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A Progesterone-Pumping Device Helps Frogs Regenerate Lost Limbs

Twenty-four hours of the hormone progesterone can kickstart over nine months of limb regeneration in amphibian amputees.

ByKatherine J. WuNOVA NextNOVA Next
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Adult African clawed frogs (Xenopus laevis) usually develop spike-like structures at sites of limb amputation. But researchers can now equip these frogs with bioreactors that help them partially regenerate their hind legs. Photo Credit: TimVickers, Wikimedia Commons

Somewhere along our evolutionary trajectory, we humans lost our regenerative powers. Sure, we can manage a hunk of liver here, the tip of a finger there—but in contrast to the salamanders, flatworms, and sea stars of the world, the spontaneous remaking of entire organs and limbs eludes humankind.

For biologist Michael Levin of Tufts University, however, “lost” isn’t the same as “nonexistent.”

Only a few million years separate humans from our closest regenerating relatives. If our animal ancestors built bone and tissue from scratch throughout adulthood, these superpowers may still lie buried within our own tissues, waiting to be reawakened. All it would take, says Levin, is the right signal.

Now, Levin and his colleagues may have taken the next steps in pinpointing that very trigger by reviving these clandestine capabilities in African clawed frogs. Their work, published today in the journal Cell Reports, kick-started the growth of partial but high-functioning hind legs in amphibian amputees after 24 hours of hormonal treatment. Though the work is still preliminary, such findings may someday inform researchers on the path to finding—and flipping—a similar switch in humans.

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Cut the tail off a tadpole, and another will take its place. But even in the amphibian world, there are sobering consequences to growing up: If a mature frog loses a leg, the best it can hope for is a frail cartilaginous spike to take its place. This ability is technically regenerative—certainly far more than what any human could accomplish—but the resulting appendage does little for a creature that relies heavily on its hind limbs to navigate its aquatic environment.

As the frog ages, its regenerative skills seem to go into hibernation—making it “a good stepping stone between animals that [fully] regenerate, like salamanders, and animals that don’t, like mammals,” Levin explains.

To rouse regeneration from its slumber, the researchers first needed to identify the right molecular alarm. The ideal candidate, they reasoned, would be a powerful chemical cue that is both naturally produced by animal bodies and capable of directing a vast array of cellular functions—a winning combination that, for the researchers, pointed to progesterone.

Every month, as a natural part of the female menstrual cycle, progesterone regenerates the entire endometrium, or lining of the uterus. “We thought, ‘It’s impossible that progesterone is only acting at this level,’” explains lead author Celia Herrera-Rincon, a neuroscientist at Tufts University.

Progesterone wields far more power than simply preparing the uterus for pregnancy. Receptors for this hormone exist all over the bodies of both male and female mammals, and in the past several decades, researchers have shown it to be a potent catalyst for wound healing, promoting nerve repair, bone remodeling, and blood vessel formation. If applied in the right place at the right time, progesterone could very well coax an injured limb onto a regenerative cascade.

“Progesterone is very useful and safe,” explains Régine Sitruk-Ware, a reproductive endocrinologist at the Population Council’s Center for Biomedical Research who did not participate in the new research. “It’s helpful for the uterus, but in many other layers of the body—in vessels, in bone, in the brain, and in the [nervous system] in general—progesterone has a positive, regenerative aspect.”

To harness the power of progesterone, the researchers designed a silicon apparatus filled with a hydrating gel and stitched it onto African clawed frogs (Xenopus laevis) whose right hind legs had been amputated three hours prior. Some of the devices also contained a dose of progesterone, which was steadily fed to the wound site for 24 hours. When the day was up, the team carefully detached the device—and waited.

Within a matter of weeks, the progesterone-treated frogs had a serious leg up on their peers. In the absence of the hormone, frog amputees produced the typical featureless rods, regardless of whether or not they’d been fitted with the apparatus. But the frogs hit with progesterone began to sprout bigger, more structured appendages. And nearly ten months out from the initial spark of progesterone, partial, paddle-like limbs had pushed their way out of the wound site.

When the researchers inspected the burgeoning limbs more closely, they found that, unlike the simple, cartilaginous spikes of typical amputees, they seemed to have the form and patterning of true legs. The limbs were thick and full of new blood vessels, nerves, and bone, and were forming what looked like tentative knees: the beginnings of actual joints. These frogs’ legs weren’t just regenerating more than their untreated counterparts—they were regenerating better. It’s a phenomenon that regenerative biologist John Barker of the Frankfurt Initiative for Regenerative Medicine praises as “smart proliferation”: the ability to form order out of the chaos of growing, dividing cells.

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Amputation of adult frogs' hind limbs usually results in the formation of a cartilaginous "spike" (left). But attaching a device that feeds progesterone to the amputation site for just 24 hours stimulates the growth of a paddle-like structure (right). The dashed line indicates the plane of amputation. Photo Credit: Celia Herrera-Rincon, Tufts University

“It was amazing that we were creating structures with only 24 hours of treatment,” Herrera-Rincon says. “I remember showing Mike [Levin] and being fascinated.”

When the frogs got the chance to stretch their legs, the untreated, spike-toting regenerates proved to be feeble swimmers and spent more time loitering in place. Meanwhile, the animals that had worn the progesterone-dosing device glided merrily by, almost indistinguishable from their unamputated kin.

Though the legs could function, the regeneration was far from complete. While the limb that appeared had clear features, the growth stopped abruptly before the frog’s hind digits would otherwise appear. And, importantly, not all the treated frogs responded to the same extent, indicating that the approach is unlikely to be a one-size-fits-all treatment. But for a first pass, Levin says, the amount of regeneration achieved was “really striking.”

“If the person grew a limb like the frogs did, no one would say it was perfect,” says Jessica Whited, a regenerative biologist at Harvard University’s Brigham Regenerative Medicine Center who frequently collaborates with Levin’s lab, but was not involved in the newest finding. “But it’s important to demonstrate something that would be better than what grows naturally.”

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Levin and his team are currently tinkering with their cocktail of apparatus-administered drugs, as well as with the duration for which the device is worn after amputation. With just a few additions and adjustments, he says, the preliminary results are already looking good.

“To rebuild any structure, you need to give the cells some signal about what they’re rebuilding,” Levin explains. “And progesterone [alone] gives an incomplete message.”

The rest of this message, Whited adds, will involve a lot of fine-tuning. When limbs grow (and regrow) naturally, they rely on a suite of complex anatomical signals that both drive the production of new tissue and bone and tell it when and where to stop. But the domino effect likely still applies: The key will be finding the right recipe of chemicals to jumpstart the process, then sit back and let nature take its course.

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Deer antlers are mammalian appendages capable of repeated rounds of regeneration. Male deer (and female reindeer) shed and sprout their antlers anew each year. Credit: USFWS Mountain-Prairie, flickr

“This isn’t ready for human trials… but it’s also not science fiction,” Levin says of the device. “I don’t think humans are barred from regeneration in any fundamental way.”

We’re not exactly a hop, skip, and a jump away from regenerating limbs in people—even partial ones. Though frogs and humans share quite a few genes and basic anatomical structures, Barker explains, we’re still fundamentally very different creatures. But, even if the packaging varies from species to species, the basic toolkit of regeneration might translate across even fairly far-flung species. There are even mammals that naturally regenerate: Deer regularly regrow their antlers well into adulthood—a process that, not-so-coincidentally, is also driven by sex hormones.

Even as technology advances, it’s unclear if humans will ever regenerate as slickly as salamanders. But maybe these able-bodied amphibians are inching us just a bit closer: One small leap for a frog may someday mean one giant step for humankind.