recipes for limb renewal

SCIENCE & TECHNOLOGY

PEOPLE WHO lose a limb to war, ac-cident, or disease can choose from a remarkable array of prosthetic replace-ments, including legs specialized for cyclists and sprinters or arms with hands that can grasp and manipulate playing cards and paring knives.

Bioengineers continue to refine pros-thetic limbs, but they still can’t replicate the entire constellation of capabilities provided by flesh and blood. So a few deter-mined scientists are pursuing a different solution: They are seeking the recipe for regrowing a missing limb.

The ability to assemble a biological limb cell by cell in a lab requires so much detailed knowledge and such advanced technical capabilities that it may be a century off, according to biologist Michael Levin, who directs the Tufts Center for Regenerative & Developmental Biology at Tufts University. “But ask-ing the host organism to build a limb is a much more achievable goal,” he

says. After all, the body already knows how to create limbs, having done so during em-bryonic development. So researchers are attempting to reactivate the body’s exist-ing biochemical programs for limb growth without micromanaging all the details of the process. Taking this shortcut “is very important for bringing this to biomedi-cal applications sooner rather than later,” Levin says.

For illustrative models, researchers in the field turn to animals that can replace body parts easily. Nonvertebrates includ-ing hydras and flatworms can grow new brains, digestive organs, nerve cords, and muscle even when huge chunks of their tis-sue are removed, Levin says.

Regenerative capabilities are rarer and much less powerful among

vertebrates. Fish can regrow fins—including skin, bone,

blood vessels, nerves, and connective tissue—as well

as kidney tissue and heart muscle. Deer routinely

sprout new antlers. And hu-mans can repair flesh wounds,

the liver, and nerves outside the brain and spinal cord, according to

Jeremy P. Brockes, who studies cellular and

molecular mechanisms that underlie regeneration at University College London.

But these fairly simple repair jobs differ considerably from “re-generating something of the com-plexity of a limb, which involves formation of the bones and the digits and the muscles and so on in a very intricate pattern,” Brockes says.

The closest that humans come to regenerating a limb is the ability of young children to regrow a finger-tip including bone, blood vessels, nerves, skin, and the fingernail.

Mice share this ability, even in adulthood, although if too much tissue is lost, neither mouse nor child can rebuild a digit.

Molecular biologist Ken Muneoka, who studies limb development and regenera-tion at Tulane University, in New Orleans, is exploring how a digit loss that can be repaired differs from one that can’t.

Working with mice, his team has discov-ered that cells release bone morphogenetic proteins (BMPs) at regeneration-capable amputation sites but not at sites that fail to regenerate. The researchers also determined that treating a normally non-regenerative amputation site with these growth factors enables the wound to grow a replacement digit. And they found that mice in different stages of development—newborn versus embryonic, for example—utilize different BMPs to promote regen-eration ( Development 2010, 137, 551). BMP production is turned on by MSX genes, whose expression increases during regen-eration of mouse digits, as well as during regeneration of tadpole tails and zebrafish fins, according to Muneoka.

An adult mouse can replace a digit tip, but the only adult vertebrate that can re-generate an entire limb is the salamander, and it completes the task in 10 weeks or less. In fact, salamanders, which include newts, “really are the champions of regen-eration among adult vertebrates,” Brockes says. “As well as the limb, they can regener-ate the tail, the jaw, ocular tissue like the lens and retina, and sections of the heart and intestine.”

AFTER A SALAMANDER loses a limb, epidermal cells move to seal the wound within about six hours. The wound epider-mis emits signaling compounds including fibroblast growth factors that attract con-nective tissue cells known as fibroblasts to the wound site, Muneoka explains. Once there, the cells revert to a less-developed state, coalescing and dividing to form a “blastema,” a mound of stemlike cells that differentiate into multiple types of cells. The blastema cells “show striking positional identity by regenerating just the structures that were removed,” Brockes says. “For example, wrist cells give rise to a hand” and not to a second forearm. Muneoka believes that Hox genes preserve this positional information in salamanders’ fibroblasts.

Blastema formation—and thus the entire process of regeneration—can suc-ceed only if the severed nerves regenerate

BODY ELECTRIC

By inducing expression of a

particular potassium ion channel in this tadpole, Levin’s team altered its bioelectrical signaling, enabling it to grow multiple arms.

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RECIPES FOR LIMB RENEWAL

Salamanders and other creatures that regrow lost body parts provide clues for ways to REGENERATE HUMAN LIMBS

SOPHIE L. ROVNER, C&EN WASHINGTON


as well, Brockes notes. A few years ago, his team discovered that both the regenerating nerves within the amputated stump and skin near the wound express a protein they dubbed newt anterior gradient (nAG). The researchers believe that the binding of nAG to the cell-surface protein Prod 1 promotes growth of appropriate cells in the stump ( Science 2007, 318, 772).

Other biochemicals that appear to help the blastema form and grow include ma-trix metalloproteinases. These enzymes may function in part by preventing scar formation, according to University of Utah neuro biologist Shannon J. Odelberg.

IT’S STILL a mystery why some animals can regenerate and others can’t, Brockes adds. Researchers don’t even know whether salamanders are unique among vertebrates in having evolved their exten-sive regenerative ability, or whether an-cestral animals possessed the ability but salamanders are unique in retaining it.

Most scientists favor this second hy-pothesis, Brockes says. They think that once they identify the factors that stop most mammals from regenerating, such as inflammation caused by immune cells or scar formation caused by connective tissue during wound healing, they can remove that block. In that way, they can ac-cess “some ancestral property, some ‘newt

within,’ that’s just waiting to be unlocked,” and reactivate regenerative capabilities, he says.

But regeneration involves the same signaling pathways, transcription factors, and other biochemical machinery used in development, tissue turnover, wound heal-ing, and other activities common to all ver-tebrates, Brockes notes. He believes that the salamander, unlike vertebrates that are incapable of regeneration, underwent evolutionary changes that modified these common pathways “to obtain a regenera-tive outcome” ( Integr. Comp. Biol., DOI: 10.1093/icb/icq022).

One example of the salamander’s repur-posing of a shared biochemical pathway involves three-finger proteins. Many dif-ferent animals utilize these proteins—which extend three fingerlike loops from a hydrophobic core—for purposes as diverse as enhancing sperm motility and instilling snake venom with toxins. But one of the three-finger proteins in salamanders is Prod 1, the protein that interacts with nAG to help orchestrate the regenerative pro-cess, Brockes says.

Further complicating the attempt to unravel regeneration is the fact that these capabilities change over the lifetime of a single organism. A tadpole, for example, can generally replace a missing tail or limb but loses this ability after its metamorpho-

sis into a frog. Likewise, higher vertebrates such as mammals can regenerate much better during their embryonic and fetal stages than after they have become adults, Muneoka says.

At least some of this loss in mam-malian regenerative capacity might be because of a change in environment after birth. “During embryonic stages, we live in a very different environment than we do as adults,” Levin says. “One of the major issues is that it’s aqueous” inside the womb. pH and oxygen exposure in the womb also differ from postnatal conditions.

Levin and his collaborator David Kap-lan, a Tufts biomedical engineer, are now testing whether a replicated amniotic environment can promote regeneration in adult mammals. Kaplan has already de-veloped a small, cylindrical “regenerative sleeve” that can be filled with an aqueous solution and fastened onto the stump of a rat’s amputated limb. The sleeve is fitted with a variety of ports and electri-cal connections so the researchers can sample and alter the container’s chemical

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“There’s a lot that we have to characterize before we can try to

manipulate the wound to cause bone regrowth or muscle regrowth.”

REGENERATION FERTILIZER A mouse can regrow an amputated digit tip, but if enough of the digit is removed—as shown in the image at left, in which the black bar indicates the line of amputation—the digit won’t grow back. However, Muneoka found that treating such an amputation site with bone morphogenetic proteins enables a mouse to grow a replacement tip (right). In these images, captured with a dissecting microscope, bones are stained red and surrounding tissue appears clear. The triangular bone on the right is 1–2 mm long.

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Maintaining the correct body plan is cru-cial for successful regeneration after an animal loses a limb to accident or disease.

But regeneration can’t even get started unless the animal can detect that its body is no longer whole, says biolo-gist Michael Levin, who directs the Tufts Center for Regenerative & Developmental Biology at Tufts University. “So an organ-ism continuously monitors its three-di-mensional shape to compare what it has now to what it’s supposed to have,” he says. “That’s a necessary process to knowing that it’s been damaged.” Blood loss, injury currents produced at the wound site, and exposure of internal cells to oxygen provide the body with indications that it has been damaged, he says.

The organism also needs to figure out how to replace the missing limb in the correct location and how to match the limb’s original shape, size, and orientation. In a salamander, “if you amputate at the wrist, the cells there somehow know to give rise to a hand,” says Jeremy P. Brockes, who studies cel-lular and molecular mechanisms that un-derlie regeneration at University College London. “If you amputate at the shoulder, they’ll give an entire arm. So there is some kind of graded property running up and down the arm which determines the identity of the structures that are going to be regenerated.”

The organism also somehow deter-

mines that it needs to “build a limb and not a tail and not a bunch of spinal cord with an eye on top of it,” Levin says. “What’s remarkable is that, for organ-isms that regenerate, they almost always regenerate exactly what they lost—no more, no less. It’s very clear that they know exactly what shape they’re sup-posed to be and they know when they’ve reached it, and then they stop growing,” he adds. “We’re studying the mecha-

nisms that underlie the storage and processing of that patterning informa-tion, that 3-D structure.”

In recent work, Levin’s team showed that it’s pos-sible to permanently reset the target pattern to which regeneration rebuilds after injury. By interfering with the normal signals provided by the central nervous system and by plasma membrane chan-

nel proteins known as innexins, the re-searchers induced amputated flatworms to grow extra heads out of their sides or out of what had originally been their tail ends ( Dev. Biol. 2010, 339, 188).

The same patterning information that guides regeneration is utilized during em-bryogenesis, when a single fertilized egg cell differentiates to form an embryo con-sisting of multiple cell types organized into a predefined morphology. This process is

remarkably forgiving: The developing organism can tell if it has deviated from the programmed pattern and will usually initiate corrective action to get back on course, Levin says.

After birth, this careful oversight must continue in order to maintain a healthy organism. During an animal’s lifetime, its cells are constantly dy-ing and being replaced, “and yet the organism continues in the same 3-D pattern, more or less, over decades,” Levin notes. “When cells stop obey-ing the morphogenetic needs of the host and revert to control at the sin-gle-cell level instead of responding to the normal pattern cues of the body,” the result is cancer, he explains. “So the body has to continuously combat that type of disorganization and try to impose order.”

Regeneration, too, is subject to checks and balances. As a result, “in a typical wound, the cells know not to sprout an extra limb,” says molecular

REGENERATION CHAMPION Salamanders, including this Notophthalmus viridescens specimen, can regrow lost limbs multiple times.


contents and also control ion flows and voltage gradients that might affect regeneration.

Bioelectric signaling—which an organ-ism detects through means including volt-age-sensing domains on proteins, changes in electrophoretic transfer of neurotrans-mitters, and movement of calcium ions—is in fact crucial for this process, Levin says ( Semin. Cell Dev. Biol. 2009, 20, 543). Changes in the electric potential of cells near the wound control differentiation and proliferation of cells in the blastema. Electrical signaling by the epithelium that covers the wound also provides a direc-tional cue for growth so that, for instance, a replacement arm grows outward rather

than into the animal’s trunk. Unlike nonregenerative or-

ganisms, salamanders that lose a limb maintain “a very strong ‘current of injury’ for weeks at the wound edge” while the new limb grows, Levin explains. The current is created by the movement of sodium, chloride, and potassium ions through

the wound epithelium. Researchers have demonstrated that applying a particular electric field to an amputated stump al-lows normally nonregenerative animals

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COPYING AN ORIGINAL

Faithful Replication Of A Lost Limb Demands Tight Process Control

THE BIONIC WAY Prosthetic limbs require sophisticated engineering to approximate the capabilities of flesh and blood. Electrical signals generated by remaining arm muscles control individual fingers in Touch Bionics’ i-LIMB Hand.

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biologist Ken Muneoka, who studies limb development and regeneration at Tulane University, in New Orleans.

Regeneration is held in check by sig-naling factors released by nerves, by the epidermis that covers the wound, and by the cells that aggregate to form the replacement limb, Muneoka says.

These signaling factors are con-trolled with finesse. For instance, cells have to deliver growth factors known as bone morphogenetic proteins (BMPs) at the right time and in the right location at a wound site. Otherwise, Muneoka says, “you’re going to get an abnormal regenerative response,” such as the growth of a toe in the wrong location or the generation of an extra finger.

Abnormal growth might also be held in check by local differences among neighboring cells, Brockes says. This model suggests that “growth stops when cells can no longer distinguish themselves from their neighbors,” for in-stance in terms of particular molecules expressed on the surface of the cells, he says. One possible example is Prod 1, a cell-surface protein that interacts with growth factor receptors such as the epi-dermal growth factor receptor.

Size control is obviously important but isn’t well understood. “A lot of work has gone into understanding how to kick-start regeneration, but almost no work has gone into learning how organ-isms stop it,” Levin notes. “Nobody wants to activate a regenerative re-sponse that gives you a 300-lb arm or a tumor at the end of a stump.”

such as adult frogs to start regrowing limbs, he says.

Levin’s team used a different, molecular genetics technique to regenerate tadpole tails—complex appendages that contain muscles and spinal cord. First, the re-searchers determined that regeneration in tadpoles requires the expression of V-ATPase, an enzyme that pumps protons out of cells at the amputation site. This pumping action alters membrane voltage at the wound and also creates a long-range electric field that promotes nerve growth into the site, Levin says.

Next, the researchers turned to tadpoles that had reached a stage of development when they are normally unable to regrow a

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tail. Levin’s group showed that V-ATPase is ineffective during this phase. In a striking experiment, the researchers nevertheless induced these tadpoles to regenerate com-plete tails by inserting proton pumps from yeast into the tadpoles’ cell membranes ( Development 2007, 134, 1323). The work showed that “any convenient electrogenic protein can be used for regenerative medi-

cine approaches, not just the ones that are natively expressed in the host,” Levin says.

Levin and Kaplan’s teams are drawing on these previous findings as they work out the optimal conditions for regrowing

an amputated rat limb with the help of their new regen-

erative sleeve. After attach-ing the device to the stump

of a rat’s limb, the researchers hope to create a regenerative

current at the stump’s sur-face by adjusting the ionic composition of the solution

inside the sleeve and by adding drugs that open or close ion chan-

nels in the membranes of the cells at the wound site.

The sleeve will offer some additional benefits. The aqueous environment it provides will prevent the scarring that normally develops in a mammalian wound exposed to air. The researchers might also use it to bathe the wound with scar-reducing compounds, immune-modulating drugs, and more traditional growth factors, Levin says.

Clearly, many details must be worked out before success with tadpoles and rats can be extended to people. In the mean-time, researchers are realizing they need to take a closer look at human patients.

THE CONVENTIONAL medical response to an amputation in humans has been to seal the wound site to prevent complica-tions, rather than to investigate the site’s growth potential, Muneoka explains. As a result, “we don’t know very much about what happens when you amputate a human limb,” he says. “We don’t know what the different stem cells are doing or what the bone is doing, or the muscles or the nerves. And there are literally hundreds of cell types there. So there’s a lot that we have to characterize before we can try to manipu-late the wound to cause bone regrowth or muscle regrowth.”

Given that a limb is so complex, it’s amazing that any organism can replace one on its own. “It’s the equivalent of forming a baby out of an embryo,” Mu-neoka says. In fact, regeneration is even harder, “because you’re working with adult tissue that is going backward in time,” he adds. “After you get the injury site organized to a certain point, the re-creation of the structure is largely a reiteration of development.” But that new structure has to connect up with the existing, adult body parts, Muneoka says. “For the most part, that interface has been ignored by researchers, and it’s as critical as being able to regenerate the new structure.” ■

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