & Circuit Formation, Self-Organization

    1. SP

      So that, that was, like, the first, and then of course, I guess I'm a developmental neurobiologist by training and, you know, I've done a lot of circuit work in early days. Of course, an obsession of mine was that especially for conditions as complex as autism and schizophrenia, we need to recapitulate some of the circuit properties of the brain, right? So we now know that, you know, probably both for schizophrenia or... and for autism, it is very unlikely, based on the evidence that we have so far, that there are cells really missing from the brain. You know, we thought for a while that maybe some cells are missing or maybe other cells are in... you know, in excess, but now the studies that have been done, especially with single cell profiling of brains of patients that have already died, showed us that the composition of the brain, of the cortex in particular, it's very, very similar. So it's unlikely that the cells are missing or, like, uh... but likely the way they are connected with each other is that makes a difference. And of course, in the beginning we were just making this clump of cells. They were all for the cortex, but they were, like, not connected to anything else.So then, uh, came the idea of assembloids, because most of the cells in the brain connect with cells across the nervous system. And in fact, even more interestingly, cells do not reside in the place in which they're born in the nervous system. We have the largest cell diversity of any other organ, almost 2,000 cell types. By the end of the first trimester, there are about 600 cell types in the human brain. You know, think about the liver, right? Maybe a couple of dozens. The brain has to make, you know, hundreds of times more. So, how do you do that? The only way is to actually make the cell types in different parts of the brain, provide local cues there, and then once the cells have been specified, let them move and find their final position. So, the first assembloid that we've actually made were of a very, uh, stereotypical canonical movement of cells in the nervous system, which has to do again with the cortex. So, the cortex, again, the outer layer of the brain, has both excitatory and inhibitory neurons. It turns out that most inhibitory neurons are not born in the cortex, but they're born deep in the brain. So essentially, all we did is we made two brain regions, the ones that has excitatory neurons and the one that has inhibitory neurons. And the plan was to put them together, hoping that at one point, you know, the cells will, like, sort of like know what to do. And in fact, that was like one of the first projects in my lab, kind of like planning that, and I remember gave to one of the students, like this very difficult task of figuring out how we're gonna fuse these two cultures. And they're about three millimeters in size, so you can see them by eye. And I thought it was gonna be very difficult to put them together. So, the student worked for, for months trying to figure out, like biological glues, you know, kind of like using various, uh, electrodes and impaling them and everything else, until somebody else came one day and said like, "Oh, it's very simple." You just put them at the bottom of a tiny Eppendorf tube, which is the tiniest like of tubes that you get. You put them there overnight, and next day they're completely fused. But they're not just fused, because now if you look inside, within a few days, the cells that are supposed to move start to actually point out towards the cortex. They literally smell the chemicals from the cortex, and they start to move in this very stereotypical way towards the cortex. And so that was the first assembloid, made around 2015. And I still remember, it was Ben, actually. Ben was so excited. Ben Barres was so excited about, like seeing the cells, he wanted to look at his movies every day. And then, um, he said... I still have this email from him, where he was very preoccupied that... He kept saying like, "This new preparation is not an organoid. It's not a steroid. It's something else. You have to find another name."

    2. AH

      He loved naming things.

    3. SP

      He loved naming things.

    4. AH

      Yeah. And he understood the importance of naming things, not just for, like, career reasons, although he understood a lot about how to build a career.

    5. SP

      Yeah, perhaps.

    6. AH

      But because naming... Like Yamanaka factor made sense-

    7. SP

      Yeah.

    8. AH

      ... to name it after Yamanaka. He got a Nobel, uh, uh, and, uh, is immortalized that way, like stem cells, immortalized.

    9. SP

      Yes.

    10. AH

      Um, but I think the naming is essential, because otherwise, um, things can get lost in, in the technical details.

    11. SP

      Yes.

    12. AH

      So, who came up with the name assembloid?

    13. SP

      So, so he kept insisting that I should find a name. So I made this long list, I still have like the, in my notebook. Like, I had a long list of about 20, and I would like keep sending Ben one, and you know, like Ben was always awake, like 24 hours.

    14. AH

      Yeah. He didn't sleep much.

    15. SP

      He never slept.

    16. AH

      No.

    17. SP

      So, I remember one... after sending many emails going back and forth, and he was just like, "No, bad name, bad name. I don't like it." And then at one point I thought, well, "oid" because it's like, and then assemble because we're assembled as circuits. So, I thought assembloid, and I sent this and he says, "Perfect. I love it."

    18. AH

      So, you named assembloids?

    19. SP

      I named assembloids-

    20. AH

      Mm-hmm.

    21. SP

      ... and Ben sort of like, uh, blessed it, like one night at like 3:00 AM. And so that was the first assembloid. And the first assembloid was for cells migrating. But then the question was, cells have to find each other and form circuits. And so within a couple of years, we started making assembloids that will have axons, so the long projections of neurons finding other partners. And, uh, you know how... I forgot who said this, must have been Rodolfo Llinas or you know, who said that the brain is sort of, um, you know, the next evolutionary step towards movement.

Episode duration: 2:23:14

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