190 | Lea Goentoro on Regrowing Limbs

0:00:00.0 Sean Carroll: Hello, everyone and welcome to the Mindscape Podcast. I'm your host, Sean Carroll. One of the fascinating things about biological organisms is their capacity for self repair. Organisms are much better at fixing themselves, healing, right, than robots or machines, at least as we currently build them. You might have a self cleaning oven, but if you break the oven, it's not gonna fix itself. Okay? But we all know that we can get a cut, a wound, even break a leg and heal ourselves. But also this healing has its limits, right? If we amputate a limb, we're not gonna grow it back. So where do those limits come from? And how can we manipulate them to get better at doing things that we couldn't have imagined doing before? Today's guest Lea Goentoro is a biology professor at Caltech. And she studies the form of animals as they develop. So similar a little bit to Michael Levin, who we had on the podcast a while back. Somehow, in ways that we don't fully understand, the genome, the DNA of biological organisms has a plan for how the grown up animal is supposed to look. Okay? And how that actually gets manifest over the course of development is very interesting and something we're trying to learn about. So what Lea's lab was actually studying was what happens to jellyfish when they lose a limb. Jellyfish have a certain symmetry, like you might have an eight armed jellyfish.

0:01:23.7 SC: And usually, if you just amputate a limb, they will not grow it back, they will sort of rearrange the other limbs to maintain some symmetry. So they'll forget that the old limb was there. But what they found in the course of this exploration was sometimes the jellyfish would grow a limb back, it would just go back to being an eight limbed jellyfish again. So scientifically, of course, when does that happen? What are the conditions? And fascinatingly, what they found is that they don't need to really coax the jellyfish into regrowing a limb, they just need to provide it the right environment and the right nutrition, right? The right chemicals around it. The cells of the jellyfish know what they're supposed to look like. And so of course, once they found that, they extended it to other animals and fascinatingly, it still works. They did it with fruit flies, drosophila, model biological organisms and even with mice, you know, just a little bit, just like a little finger of a mouse. But they found they could coax the mouse into regrowing its fingers.

0:02:22.3 SC: Lea will go into the details about this, as I always like to emphasize, I am not a biologist here. But the fascinating prospect is that we do have within all of us the capacity for not just knowing what kind of limb we want to regrow, but actually biologically doing it if we're just put in the right circumstances. It's a long way to go before we're talking about doing this to human beings. But it's not completely crazy talk. And that would be a true game changer in a lot of ways. This is still very much at the stage of very basic science. It's nowhere near being practical medicine, but it's a little peek at the frontiers of what science and biology are able to do. Let's give you an occasional reminder that here on Mindscape, we have a web page preposterousuniverse.com/podcast, where you can find show notes, links, so I'll give you for example the link to Lea's group's most recent paper, including her students and so forth. And also we have full transcripts for every episode and they're all searchable. So you can search all the transcripts from Mindscape almost 200, actually over 200 episodes now if you include specials and AMAs and things like that. So a treasure trove of cool stuff that you can find on the website preposterousuniverse.com/podcast. And with that, let's go.

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0:03:54.4 SC: Lea Goentoro, welcome to the Mindscape podcast.

0:03:54.7 Lea Goentoro: Thank you for having me.

0:03:57.6 SC: We're gonna be talking about kind of provocative big ideas, right, about regenerating limbs. This sounds very science fictiony, but I know that you do a lot of different things in your lab and I know that, I think, I get the impression that this particular work stemmed from previous work with jellyfish, you seem to really like jellyfish and experimenting on them. So maybe if we you could just sort of start softly by saying what is it about the jellyfish that got you so interested in thinking about that as a model organism?

0:04:26.9 LG: Well, to be honest, it was a little side project in the lab, in the beginning. I came to Caltech and then I think I was introduced to the research that John Dabiri did, was doing on jellyfish and that got me interested in just thinking about jellyfish. For me it's a couple of things. One is just the evolutionary position of jellyfish enables us to make evolutionary, ask evolutionary questions. And the other thing is every model, every animal has a unique biology that can give us new insights. So with just studying more and more different animals, I believe we can get more a diverse and complete picture of biology.

0:05:20.3 SC: It's interesting to me 'cause I'm totally not a biologist, as I'm sure I'm gonna end up saying several times during this podcast, I am not a biologist and ask very elementary questions, but I'm interested both in the fact that, you know, animals and plants and other life forms are so different, but they're also similar. They do share things. I did a whole podcast recently with Arik Kershenbaum about his idea that aliens won't be that different from us, because they're still biological organisms and they'll still have to solve the same problems. But one of the papers I liked that you wrote was about the fact that jellyfish sleep, even though they don't have brains, right? It is sort of a thought provoking idea that even something as simple as a jellyfish has to go to sleep sometimes.

0:06:06.6 LG: Yes, yes. And this is, I have to mention the students who really...

0:06:13.3 SC: Please.

0:06:14.5 LG: Pushed this project, Michael Abrams and then Claire Bedbrooks and Ravi Nath and they really... It was actually a very fun project and very fun the way it started, because it was really the students who made the observation and then designed the experiments and then pretty much just told the three of us Viana, Paul and me that they were going to do this project. [laughter]

0:06:41.0 SC: And what did you learn from that? Why is it that jellyfish sleep anyway?

0:06:44.4 LG: Well, we don't know why, although my colleague Elliot likes to speculate whether they dream.

0:06:50.0 SC: Ah okay good.

0:06:52.5 LG: If they sleep. But that really... The finding that they sleep means that sleep could be an ancestral property of animals, all animals sleep and that raises new questions about what sleep does in animals without a brain.

0:07:17.4 SC: What does it even mean in animals without a brain, I think of sleep as sort of turning my brain off and letting my body recuperate, but if I didn't have a brain, do I just stop moving?

0:07:26.9 LG: Exactly, so that suggests that sleep may have very ancient properties that even just a primitive nervous system sleep or show sleep behavior.

0:07:40.2 SC: So a jellyfish...

0:07:41.2 LG: So beyond that... Yeah.

0:07:42.9 SC: A jellyfish has a nervous system, but not a brain?

0:07:45.2 LG: Exactly. Yes.

0:07:46.2 SC: Okay, got it. And is it still floating in the water when it's sleeping, or does it go to the ground, does it take a nap on the bed?

0:07:54.5 LG: In the species that we studied, it stays on the ground, well the species is stationary, it does not float so, but what happens is that when it sleeps and it sleeps at night, it pulses less frequently.

0:08:12.8 SC: Okay and probably this has some sort of regenerative function of some sort right, catching up, repairing itself?

0:08:21.4 LG: Yes. We don't know exactly what.

0:08:25.0 SC: You don't know what. [laughter] This is good, this is a great insight for the audience into how science goes, like sometimes you just discover something cool and you don't know why or what function it has, but leave that for the next generation, right?

0:08:40.9 LG: Yes and in particular, my student, Michael Abrams is continuing studying this process in jellyfish.

0:08:46.6 SC: Okay, very good. And I guess, tell me how that led into the study of what happens when jellyfish lose a limb of some kind, 'cause there was an original study you did about symmetrization, which is itself interesting even before we get into regeneration.

0:09:03.1 LG: Right, so I think the study on symmetrization is what led us to eventually studying regeneration. So that was, again, it started as a side project and we made an accidental observations that when we cut, when we injure jellyfish, instead of regrowing body parts, which was what we expected from cnidarians, the jelly simply rapidly reorganized existing body parts and regained body symmetry and this occured really fast in two or three days.

0:09:43.9 SC: So how many legs does one of these jellyfish have?

0:09:47.7 LG: A jellyfish typically has eight legs, arms, appendages.

0:09:54.6 SC: Appendages.

0:09:54.9 LG: They're really swimming appendages.

0:09:56.7 SC: And you by mistake cut one off or intentionally cut one off, how did by mistake, you hurt the appendage of a jellyfish?

0:10:06.2 LG: Yeah, obviously we don't by mistake cut them off. And this was Michael's rotation project and what we did was we just wanted to get comfortable culturing them in the lab and Michael had a fun experiment, decided to do a fun experiment and did amputations just to get used to handling them and then two or three days later, he observed that they...

0:10:35.4 SC: It's symmetric again.

0:10:35.7 LG: Become symmetrical again. That's right.

0:10:37.4 SC: So because we don't have the pictures to show the audience, right, it's an audio only podcast, so you have eight appendages and they're symmetrically dispersed around the jellyfish and you cut one off and sort of it squeezes the other ones or redistributes its seven, so that those seven are now symmetrically distributed, yeah?

0:10:56.7 LG: Yes, yes, you described it really well and it works with any number of arms cut off, so you can cut two, you can cut three or four, half the animal and its cut part reorganize within two or three days and becomes symmetrical again.

0:11:15.6 SC: This is a very unfair question, but why does that happen? Do we know why?

0:11:22.8 LG: Again, it's hard to answer why, but in biology the way we answer why is to just observe what it does, so the animals that regain body symmetry, regain propulsion and that allows them to feed, to eat.

0:11:45.6 SC: I see, so...

0:11:47.0 LG: While the animals... Yes.

0:11:48.1 SC: So these jellyfish use their appendages to zip around underwater and if they were asymmetrically distributed, they would just be spiraling away instead of going in the straight line they wanna go in?

0:11:58.2 LG: Exactly.

0:12:00.5 SC: Okay and yet, so that does kind of make sense, even if we don't know sort of the complete story there, but then you also discovered and apparently this was just serendipitous, but tell me if I'm wrong again, sometimes the jellyfish did regrow or at least partly regrow an appendage?

0:12:18.4 LG: Right. So in the course of just amputating hundreds and hundreds of these animals to study the process of symmetry recovery, we started noticing that in very few animals, there's a little buds growing from the amputated side, so that made us wonder, even though we don't normally see them regrow their appendages, if they may have a latent ability to regrow appendages.

0:12:52.3 SC: So yeah, so was it predictable, like what kind of circumstances under which they would regrow versus symmetrize?

0:13:00.9 LG: Right. That was the long journey...

0:13:03.2 SC: Yeah, okay, well, let's get there.

0:13:05.4 LG: To discovery.

0:13:06.5 SC: In fact let me un-ask that question. Let me ask it later because we're at the position where we say, "Okay, under the right circumstances, whatever they may be, the jellyfish will regrow a limb," so we need to fill in the background about animals regrowing limbs or other kinds of species regrowing limbs. What's the state-of-the-art more general biology, about regenerating lost appendages? Is that something for which there's some kind of systematic theory or at least little bits of knowledge?

0:13:37.6 LG: Right. So I think we're at an exciting place right now where the framework is emerging, I believe, of how to think of that question. So that's the big question, which is that if you look at all animals around us, we know there's this few animals that can regrow missing body parts spectacularly well. Axolotl is a good example of that. They can regrow a limb, the spinal cord.

0:14:07.2 SC: Sorry, what is that? What is that animal?

0:14:09.2 LG: Salamanders.

0:14:13.8 SC: Oh, okay. [laughter] Physicist, remember. [laughter]

0:14:15.5 LG: Or axolotls. [laughter]

0:14:16.6 SC: Okay.

0:14:17.1 LG: Right. But, yes, salamanders and axolotls could regrow a limb. Certain kind of flatworms called planarians can regrow a missing head or a missing tail.

0:14:30.2 SC: That's very impressive.

0:14:32.2 LG: That's right and hydras, which is a close cousin of jellyfish, can regrow any lost body parts.

0:14:39.3 SC: Okay.

0:14:39.9 LG: So some animals seem to be able to spectacularly regrow lost body parts. The rest of us are not as lucky.

[laughter]

0:14:50.1 SC: And what's weird about that collection you just went through is that it's not like they're all closely related to each other.

0:14:56.2 LG: That's right, that's right. So then the question is that when we look at animals that cannot regenerate, is it because they never evolved the ability to do so, or could it be, some biologists are beginning to think, that maybe all of us can regenerate, it's just that in some of us, the ability has been inactivated.

0:15:20.5 SC: And am I right that lobsters and crabs can regrow claws?

0:15:25.1 LG: That's right.

0:15:26.1 SC: Okay, yeah.

0:15:26.9 LG: That's right, yes.

0:15:27.9 SC: Yet another very different set of organisms.

0:15:30.4 LG: Yes.

0:15:31.4 SC: And so once you say that and again, I'm getting the feeling that I'm gonna ask you a whole bunch of unfair questions, but you never know whether the answers are known, but in physics, we know a lot of things and we know exactly what we don't know, but in biology, there's so much we don't know that we keep stumbling across things. So from an evolutionary point of view, why can't we just regrow limbs? [laughter] That would seem to be a very useful thing to be able to do if I lost a finger or a hand, just to regrow it. Is there some story we can tell about why most organisms don't regrow limbs? Why isn't that the conventional usual story?

0:16:11.8 LG: Right. A lot of biologists are also asking that question, because the ability to regrow lost body parts seems to... It seems that it should be useful.

0:16:21.6 SC: Yeah, I would think. [laughter]

0:16:23.4 LG: Yeah. [chuckle] So the thinking is, there might be a trade-off. We don't know what the trade-off is, but perhaps the ability to regenerate is coupled with some other maladaptive side effects, but we don't know that, but the hope is that if we know what governs our latent ability to regenerate, then we can start speculating.

0:16:50.5 SC: Right. So speaking of speculating, let's just do it. What might be these trade-offs? Does it cost too much energy or food maybe to regenerate a whole limb?

0:17:01.0 LG: Right. Well, I can say what... I can speculate based on what we find. Right. But that will mean that I will have to describe what we find.

0:17:12.8 SC: Please. Please describe what you found out. Don't hold back on my account.

0:17:17.3 LG: Well, like you said, in the jelly, we first found that they don't typically grow lost appendages, instead they just regain body symmetry. What we subsequently found after long three years of screening is that under certain very specific conditions, they can grow lost appendages and it's not always perfect, but the idea is that we can kickstart, coax latent ability to regrow appendages to emerge. And then to quickly summarize and then I can explain later, is that the same strategy that we found worked in jellyfish, worked as well in the fruit fly, drosophila, in coaxing limb to regrow in fruit flies and finally in mouse.

0:18:25.1 SC: Okay. Now we're talking, right? I mean, [laughter] mice are pretty close.

0:18:29.8 LG: That's right, that's right. And the mice... I have to clarify that the mouse digit is our model to explore how to induce, if we can induce, limb regeneration in humans.

0:18:41.7 SC: Sure, sure. So, we have a situation where... Is it... You said it's like under some circumstances that these limbs do regenerate versus just symmetrization or something? Like a mouse doesn't even symmetrize, right? We just sort of heal over the wounds or whatever. But you said for the jellyfish and presumably for the flies and for the mice, the conditions have to be right. So, what do those conditions have to be?

0:19:07.7 LG: Right. And that was part of the surprise of the finding, is that, first of all, there is no guarantee that we can induce appendage or limb regeneration. If you go to animals, they don't regenerate. There's not a guarantee that the ability is there. So the first surprise is the fact that we could observe it at all and the second surprise was the conditions, the conditions was surprisingly simple. In jellies, the factors that we needed to present to them to coax appendage regeneration are simple nutrient factors. One is just a very high amount of food, we think more than they normally see [chuckle] and then the growth hormone insulin and the amino acid leucine, the same factors also worked in flies, we just mixed the amino acid leucine and the growth hormone insulin in their food. Same thing in mice, we mixed leucine and in the case of mice, we can't give insulin orally because just like in humans, insulin cannot be administered orally, it gets digested in our guts, so instead of insulin we just give the mice sugar to induce endogenous production of insulin. So, just simple amino acid leucine and insulin mixed into drinking water and we can coax digit regrowth in about 20% of the mice.

0:20:43.4 SC: And these are, again, just for people who know even less biology than me, these amino acids, the insulin and the leucine, they're kind of universal, like all life forms make use of these already, it's not something that is specialized to these organisms.

0:20:56.6 LG: That's right, humans, our bodies can make some amino acid and we need some amino acids from our food. So leucine is one of those that we intake from food, it's called essential amino acids. And actually leucine is something that should be familiar to all of us, because if you have a friend that's into body building, leucine is one of the supplements that they take to promote muscle growth, so yes, it's in us too, the same processes.

0:21:26.1 SC: And maybe let's be very, very specific about what the results were, 'cause you kind of alluded to them a little bit, both what percentage of the time did you get limbs to regenerate and when they did regenerate, did they go all the way, or were they like stunted little things. What did they look like?

0:21:42.9 LG: Right, so it varies in different organisms, so I think in mice, as I said, 20% of them show regrowth and digit regrowth is of various extent from moderate regrowth to almost perfect regrowth. But the most exciting thing is that it happens. Yes, it's not full, it's not complete all the time, but it happens and we have toeholds. So, now the question is, how can we make them more perfect and stronger.

0:22:19.5 SC: And now, is this the first example that you know of, of limb regeneration other than species that do it automatically? Did you coax them along to doing it?

0:22:31.0 LG: No, so thank you Sean, for clarifying that. So, what's new about this? So people have studied in some animals how... People have studied animals and asked how to make them regenerate. So, most of studies of inducing regeneration, it's been done in frogs. And also more recent studies in mouse digits, so in frogs for instance it's like, it piqued this question of how to induce regeneration, piqued in early 20th century. And then there was just these really wonky discoveries, people found all kinds of things that could make frogs regenerate. So, let me clarify first frogs, so why is frog an interesting system? Frog is an interesting system, because as a tadpole they can regrow limbs but then this ability is dramatically lost upon metamorphosis. So tadpoles can regrow limb and frogs cannot. So then the question is, can you reactivate regeneration that's been lost upon metamorphosis? So notice how specific I'm phrasing the question.

0:23:40.9 SC: Yes, yes.

0:23:41.8 LG: Okay. And then people found all sorts of things, electrical signals can coax limb regrowth in adult frogs. What else did you have? Salt, just simple salt administration and then I think osmotic shock as well, just all kinds of stimuli and the studies kind of peter off because they're... It didn't lead to a lot of mechanistic insights.

0:24:11.9 SC: Okay.

0:24:13.4 LG: So, it's not clear yet, so that's in frogs... Yeah.

0:24:13.5 SC: But these are all in species where, like you just said, just to clarify, they already at one point in their development had the ability to regrow and so maybe you're more optimistic and you zapped them in the right way and the adult frog would regrow, but it wasn't... You weren't able to generalize that to other species or anything like that.

0:24:36.9 LG: Exactly, so I think that was the key and that's why I said earlier, that I think we're at an exciting place where a general framework might be emerging.

0:24:47.5 SC: Is it generally true that younger organisms are better at regenerating or things like regenerating than older ones are?

0:24:55.5 LG: Exactly. So, I think you brought up different points, but you mentioned earlier how a lot of... Some animals that can regenerate, they don't all come from the same lineages, so the opposite of that... Let me rephrase that. So infact, the more we study regeneration and in particular in the past couple of decades, the more animals we study and ask which animals regenerate, which animals don't regenerate? And well it's a striking insight from studies in the past couple of decades, is that one, like you said, Sean, the animals that can regenerate don't all come from evolutionarily close lineages, they're all scattered across the animal phylogeny. So that's one. And then two is that, in fact, the closer we look at different animals, the more we realize that actually, young animals, the young stages across animal species have greater regenerative capacity than the older ones, like the embryos obviously have an amazing ability to...

0:26:11.8 SC: [chuckle] They have to generate their whole body in the first place, yes.

0:26:13.7 LG: Exactly, that's the embryos. But then newborns, tadpole's juvenile stages have greater regenerative capacity than adults, right? Mice for instance can... Newborn mice can regrow injured hearts, but this ability is rapidly lost upon a couple of days, I think after birth.

0:26:38.4 SC: It does surprise me a little bit, again I mean, all these animals can heal when they get a wound, right? So there's some decision... I know this is true anthropomorphic, but there's some decision that evolution made to stop older animals from being able to regenerate even though, number one, the younger ones could and number two, the older ones can still heal minor or major wounds, that seems like, there has to be a reason why evolution prevents us from doing this.

0:27:06.3 LG: Right. And you keep going... You keep touching back to the speculation, which I will divert at the very end before I speculate.

0:27:14.1 SC: Yeah, okay.

0:27:15.0 LG: But I'm going to add another... A third point to the intriguing picture which is that, not only that, all of us have capacity for what we call tissue repair or maintaining tissue homeostasis with our skin cells completely are rapidly turned over, continually turn over during lifetime. We can repair minor injuries to the bones, to muscles, so we have this baseline ability to replace cells, to repair minor injury to tissues. So all... Yeah.

0:27:57.8 SC: Well, I wanted to ask more about that because, again, I'm not a biologist, but we all know that cells can divide, right? Cells can grow a little bit and divide, but I usually when am told that story, think about unicellular organisms, I don't actually know that much about the cells in my body, are typical cells in my body dividing all the time? And some of them must be dying off, otherwise I'd be growing even faster than I am. [chuckle]

0:28:27.5 LG: It varies across organs.

0:28:30.0 SC: Okay.

0:28:31.7 LG: Yeah. The cells turn over in some organs faster than others and in some other organs maybe very, very slowly. But it's pretty amazing because our skin cells, for instance... Men, can you believe this? Okay, the cells that make up our skin are replaced every two or three weeks, so that means you're a completely different person.

0:29:00.8 SC: Yeah, by the skin.

0:29:03.1 LG: Appearance cell-wise, every few weeks or so.

0:29:08.7 SC: So, how does this happen because, is it that the skin cells divide or they just die off and are replaced with stem cells or something from somewhere else?

0:29:18.7 LG: The point being that, our body has a baseline capacity to continually replace cells or to mend mild injuries, so the question is, since our young self has greater capacity to re-grow lost parts and even as adult, we continue to maintain this basal ability, physiological ability to replace cells and fix tissues, then how far can we push this?

0:29:49.0 SC: Sure, sure.

0:29:50.2 LG: How far can we stretch this ability?

0:29:53.3 SC: And I don't even know that much about the process of healing wounds, to be honest, is that mostly a process where you're introducing new healthy cells to replace the damaged ones, or does the body fix cells at that kind of level or take cells and sew them together in more functional ways?

0:30:12.5 LG: It's a mix of both.

0:30:14.0 SC: Okay.

0:30:15.0 LG: Yeah. So, a little bit of cells replacement, but primarily just covering the wound, epithelialization to cover the wound.

0:30:26.1 SC: With new cells?

0:30:28.0 LG: With new cells, yes.

0:30:29.3 SC: Right, okay. Good, sorry, we got off track, but let's go back to the regeneration here with the jellyfish. So you mentioned that there were a few factors that were important to the regeneration, the energy and the amino acids, but you didn't need to give them electrical shocks or anything like that. Like, we picture the frogs or Frankenstein or whatever shocking organs or tissues. Did you sort of go through a whole bunch of different things that you could try to do before you homed in on the ones that actually seemed to be relevant?

0:31:02.6 LG: Yes and I like to tell these stories because when you tell people that, "Oh, the regeneration can be coaxed," it seems so easy after the fact but it was actually really, really hard. [laughter] It's actually really, really hard to find the conditions that...

0:31:22.4 SC: Sure.

0:31:23.7 LG: Yeah, could make them regenerate. And so, we spent almost three years and just screen through many, many conditions, try as many molecules as we could get a hold of to just see what we can find. We have starting hypothesis, of course, we tested processes that are often implicated in studies of regeneration in for instance, salamanders and planarians and hydras, but yeah, we just tested everything that we could think of. And what we found was something that we didn't expect and that was... I guess, what makes science fun.

0:32:04.5 SC: Absolutely. Do I remember correctly also, I forget it was... You mentioned it, but I think I remember reading that low oxygen levels was helpful.

0:32:13.4 LG: That's right. That's another thing that we haven't followed up. I think we've focused so far in the nutrient factors and we tested the nutrient factors in flies and in mice, but we haven't followed up on the hypoxia, it's actually a very strong effector of regeneration. And...

0:32:35.2 SC: Which seems... Go ahead, go ahead.

0:32:37.0 LG: Oh no, please do.

0:32:37.8 SC: It seems weird to me 'cause...

0:32:38.9 LG: And we don't know why. [laughter]

0:32:41.0 SC: Yeah. Well, we don't know why. Good. I could have guessed. That's why it's good to get out on the ground floor, like, I always want to use this podcast to inspire young students to go follow up on these things. It just seems weird to me, 'cause oxygen is important for life, for animal life anyway, but I guess, if this kind of capacity does originate way back there in evolutionary time, maybe it comes from a time when oxygen was less important or even poisonous.

0:33:09.9 LG: Yes, that is actually a good idea. And I haven't thought much about that connection, honestly. And I will now.

0:33:18.6 SC: Okay, good. [laughter]

0:33:19.3 LG: But what we do know, from the three factors that promote... That we found so far that promote regeneration in the jellyfish and this includes reduced oxygen level is that, they all promote growth. Yes, so the jellyfish grows much faster under these conditions.

0:33:41.7 SC: Right. And the body builders like this leucine, so there's something about the regeneration of a limb, which is basically supercharged growth of the organism, right?

0:33:54.0 LG: Exactly. So, we don't know yet whether it's a parallel, or whether it has some causal connections, the coincidence that the two conditions are related.

0:34:05.8 SC: And then, so it makes me wonder and again, I'm gonna speculate and don't worry about giving away secrets, they're gonna appear later on, but growth has its ups and downs, right? I mean, cancer is a kind of growth that can get in the way and maybe the reason why evolution shuts off our ability to regenerate limbs is because keeping it on would open us up to other vulnerabilities, like getting cancer too easily.

0:34:28.7 LG: Exactly. So, I think you're nicely leading us to making the speculations. [laughter]

0:34:35.4 SC: Please speculate away.

0:34:37.9 LG: Yeah. So, I think that's... So, that's what I meant earlier, it's like the more we know about the process, the more we're able to form a hypothesis, a responsible hypothesis as to why animals, not all animals regenerate. And in this case, based on what we find, that simple nutrient factors, the high amount of nutrient factors can coax late regeneration to emerge, suggest that, yes, it might be some energetic trade off. So, perhaps the conditions that we require, that is permissive for regeneration is also a condition that is permissive for cancer development, for instance.

0:35:30.2 SC: Right.

0:35:30.6 LG: Of course, we don't know this for sure, but we can start thinking, making hypotheses along this direction.

0:35:35.7 SC: And then I mean, certainly we can imagine there's good evolutionary reasons why you and I stop growing at a certain age, right? Like if we just linearly grew in size, that would be bad, we'd soon... Our metabolic needs would get too large, so could there be some kind of side effect thing going on where the evolution wants us to stop growing and that process also stops us from being able to regenerate?

0:36:00.9 LG: Right, right. That's a very good point. So a couple of things. So before I get to that, I should mention that, yes, the speculations have been made, we're not the first one speculating that regeneration has a trade off with cancers, but I think that what we're finding allows us to anchor that with... Following up that hypothesis with perhaps identifying specific pathways where the trade off might occur. So, that's what makes me excited about, "Ah, okay, this is along what people have thought about, but now we can get specific pathways." And the other thing that you said is just true, it might not just be side effect, but also because of the way we grow. When we grow, we stop growing, our metabolic state actually changes from utilizing specific pathways that are focused on making materials, building blocks for making new cells, new tissues, we switch to a different pathway that allows us to make energy more efficiently. So then one question is like, is that switch, might that have something to do with decrease in our ability to regenerate?

0:37:16.1 SC: Could you go into a little bit more details about that? The audience likes a little bit of details. What are these two different ways of generating energy we're thinking of?

0:37:25.0 LG: Right. So in one metabolic pathway, that's more associated with growing state, we utilize, we breakdown food, we utilize carbohydrates using this pathway called glycolysis and this is a pathway that is intriguingly... Is also heavily utilized by cancer cells.

0:37:47.6 SC: Ah, okay.

0:37:48.1 LG: Correct, yes. And then as we grow, we switch from glycolysis heavy to another pathway of breaking down carbs that utilizes what's called the oxidative phosphorylation and this pathway is a lot more effective in making energy from every molecule of glucose. So it really is the difference between way of processing food that's focused on making building blocks, versus way of processing food that's focused on thriving energy.

0:38:21.1 SC: And for something like a human being, when does this transition happen?

0:38:24.8 LG: Oh, that's a good point. I don't know the exact transition point.

0:38:28.0 SC: But are we thinking like...

0:38:29.0 LG: Or whether it's correct.

0:38:30.8 SC: Three weeks, or 18 years, or 50 years?

0:38:35.2 LG: Well, we're still growing as teenagers.

0:38:37.4 SC: Right. Okay.

0:38:38.7 LG: So my guess is it's gradually switching, switching. I also don't know whether... How much this has been characterized...

0:38:46.1 SC: Okay.

0:38:46.7 LG: In quantitative way.

0:38:48.9 SC: I mean, both this and other podcasts I've done on biology really drive home the fact that we're not intelligently designed, we organisms, we human beings, right? Like it's a constant set of trade offs between this and that. And when you say, well, why can't I do this, there's always a reason why well, that would lead to this bad effect somewhere else. [chuckle]

0:39:09.8 LG: Yes. I like that idea, I like that point that you're making.

0:39:16.8 SC: Let me get a little bit more detailed about what we see when we see these limbs regenerating. I mean, is it like literally slow growth of layers of cells lying on top of each other just, or how similar is it to a baby growing? I mean is it a completely different kind of thing, or is it just you're remembering, you're a jellyfish, or you're a fly, is remembering what it was like to grow that limb in the first place?

0:39:44.0 LG: Right, right, right. So it is like, if you look at... If you look at it morphologically it is like growing a limb.

0:39:54.4 SC: In the first place.

0:39:54.8 LG: So that's why a lot of people often think of regeneration as recapitulation of development.

0:40:00.7 SC: I did do a podcast with Michael Levin, I don't know if you know him from Tufts.

0:40:04.9 LG: Oh, yes. Yes. And I'm a big fan of his.

0:40:07.8 SC: Yeah. He has wonderful ideas about how the organism kinda knows what shape it wants to be and sort of moves its face in the right direction. And I guess that's a trade off, because when you say... I'm making this up and Michael's not here to correct me, but when you say the organism knows what it wants to be, it doesn't wanna be symmetric like that... And that's a fact that the jellyfish had and they sort of fix that when they lost a limb just by rearranging the other ones or it doesn't wanna have eight limbs, in which case, you wanna regenerate. So there're sort of different ways to fulfill that goal of being what you wanna be.

0:40:44.2 LG: Right. And I'm gonna speculate even more.

0:40:46.6 SC: Please.

0:40:47.2 LG: Which is that, well, first of all, it's still a question how we know the shape that we'll eventually become.

0:40:57.4 SC: Yeah.

0:40:58.4 LG: Right. And in regeneration specifically, I think it's a different end of the spectrum of questions. There's how to kickstart regeneration.

0:41:09.7 SC: Right.

0:41:10.4 LG: Which is what we're interested in and I think Michael is also interested in that as well, in his work in frogs. And then, how does it know when to stop? Because if you don't know... [laughter]

0:41:22.5 SC: Right.

0:41:23.4 LG: Which is this question, is like it knows the shape that it wants, but how does it know? How does it know? Because if you don't know how to stop, then you have uncontrolled growth? And that is, of course, cancer.

0:41:36.4 SC: Yeah. No, I mean, actually, it's a very good way to put it. You're clarifying it to me, because I feel a little bit insulted that my body isn't smart enough to regrow a limb if I lose it. But on the other hand, I don't want my existing limbs to just grow to be 20 feet long. I want them to grow to the right length and then stop. And so it's not that hard to imagine that the same thing that stops my actual arms from growing, stops me from growing new ones when I lose this one.

0:42:01.4 LG: Yeah. Yes, exactly. And I think that question of how does the organism know the shape that it wants to be or doesn't want to be in a certain shape to begin with? That is a fascinating question in itself.

0:42:16.4 SC: That's a big picture question, yes. Maybe we can get more clues. So we have the jellyfish but the flies and the mice... Somehow this is my human parochialism, it seems more astonishing to me that you can get them to regrow limbs. Jellyfish it's like, it's jelly, how hard can it be? [laughter] But is the thing that regrows on the flies and the mice, are they just as functional, or is it a stunted little thing?

0:42:46.2 LG: Right, right, right. So first of all, yes. We tend to think that humans are more complex than other animals. [laughter] A lot of us often like to pose this questions like, "Oh, how true is that statement? Are we really that much more complex than a bacterium for instance?" Bacteria are pretty fascinating too and can like we know, some of them can upend our whole lives. [laughter]

0:43:13.5 SC: Yeah. Yeah, yeah, yeah.

0:43:16.3 LG: But yes. So, to answer your question, is it functional in jelly so we can make movies and we can show that yes, they can pulse synchronously the new appendages, pulse synchronously with the existing arms? Same thing in flies too. We can make little videos and we know that they work.

0:43:34.1 SC: Okay. [laughter]

0:43:34.6 LG: With a little imperfect limb that they're regrowing. So in fact, that we're considering some... We're trying to test whether the walking is necessary to coax further regrowth, the use, using it stimulates just like our muscles, how we lose them from disuse for instance. And then...

0:43:56.7 SC: Yeah and the audience, you don't know, but Lea is mimicking a fly walking around. So just imagine in your head, a little fly squirming around.

[laughter]

0:44:05.6 LG: And in mice, we haven't done... We're still trying to... We've done bone staining the way we assessed regrowth in mice, not only by taking photograph of the digit we can see the nail tissues are regrown. But we also can stain the bones and we can show that here's where the amputation is and there's new bones being grown from the amputation plane. And then we're still trying to think of a design, maybe a climbing design where we can test how well they use the new digit segment that's regrown. So that's part of the fun to state.

0:44:46.2 SC: So if a known biologist such as myself were to see one of your flies that had regrown a limb, would I be able to tell that it regrown a limb or would it look just like a regular fly?

0:44:58.1 LG: Yes and no. One is that because it's not perfect so you know that that leg has been amputated.

0:45:02.4 SC: Okay.

0:45:02.5 LG: In fact, the fly is really challenging for us because we have not been able to fully regrow the limb. In mice we've... Actually more dramatic in mice than in flies. But it's pretty dramatic too in flies in the sense that it is partial still, but it's multi segments. So we can grow multiple segments of the legs.

0:45:23.6 SC: Oh, I see. I see.

0:45:24.9 LG: Yeah. Or we in the sense that the flies can grow multiple segments of the limb.

0:45:28.6 SC: The flies do it. This is part of the wonderful thing here, is that you're not training it. You're just putting it in the right situation, it knows what it wants to do, right?

0:45:37.3 LG: Yes. Yes. Yes. I think it's like... That's a good point. In the sense that, when we all started this is... A lot of talented students are involved in this project... Also courageous, [laughter] 'cause I told them that, "Hey, [chuckle] let's try this." And like I said, there is no guarantee it could happen. And the way we currently imagine of how we will eventually coax limb regeneration in human, some people dare to imagine, like you said, it almost sounds like science fiction, but the way we currently imagine that is that we're going to have to mimic development, reconstitute development, give a certain... Specific signals at different times, specific combinations of signals, maybe form a gradient so it's really controlling the dose, the timing, the different types of molecules that are administered at different times, combined with some biophysical stimuli, perhaps electrical as well as mechanical stimuli. So it's a very complex process that we're imagining. So part of the surprise here is that, ah, first of all, [laughter] the latent ability is there and it gets a simple tilt, can start... Can coax the process to start. So that brings just a little bit more hope too perhaps.

0:47:04.6 SC: Right. So that process that you just... That sort of complicated procedure you just outlined is, you're saying that's what we might have guessed would be required, but in fact, the body knows what limb it's supposed to have and it's more a matter of coaxing it to take advantage... Not take advantage even, but to realize that goal that it's had all along and the body will figure out what to do at what stage in the process?

0:47:31.5 LG: Yes, that's what I'm optimistically excited about.

0:47:35.0 SC: Optimistic, yeah. [laughter] But how... We can speculate here. It's late in the podcast, how close are we? I mean, mice seem pretty close. And you said that the mice did it a little bit better than even the flies did. I mean, what are the steps in between here and getting to people?

0:48:00.3 LG: Ah, [laughter] that's always something that one has to be... A biologist has to be really cautious about. [laughter]

0:48:05.8 SC: Sure. I won't tell anyone. [laughter]

0:48:10.2 LG: But I think the optimism serves just to motivate the experiments. We do the experiments, all right? So that's what's in defense of optimism.

0:48:19.7 SC: Yeah.

0:48:20.2 LG: But I think it will take a lot of efforts, a lot of courageous students. I want to emphasize right now, our model is the mouse digit. It's a digit.

0:48:30.4 SC: Right. One digit, one thinker, yeah. [laughter]

0:48:32.2 LG: One thing at a time. [laughter] And it's a digit because it's a gentler amputations. We don't want to... Without stronger hypothesis, we don't wanna start experimenting on limb directly, right? But that's the hope, is that now what we have is, like I said, a toe-hold into the problem and now we can start understanding the mechanisms. If we understand the mechanisms, then we can engineer it to be better. That's the hope. And if we're lucky, then maybe we can make leaps ahead, but...

0:49:06.2 SC: Right. But what would the steps be? I'm not saying they're gonna be possible, they might not work, it might never happen for human beings.

0:49:13.5 LG: I see.

0:49:14.4 SC: But if you get better at the mice, do we then move on to chimps, or is it some intermediate stage where it's like half prosthesis and half growing a little bit? I don't know. What do you imagine? None of this is real, so we're not under any obligation to predict correctly.

0:49:33.6 LG: Right. So I think a couple of things. One is that limb, in some way, is a great model because ironically, the procedure to test it is simpler than, let's say, resecting an organ and also the outcome is extreme and clear. So that's why we start with the limb, right? Ironically is simpler [chuckle] to assess. So how do I envision getting there if we can get there? One is that at the moment... There are several time scales here. At the moment, limb, like I said, is simpler, but also it's a great model, because being able to regrow a limb represents the complex challenge of regrowing complex organs, so it needs generating many new cells, patterning the cells in space and time and then building tissues and the new cells. But a limb has multiple tissues, so if you know how to regrow limb, know how to enhance regrowth of muscle, bone, joint, etcetera, so all those can already have, optimistically speaking, perhaps usefulness. So in the intermediate scale, maybe we're not regrowing a full limb, but what we learn from regrowing a limb can help us with enhancing the regeneration of multiple tissues.

0:51:05.7 LG: And then, like you said, well, if we want to know the mechanism, because then we can approach this in a more rational design, manner, that's the dream in biology, is like to make it more like physics, where it's more theory-driven rather than observation-driven, where we are right now. And then once we have that, the ability to predict, right? So ideally you would have a mathematical model coupled with mechanistic understanding of the process, then I think that would be empowering to go to the more different...

0:51:47.1 SC: I see.

0:51:47.7 LG: We have to test it in two or three more species before humans.

0:51:53.9 SC: But you're raising a very good point, which I would have figured out if I were a biologist, which is that, of course, our imaginations go to something dramatic, like regrowing a limb to someone who's lost a limb, but there's a lot of potential cases where there are wounded or damaged or dying organs in our bodies or something like that and that might eventually be an even sooner realistic use for this kind of process, yeah?

0:52:22.2 LG: Yes, that's right. So what we're studying now is like... Every contribution is just... I think it's just a very exciting feel, the feel of regenerative medicine. And it's also... Basic biology-wise, it's like, it's amazing of how much we can push our body to do. [laughter] But like you said... I also wanna... Another thing that I find really exciting is the fact that all right... So our assay's the limb, but then we administer leucine or insulin or sugar through diet, the animals eat them.

0:52:58.3 SC: Okay. That's pretty easy, pretty simple.

0:53:01.5 LG: Pretty simple, in a way. And so the idea is like it's systemic, the factors...

0:53:08.7 SC: Are all over.

0:53:09.8 LG: Are ingested systemically, the effects so far that we have assayed is very specific, the limb, but then the question that I'm excited about is like, well, actually, how systemic is the effect?

0:53:22.6 SC: Ah. [laughter]

0:53:23.1 LG: Generally. [chuckle]

0:53:24.9 SC: No, I mean, that's a very, very good point. So now I'm thinking about it. So because when... It's a different visualization when it's a jellyfish versus a mouse, so for this mouse, it's regrowing a digit, but the things you're doing to it you're doing to its whole body. [laughter] And so, on the one hand, you worry, or maybe you're hopeful that it's fixing everything in its body or just growing in crazy ways. But on the other hand, is it, it seems to be smart enough to know where the lost digit is and concentrate its efforts on growing that back? Is that right?

0:53:58.7 LG: Exactly. That's right. So on a practical level, if you think of translation, it's worrying because well, you don't wanna affect the whole body. So maybe you will have to design something more specific. But biology-wise, it's like, it's fascinating that you tweak the whole body and yet it knows specifically to... Yeah, repairs.

0:54:22.7 SC: I mean, I think overall, again, I'm not constrained by the standards or ethics of being a biologist, so it seems very optimistic to me what you're saying is that the body has enormous latent capacity to fix itself, as long as we give it the right raw ingredients. So we don't need to go in there with a scalpel and a micrometer or whatever and carefully fix it cell by cell, just give it food and stand back and the body will fix itself.

0:54:51.7 LG: Yes. That's actually a very good way of saying what we found.

0:55:00.3 SC: Yeah, optimistic sales fit.

0:55:01.7 LG: For me that's the most... I think that it's not optimistic, I think that it really is what we're seeing along with what the field is... Also, other studies in the field are beginning to see as well. So it's, I think that's just an amazing insight in my mind that... And especially that the same thing works across multiple species, which means that if you look out your windows, it's just whatever animals you're seeing, the birds, the squirrels, the beetles, we all have this amazing ability to repair our body, regrow a limb or any body parts, you just need a little tilt to...

0:55:40.9 SC: And this relates to something that we were hinting at before, maybe you've already said it out loud. But the potential existence of some kind of ancestral ability here, something that arose, this ability arose way back when in the evolutionary tree like maybe when multi-cellularity arose, right? When we first started differentiating organs of our body in the first place.

0:56:05.6 LG: Right. That's right. So right, like, yeah, so we discussed earlier that there is two differing opinions, historically more biologists that some feel that or think that the ability to regenerate is something adaptive, so you evolve it specifically for specific body parts, right? But then some other biologists think that it's something inherent in all of us, just like cell dividing is something inherent that the cell does. So we don't know which one is right. But if all animals have it, then it raises the possibility there is something ancestral in animals. And if it's something ancestral, maybe it's really tying in to the very basic property of biological cells, the fighting, growing.

0:57:00.1 SC: It does seem... Right, right. I'm gonna show my ignorance once again. But plants and animals seem very different in this way. I mean, to me, when I look at a tree with all of its branches, I don't think that the specific locations of all those branches were built into its DNA, right? There were some accidents of history that made that happen. Whereas when I look at a person with two arms and two legs, I think that was built into the DNA, am I right or wrong to think that plants are sort of more plastic in this way and animals are a little bit more rigid in their design?

0:57:32.8 LG: Right. Yes, I think so. Because the shape of an animal is more... The way I think about it, is more closely tied to the functions.

0:57:44.2 SC: Yeah, more differentiated in functions.

0:57:45.0 LG: Exactly, yeah. Or like specific shapes of animals. That's right. But deep regulation between growth and repair could be conserved at that deep level.

0:57:57.7 SC: Okay. That makes sense. I guess another... As we're winding down here, another big potential implication for this. I mean, we started with regeneration, which is already pretty big. But other regenerative healing is fascinating. There's a whole bunch of other things that go wrong with our bodies like Alzheimer's disease or just aging in general. These are also situations where our bodies or brains sort of know what they're supposed to do and kind of give up in some way. They're like, "Yeah, we've done this for long enough. We've outlived our reproductive usefulness, we don't need to keep it up." Do these new techniques or technologies potentially open a way to extend our lifespans or our alertness lifespans?

0:58:45.1 LG: That's the hope. That's the hope. It is a very active field, aging and neurodegenerative diseases. It's hard to speculate responsibly in that direction, but I think the broader hope of regenerative medicine is exactly that, which is that if we can maintain our tissues and working conditions and fix our brains or something goes wrong, then it can improve our lifespan, it can improve our life quality, I should mention there's also trade-offs speculated between lifespan and regenerative capacities and cancer, so that's a whole new discussion, but I wonder sometimes whether these processes are tied in and so the big challenge is to identify the specific processes that...

0:59:45.7 SC: That's right.

0:59:46.6 LG: Sit at the trade-off.

0:59:47.5 SC: And part of the good news that you have is that, again, well you said this already, but it's so amazing to me I am just gonna repeat it, that you're not in there micro planning cell by cell, you're just giving some resources to the body. So the ideal utopian future for combating aging or neuro degeneration is take the right pills or the right shots or eat the right food and let the body do it itself. That's the crazy utopian hope, right?

1:00:16.1 LG: That's right, that's right. And like you said, take the right pills, that means... Are you thinking about pre-emptive regenerative treatment? Before the injury even happens we can prepare the body.

1:00:29.1 SC: Right. And the potential downside, not to end on a down note, but the potential downside is making it much more likely that we get cancer or are subject to some other catastrophic failure because it's like super charging the engine of a car, right? Like it goes faster...

1:00:43.9 LG: Yes.

1:00:43.9 SC: But it might blow up. [laughter]

1:00:46.3 LG: Exactly, that's right. So from application side of it, this is still a long path ahead but from basic biology point of view, it's fascinating that it ties in to all these different processes, for instance, sugar obviously promotes diabetes and wound healing is disrupted in diabetic state, so then what is the connection between these different...

1:01:11.9 SC: Right, sure.

1:01:12.7 LG: Processes.

1:01:13.9 SC: Let me end with this just a little bit of a meta discussion, I mean I know that I'm at Caltech, but it's a complicated place. What department are you in at Caltech? What kind of biology... What is the label we put on for this kind of thing?

1:01:29.6 LG: Well, I'm in division of Biology and Biological Engineering and that's all of us doing research in different questions in biology so we are all just one division.

1:01:45.4 SC: Okay. Yeah, I know, Caltech, we have big divisions, but then smaller departments within the division, like I'm in Physics, Math and Astronomy, but Physics within that.

1:01:53.9 LG: Yes, I think it's structured a little differently in Biology.

1:01:58.7 SC: I see. Okay.

1:01:58.9 LG: In BBE.

1:02:00.2 SC: I mean I'm thinking...

1:02:00.4 LG: Yeah in a way that people...

1:02:00.5 SC: Of students who wanna do this kind of thing, if they're looking at different places to apply, what is the kind of biology that this is?

1:02:08.9 LG: I see. It's Regeneration Biology would be the field that I associate but then we ask also evolutionary questions.

1:02:21.0 SC: Sure.

1:02:21.3 LG: And we're also approaching it somewhat from engineering point of view...

1:02:25.9 SC: Right, that is totally fine. Yeah.

1:02:29.3 LG: How can we make something happen, yes.

1:02:29.4 SC: Yes, yeah.

1:02:29.5 LG: That's right. So Bio-Engineering is another part or of sub-department that I associate with, but it really is a lot more amorphous than that.

1:02:41.3 SC: It is. And Caltech doesn't have a medical school either, but probably some other places do, if people are really interested in the potential therapeutic applications of this down the road.

1:02:51.5 LG: Right, yes. Although, if I were to position myself, I think we're still more in the basic...

1:02:55.9 SC: Basic research.

1:02:56.1 LG: Basic research side of things.

1:03:00.0 SC: That's the fun side of things to be on, I think that's a perfectly good place to be. [laughter] Alright, it's extremely exciting. I once had a friend of mine who's a biologist tell me that we are in the last generation of people who might die of old age [laughter] and some day we'll figure it all out. [laughter]

1:03:19.1 LG: See I think I'm pretty optimistic of it, but that's still a little too optimistic for me.

1:03:23.1 SC: Never.

1:03:23.5 LG: I certainly hope that it will be true.

1:03:25.7 SC: You can never be too optimistic. Lea Goentoro, thanks so much for being on the Mindscape podcast. This was great.

1:03:31.0 LG: Thank you again, Sean.

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