Frog

Science Fiction Made Real

At any given moment, researchers around the world are toiling away in labs, sciencing every single day. The process is slow. Scientists chip away at their pursuit, conducting studies that can appear to be banal to the untrained eye. It’s a rare day when a truly transformational discovery is made.

But sometimes the pieces of knowledge come together in an extraordinary way. A major piece of a seemingly-impossible scientific puzzle and the birth of a new perspective on how the body works were recently uncovered by Dr. Michael Levin, PhD; Dr. David Kaplan, PhD; and their research team at Morphoceuticals, a portfolio company of Juvenescence, working out of Tufts University in Boston, MA. The finding — and the underlying theory behind it — has life-altering potential. Their discovery may not only play a powerful role in healing from injury and disease, but reveals a new paradigm of physiology that could transcend into all areas of medicine.

Reimagine a lifetime: organ regeneration

Dr. Levin and Dr. Kaplan’s ground-breaking experiment, published in the journal Science Advances, describes how they found a way to spur the complete regeneration of hind legs in the African clawed frog (Xenopus levies.) While a few organisms like some worms and lizards, as well as tadpoles, Zebrafish, and salamanders (axolotls) can regenerate body parts, humans and other vertebrates — including adult frogs — do not. Dr. Levin’s team demonstrated that an animal who normally is not able to recreate a lost limb could, in fact, be triggered to do just that. “We showed how animals that do not normally regenerate their limbs, can be made to regenerate their limbs,” says Dr. Levin.

In frogs, as in humans, when an injury occurs, the body’s mending mechanisms quickly kick in. Healing is a complex process with multiple phases. In the immediate moments after an injury, bleeding is stopped by the constriction of blood vessels and an increase in clotting factors that plug up the wound. Then a variety of cells migrate to initiate an inflammatory process — some rush in to kill bacteria and others to remove damaged tissue. During the next phase, new tissues and blood vessels start appearing. Collagen forms and eventually scar tissue develops to protect and seal the wound.

Of course, the very action that can save life by stopping bleeding and preventing infection, also stops the possibility of the injured tissue repairing in such a way that full form and function is restored. A leg heals into a stump. Cancerous or burned tissue scars over, usually leaving a disfigured surface. Failing organs continue to operate at half-mast, and most likely continue to deteriorate. br>
In Dr. Levin and Dr. Kaplan’s experiment, something entirely different happened: the frogs grew fully functioning new legs that included bone, muscle, blood vessels, and — most unexpected — nerves. Wounds weren’t just healed: frogs could swim and jump and feel sensations in their regrown limbs. And the treatment wasn’t specific to legs — the same principles could be used elsewhere in the body. The implications for future medical practice are enormous: a diabetic might reclaim a diseased foot or leg, failing kidneys might be regrown, cancerous lungs might be replenished with healthy tissue.

One might assume that the undertaking involved complex procedures implanting and manipulating stem cells or trying to rearrange DNA by programming genes to be expressed in different ways. But Dr. Levin and his team took a surprisingly hands-off approach — they merely stimulated the process. “We showed that you don’t have to micromanage it,” he says. “In the first 24 hours after an injury, cells can make a decision: they can embark on a restoration process or they can scar. We showed that we can intervene in that decision process by providing the stimuli to kick-start the activity in the direction we want — towards regeneration. We showed that we could trigger the cells to do all kinds of stuff that we didn’t have to control — and that’s very powerful technology.”

Unlocking the cellular code

The ability to orchestrate an approach to healing that doesn’t just seal a wound, but actually repairs and renews it is noteworthy, to say the least. Dr. Levin and his team were able to do it because they are beginning to unravel how cells communicate and work together.

Much like the groundbreaking and life-saving discoveries of insulin and penicillin, this finding has the potential to rewrite medicine as we know it. One can easily imagine where this scientific research might lead: wounded warriors could regrow lost legs or arms, cancer patients could replace parts of the body obliterated by the disease, burn victims might restore healthy skin, and organs damaged by a variety of health conditions could be recreated. More importantly, these regrown body parts would operate normally.

“We know that this is physically possible in a human because it already happens in embryonic development — half of the human population — females — can regenerate a whole person out of one cell. Embryonic cells build legs and other tissues to create a complete body,” says Dr. Levin, who is the director of the Allen Discovery Center at Tufts University in Medford, MA.

While this type of renewal seems to stop once a body is formed, there remain some signs that the innate ability to execute this still lingers at later life stages. “Children up to the age of 11 can still generate their fingertips, and adult livers can partly regenerate. There's nothing impossible about that,” explains Dr. Levin. Somewhere in the physiological system the know-how to build a body is intrinsic to cells. “And there's no reason to think that that information has disappeared,” says Dr. Levin. “I think it’s latent, but that potential is definitely there. We just need to unlock it. And the key is to understand the decision making of cells and tissues: why do they build one thing rather than another at any point in time?”

THE IMPOSSIBLE MADE POSSIBLE

Dr. Levin’s team made the impossible happen by identifying key factors that could enable repair and regeneration over the default healing mode seen in typical wound closure. Lesions or lacerations in mammals that are exposed to dry air typically initiate a scarring response to protect against infection and blood loss. But that fibrotic tissue in scars also stops the possibility of regeneration. So, Dr. Levin, Dr. Kaplan, and their team created a thin, sleeve-like device made of silicon rubber and filled with a hydrogel, called a BioDome. This cap provides an aqueous environment, thought to be a more optimal milieu to initiate healing without scarring. This enabled the cells to take the alternative path — away from wound repair and towards regeneration.

The BioDome was surgically fitted on the wound and infused with a cocktail of five potent compounds, each with specific roles designed to shift the healing into regrowth mode. Growth hormone is a well-known activator of tissue synthesis. Brain-derived neurotropic factor (BDNF) promotes nerve growth. Retinoic acid, an offshoot of vitamin A, is a morphogen, or signaling molecule, that spurs tissue, bone, and limb development. Resolvin D5, a fatty acid derivative, is anti-inflammatory. And 1,4-DPCA inhibits excess collagen deposition, which is a feature of scarring and wound closure.

The treatment was super short, but astoundingly effective: researchers exposed the leg wound to the medical-cocktail-infused BioDome for a mere 24 hours. Then the frogs were left on their own, with no further medical intervention designed to manipulate the response. Although regrowth was slow, signs of new tissue formation were seen within two weeks. And a fully- formed new leg – with blood vessels, muscles, bone, nerve cells, and all —developed by one- and one-half years. Dr. Levin’s team only provided the stimulus — the frog’s body did the rest.

Magical, mysterious science

How could such a minimalist approach yield such intricate results? Dr. Levin and team not only opened a window into how cells communicate with each other, they figured out how to communicate with the cells to coax them to do their thing. They prodded the cells, and the cells responded, kicking into high regeneration gear with no further intervention. Earlier, Dr. Levin had reasoned that cells contain an inherent ability to coordinate with each other to generate all the tissues required to create a body part. So, he and the team gave them the signal that they should.

The simplicity of this experiment —and, more importantly, the insights into how to facilitate regrowth —mirrors other major scientific discoveries. If an underlying principle is discovered, the details will sometimes work themselves out.

“If you think about it, all of science is about taking a bunch of different observations and coming up with one symmetry to them, recognizing the beauty in the fact that all those observations stem from one thing,” he says. “You can watch what happens if you throw a baseball, lob a missile, or watch an apple fall from the tree. If I’m Isaac Newton, I’m going to say, I recognize what’s underneath all this.” (Sir Isaac Newton is known for discovering the laws of gravity.) “That’s the beauty of scientific theories. They compress a ton of disparate observations into one underlying law — all these different things happening are driven by just one thing,” Dr. Levin explains.

The intrinsic collective intelligence of cells

With Dr. Levin and Dr. Kaplan’s experiment, the “thing” was that they found a way to activate the cells’ code to coordinate as a collective and rebuild the tissues. That’s how, with arguably the most minimalist intervention one can imagine, the regrowth of the frog’s legs was possible.

Scientists have traditionally identified brain cells, or neurons, as the “smart” cells in the body that direct everything that happens, and DNA is viewed as the library where all information about a body is stored. But Dr. Levin believes that cells all over the body have a collective intelligence. “I'm very interested in cognition and where minds come from, and how minds come to operate within bodies? What kind of physical structure do you require to have memories and communicate? This is what I think about all the time,” says Dr. Levin. “It’s not just the neural networks of cells that get to do certain things. I think you can find some degree of intelligence in very unfamiliar guises.” In other words, Dr. Levin posits that all cells, and especially groups of cells, are smarter than has previously been recognized.

Although the theory has been debunked, scientists are often accused of being “left-brained,” or stuck in a mode that’s high in logic and math but closed to creativity and intuitive open-mindedness. But Dr. Levin most surely is not. Followers on his Twitter feed (@DrMichaelLevin) get glimpses at how his brain ticks. He regularly posts about books that he has read that currently reside in his vast wall-to-wall, floor-to-ceiling home library. Some recent titles: Memories and the Flow of Time, Mental Reality and Intentionality – An Essay in the Philosophy of Mind.

Despite his forays into the loftier and fuzzy aspects of existence, Dr. Levin is quick to emphasize that there is a distinction between consciousness and cognition — he calls consciousness a “slippery beast.” But he believes that investigating how these concepts might apply to cells is an important driver of how to approach regenerative medicine. “This idea of cognition and the idea that there’s intelligence, all the way down to the sub-cellular level, I take that very seriously,” he says. “What do cells and the tissues know? What do they expect? What do they learn? What preferences do they have?”

Drs. Kaplan and Levin’s backgrounds also contributed to their out-of-the-scientific-box thinking. Dr. Levin has dual degrees in computer science and biology from Tufts and Harvard Universities and worked as a software engineer. Dr. Kaplan is the chair of biomedical engineering at Tufts University and has published over 900 peer-reviewed studies.

Many people assume that how the body works all boils down to genes and DNA. But it goes beyond that, according to Dr. Levin. He’s likened how cells operate to computer hardware and software. “The genome specifies the micro hardware of every cell. It tells you what proteins each cell has to play with, which determines the cellular hardware that can exploit the laws of physics and computation to do remarkable things,” he says. But it’s the software is what makes things work. And Dr. Levin says it’s possible to get a glimpse of cellular software by looking at bioelectrical signals.

Trying to orchestrate changes in the body by manipulating DNA is not an effective path, according to Dr. Levin. “Do you want to be trying to tell millions of cells which of their 1000s of genes they need to turn on and off at any given moment? Wouldn’t it be much more efficient to exploit the high-level control signals, the way we train a rat to perform a trick, rather than playing all of its neurons like a puppet on strings?” he says.

Dr. Levin and team have identified that all cells in the body communicate with each other via electrical signals. In what seems like a scientific Avatar-remake, Dr. Levin and team have started to map out actual conversations between cells as reflecting the computations of a tissue-level collective intelligence. They have started tracking these cellular chats which seem to be conducted through electrical signals. “We're going to need to crack the bioelectric code,” he says. “So that means we need to understand what kind of electrical patterns mean what to the system.” Similar to how brain researchers have used MRIs and other instruments to measure electrical activity and waves in neurons, Dr. Levin’s team has recorded slow electrical patterns between cells all over the body that constantly change and seem to contain messages.

Learning the language of cells may be the holy grail of being able to trigger the body to perform different actions. “How do we communicate to groups of cells what they should be doing? We want to be able to specify what outcome we want and try to facilitate them to reach it,” he says. He describes these electrical patterns as cellular conversations. “What we need to do is read and write electrical pattern memories into the collective intelligence of the cells,” he explains. “We're going to need to figure out which electrical states correspond to specific things like ‘make an eye’ or ‘make a limb’ or ‘I want four fingers, or five fingers — not 2 or 17’.”

This electrical communication between cells is not the same as physical electricity. So, interacting with these signals doesn’t mean doling out electric shocks, an assumption that some people make when they hear about “electric signaling.” In cells, bioelectrical signals are conducted in the fluid environment of cellular tissue, through ion channels.

Dr. Levin says that to intervene in these electrical conversations, ion channels in various tissues need to be identified and then opened or closed, probably with what are known as “ion channel” drugs (many of which are already approved for human use for other medical reasons). Drs. Levin and Kaplan’s experiment represents the first foray into this type of medical intervention. And what’s fascinating is that this type of medical treatment typically would not require surgery. “If someone got injured and lost their limb a long time ago, and now have a stump, you may need to open the stump so the wound is fresh if you want to regenerate,” says Dr. Levin. “But this approach would not necessarily require deep local application to make sure that the drug is right where it needs to be. It can be a global — systemic — approach, as long as you're targeting the right ion channels.”

And the possibilities are turning science fiction into fact. Not only might damaged cells, and tissues be replaced, but whole organs and body parts. Perhaps aging could be stalled as new tissues are regenerated to replace those that are wearing out.

The laws of regeneration

Dr. Levin’s approach stems from stepping out of the weeds to gain insights by taking a macro perspective. “When I talk to my students, I remind them to look at physics in the late 1800s and early 1900s. People were trying to figure out how steam engines work. It became clear that we were not going to be able to follow every molecule of gas and figure out what happens to it and then direct each molecule. So, developed thermodynamics — a science of order and disorder,” says Dr. Levin. “Understanding this didn’t just help them learn how to make better steam engines, it helped them understand how things grind to a halt, and even how the universe was formed. This revolutionized physics in a deep way, impacting almost every part of that subject.”

Traditional, reductionist approaches to science entail identifying every gene, molecule, every cell, every enzyme, different body compounds, and the like and manipulating them in endless ways to see what happens. “Imagine if, at that time, people started to get the idea that we actually could track every molecule of this gas. If they believed that, then we might have missed this really beautiful theory of thermodynamics because we would have been paying attention to the details and not trying to abstract the laws underneath, like entropy” says Dr. Levin. “I sometimes feel like biology is in danger of that, where people are so excited about tracking the details — because now it's starting to look like you might be able to track every single protein in the cell and every gene expression at every moment — but then you make these gigantic datasets at the expense of the deeper insight.”

Dr. Levin adds, “Of course, reductionism sometimes gets a bad rap in the sense that, if, if you don't try the reductionist version first, you end up not being able to say anything, because you're overwhelmed with the complexity. Whereas somebody who takes a super simplistic view says, I'm going to isolate this thing into pieces and see how far we can get looking at what each piece is doing. That is an essential part of scientific progress.” But delving into the minutiae has its limitations. “Inevitably, they come to a point where you've picked all the low-hanging fruit of what the individual things do. Now, you've got to understand the system as a whole. But they got us to that point in the first place. So, I feel like reductionism is incomplete, but it's where you have to start. Otherwise, you never get off the ground — that’s what the history of science tells us.”

Drs. Levin and Kaplan have joined with Juvenescence Ltd to form Morphoceuticals Inc., a research company whose mission is to elucidate the underlying biophysical mechanisms that contribute to limb regeneration and to use bioelectric dynamics to spur the regrowth of limbs and other tissues in organisms who do not normally do so. “In biology, we need to understand the deep laws of form and morphogenesis, even though a lot of technology is moving in the direction of learning more and more about the particulars,” says Dr. Levin. Drs Levin and Kaplan have revealed that cells communicate and that their language can be parsed out through a capturable pattern of electrical waves. In their leg regeneration study, they’ve demonstrated that it’s possible to intervene in this system by providing a stimulus that communicates with the cells and activates them to work together to fix the problem.

Dr. Levin recently shared a quote from German-American poet and novelist, Charles Bukowski, on Twitter. “The way to create art is to burn and destroy ordinary concepts and to substitute them with new truths that run down from the top of the head and out of the heart.” He added: “Some science too – burn and destroy ordinary concepts.”

Magical, mysterious science

How could such a minimalist approach yield such intricate results? Dr. Levin and team not only opened a window into how cells communicate with each other, they figured out how to communicate with the cells to coax them to do their thing. They prodded the cells, and the cells responded, kicking into high regeneration gear with no further intervention. Earlier, Dr. Levin had reasoned that cells contain an inherent ability to coordinate with each other to generate all the tissues required to create a body part. So, he and the team gave them the signal that they should.

The simplicity of this experiment —and, more importantly, the insights into how to facilitate regrowth —mirrors other major scientific discoveries. If an underlying principle is discovered, the details will sometimes work themselves out.

“If you think about it, all of science is about taking a bunch of different observations and coming up with one symmetry to them, recognizing the beauty in the fact that all those observations stem from one thing,” he says. “You can watch what happens if you throw a baseball, lob a missile, or watch an apple fall from the tree. If I’m Isaac Newton, I’m going to say, I recognize what’s underneath all this.” (Sir Isaac Newton is known for discovering the laws of gravity.) “That’s the beauty of scientific theories. They compress a ton of disparate observations into one underlying law — all these different things happening are driven by just one thing,” Dr. Levin explains.

The intrinsic collective intelligence of cells

With Dr. Levin and Dr. Kaplan’s experiment, the “thing” was that they found a way to activate the cells’ code to coordinate as a collective and rebuild the tissues. That’s how, with arguably the most minimalist intervention one can imagine, the regrowth of the frog’s legs was possible.

Scientists have traditionally identified brain cells, or neurons, as the “smart” cells in the body that direct everything that happens, and DNA is viewed as the library where all information about a body is stored. But Dr. Levin believes that cells all over the body have a collective intelligence. “I'm very interested in cognition and where minds come from, and how minds come to operate within bodies? What kind of physical structure do you require to have memories and communicate? This is what I think about all the time,” says Dr. Levin. “It’s not just the neural networks of cells that get to do certain things. I think you can find some degree of intelligence in very unfamiliar guises.” In other words, Dr. Levin posits that all cells, and especially groups of cells, are smarter than has previously been recognized.

Although the theory has been debunked, scientists are often accused of being “left-brained,” or stuck in a mode that’s high in logic and math but closed to creativity and intuitive open-mindedness. But Dr. Levin most surely is not. Followers on his Twitter feed (@DrMichaelLevin) get glimpses at how his brain ticks. He regularly posts about books that he has read that currently reside in his vast wall-to-wall, floor-to-ceiling home library. Some recent titles: Memories and the Flow of Time, Mental Reality and Intentionality – An Essay in the Philosophy of Mind.

Despite his forays into the loftier and fuzzy aspects of existence, Dr. Levin is quick to emphasize that there is a distinction between consciousness and cognition — he calls consciousness a “slippery beast.” But he believes that investigating how these concepts might apply to cells is an important driver of how to approach regenerative medicine. “This idea of cognition and the idea that there’s intelligence, all the way down to the sub-cellular level, I take that very seriously,” he says. “What do cells and the tissues know? What do they expect? What do they learn? What preferences do they have?”

Drs. Kaplan and Levin’s backgrounds also contributed to their out-of-the-scientific-box thinking. Dr. Levin has dual degrees in computer science and biology from Tufts and Harvard Universities and worked as a software engineer. Dr. Kaplan is the chair of biomedical engineering at Tufts University and has published over 900 peer-reviewed studies.

Many people assume that how the body works all boils down to genes and DNA. But it goes beyond that, according to Dr. Levin. He’s likened how cells operate to computer hardware and software. “The genome specifies the micro hardware of every cell. It tells you what proteins each cell has to play with, which determines the cellular hardware that can exploit the laws of physics and computation to do remarkable things,” he says. But it’s the software is what makes things work. And Dr. Levin says it’s possible to get a glimpse of cellular software by looking at bioelectrical signals.

Trying to orchestrate changes in the body by manipulating DNA is not an effective path, according to Dr. Levin. “Do you want to be trying to tell millions of cells which of their 1000s of genes they need to turn on and off at any given moment? Wouldn’t it be much more efficient to exploit the high-level control signals, the way we train a rat to perform a trick, rather than playing all of its neurons like a puppet on strings?” he says.

Dr. Levin and team have identified that all cells in the body communicate with each other via electrical signals. In what seems like a scientific Avatar-remake, Dr. Levin and team have started to map out actual conversations between cells as reflecting the computations of a tissue-level collective intelligence. They have started tracking these cellular chats which seem to be conducted through electrical signals. “We're going to need to crack the bioelectric code,” he says. “So that means we need to understand what kind of electrical patterns mean what to the system.” Similar to how brain researchers have used MRIs and other instruments to measure electrical activity and waves in neurons, Dr. Levin’s team has recorded slow electrical patterns between cells all over the body that constantly change and seem to contain messages.

Learning the language of cells may be the holy grail of being able to trigger the body to perform different actions. “How do we communicate to groups of cells what they should be doing? We want to be able to specify what outcome we want and try to facilitate them to reach it,” he says. He describes these electrical patterns as cellular conversations. “What we need to do is read and write electrical pattern memories into the collective intelligence of the cells,” he explains. “We're going to need to figure out which electrical states correspond to specific things like ‘make an eye’ or ‘make a limb’ or ‘I want four fingers, or five fingers — not 2 or 17’.”

This electrical communication between cells is not the same as physical electricity. So, interacting with these signals doesn’t mean doling out electric shocks, an assumption that some people make when they hear about “electric signaling.” In cells, bioelectrical signals are conducted in the fluid environment of cellular tissue, through ion channels.

Dr. Levin says that to intervene in these electrical conversations, ion channels in various tissues need to be identified and then opened or closed, probably with what are known as “ion channel” drugs (many of which are already approved for human use for other medical reasons). Drs. Levin and Kaplan’s experiment represents the first foray into this type of medical intervention. And what’s fascinating is that this type of medical treatment typically would not require surgery. “If someone got injured and lost their limb a long time ago, and now have a stump, you may need to open the stump so the wound is fresh if you want to regenerate,” says Dr. Levin. “But this approach would not necessarily require deep local application to make sure that the drug is right where it needs to be. It can be a global — systemic — approach, as long as you're targeting the right ion channels.”

And the possibilities are turning science fiction into fact. Not only might damaged cells, and tissues be replaced, but whole organs and body parts. Perhaps aging could be stalled as new tissues are regenerated to replace those that are wearing out.

The laws of regeneration

Dr. Levin’s approach stems from stepping out of the weeds to gain insights by taking a macro perspective. “When I talk to my students, I remind them to look at physics in the late 1800s and early 1900s. People were trying to figure out how steam engines work. It became clear that we were not going to be able to follow every molecule of gas and figure out what happens to it and then direct each molecule. So, developed thermodynamics — a science of order and disorder,” says Dr. Levin. “Understanding this didn’t just help them learn how to make better steam engines, it helped them understand how things grind to a halt, and even how the universe was formed. This revolutionized physics in a deep way, impacting almost every part of that subject.”

Traditional, reductionist approaches to science entail identifying every gene, molecule, every cell, every enzyme, different body compounds, and the like and manipulating them in endless ways to see what happens. “Imagine if, at that time, people started to get the idea that we actually could track every molecule of this gas. If they believed that, then we might have missed this really beautiful theory of thermodynamics because we would have been paying attention to the details and not trying to abstract the laws underneath, like entropy” says Dr. Levin. “I sometimes feel like biology is in danger of that, where people are so excited about tracking the details — because now it's starting to look like you might be able to track every single protein in the cell and every gene expression at every moment — but then you make these gigantic datasets at the expense of the deeper insight.”

Dr. Levin adds, “Of course, reductionism sometimes gets a bad rap in the sense that, if, if you don't try the reductionist version first, you end up not being able to say anything, because you're overwhelmed with the complexity. Whereas somebody who takes a super simplistic view says, I'm going to isolate this thing into pieces and see how far we can get looking at what each piece is doing. That is an essential part of scientific progress.” But delving into the minutiae has its limitations. “Inevitably, they come to a point where you've picked all the low-hanging fruit of what the individual things do. Now, you've got to understand the system as a whole. But they got us to that point in the first place. So, I feel like reductionism is incomplete, but it's where you have to start. Otherwise, you never get off the ground — that’s what the history of science tells us.”

Drs. Levin and Kaplan have joined with Juvenescence Ltd to form Morphoceuticals Inc., a research company whose mission is to elucidate the underlying biophysical mechanisms that contribute to limb regeneration and to use bioelectric dynamics to spur the regrowth of limbs and other tissues in organisms who do not normally do so. “In biology, we need to understand the deep laws of form and morphogenesis, even though a lot of technology is moving in the direction of learning more and more about the particulars,” says Dr. Levin. Drs Levin and Kaplan have revealed that cells communicate and that their language can be parsed out through a capturable pattern of electrical waves. In their leg regeneration study, they’ve demonstrated that it’s possible to intervene in this system by providing a stimulus that communicates with the cells and activates them to work together to fix the problem.

Dr. Levin recently shared a quote from German-American poet and novelist, Charles Bukowski, on Twitter. “The way to create art is to burn and destroy ordinary concepts and to substitute them with new truths that run down from the top of the head and out of the heart.” He added: “Some science too – burn and destroy ordinary concepts.”

REGENERATION

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