MEDICINE Regrowing Human Limbs Progress on the road to regenerating major body parts, salamander-style, could transform the treatment of amputations and major wounds 56 SCIENTIFIC AMERICAN © 2008 SCIENTIFIC AMERICAN, INC. April 2008 Regrowing Human Limbs By Ken Muneoka, Manjong Han and David M. Gardiner salamander’s limbs are smaller and a of a salamander, but soon afterward the human bit slimier than those of most people, and amphibian wound-healing strategies diverge. Abut otherwise they are not that differ- Ours results in a scar and amounts to a failed ent from their human counterparts. The sala- regeneration response, but several signs indicate mander limb is encased in skin, and inside it is that humans do have the potential to rebuild composed of a bony skeleton, muscles, liga- complex parts. The key to making that happen ments, tendons, nerves and blood vessels. A will be tapping into our latent abilities so that loose arrangement of cells called fibroblasts our own wound healing becomes more salaman- holds all these internal tissues together and derlike. For this reason, our research first gives the limb its shape. focused on the experts to learn how it is done. Yet a salamander’s limb is unique in the world of vertebrates in that it can regrow from a stump Lessons from the Salamander after an amputation. An adult salamander can When the tiny salamander limb is amputated, regenerate a lost arm or leg this way over and blood vessels in the remaining stump contract over again, regardless of how many times the quickly, so bleeding is limited, and a layer of skin part is amputated. Frogs can rebuild a limb dur- cells rapidly covers the surface of the amputation ing tadpole stages when their limbs are first site. During the first few days after injury, this KEY CONCEPTS growing out, but they lose this ability in adult- so-called wound epidermis transforms into a n The gold standard for limb hood. Even mammalian embryos have some layer of signaling cells called the apical epithelial regeneration is the sala- ability to replace developing limb buds, but that cap (AEC), which is indispensable for successful mander, which can grow capacity also disappears well before birth. regeneration. In the meantime, fibroblasts break perfect replacements for Indeed, this trend toward declining regenerative free from the connective tissue meshwork and lost body parts throughout capacity over the course of an organism’s devel- migrate across the amputation surface to meet its lifetime. Understanding how can provide a road opment is mirrored in the evolution of higher at the center of the wound. There they prolifer- map for human limb animal forms, leaving the lowly salamander as ate to form a blastema—an aggregation of stem- regeneration. the only vertebrate still able to regrow complex like cells that will serve as progenitors for the body parts throughout its lifetime. new limb [see box on next two pages]. n The early responses of tis- Humans have long wondered how the sala- Many years ago studies in the laboratory of sues at an amputation site are not that different in mander pulls off this feat. How does the regrow- our colleague Susan V. Bryant at the University salamanders and in ing part of the limb “know” how much limb is of California, Irvine, demonstrated that the humans, but eventually missing and needs to be replaced? Why doesn’t cells in the blastema are equivalent to the cells human tissues form a scar, the skin at the stump form a scar to seal off the in the developing limb bud of the salamander whereas the salamander’s wound as it would in humans? How can adult embryo. This discovery suggested that the con- reactivate an embryonic salamander tissue retain the embryonic poten- struction of a limb by the blastema is essentially development program to tial to build an entire limb from scratch multiple a recapitulation of the limb formation that took build a new limb. times? Biologists are closing in on the answers place during the animal’s original development. n Learning to control the to those questions. And if we can understand An important implication of this insight was human wound environ- how the regeneration process works in nature, that the same genetic program is involved in ment to trigger salaman- we hope to be able to trigger it in people to regen- both situations, and because humans make derlike healing could make N A erate amputated limbs, for example, and trans- limbs as embryos, in principle we should already it possible to regenerate form the healing of other major wounds. have the necessary programming to regenerate large body parts. The human body’s initial responses to such a them as adults, too. It seemed, therefore, that all — The Editors RON GOODM RON A A serious injury are not that different from those scientists needed to do was figure out how to www.SciAm.com © 2008 SCIENTIFIC AMERICAN, INC. SCIENTIFIC AMERICAN 57 [THE GOLD STANDARD] Perfect REGeneration Salamanders are the only vertebrates able to regrow lost limbs, as a rush of cells from stump tissues to the amputation site. The next well as many other body parts, throughout their lifetimes—and they stages involve reversion of those cells to an embryonic state and can do it repeatedly. Studies of how a limb forms on the salamander their building of a new limb following the same steps as in have revealed that the process begins with rapid wound closure and embryonic development. Blastema Apical epithelial cap Embryolike cells Wound epidermis Bone Fibroblasts Nerve Muscle WOUND CLOSURE HEALING SIGNALS RETURN TO THE WOMB Within hours of a leg amputation, Epidermal cells form a ridge known as an apical Cells that migrated to the wound revert to a epidermal skin cells migrate across epithelial cap, which generates critical signals that less specialized embryonic state and begin the wound to seal it, forming a guide the behavior of other cells. Fibroblasts and dividing to populate the bud of a new limb, wound epidermis. muscle cells start migrating toward the wound site. called a blastema. induce an amputated limb to form a blastema. factors provided by the nerve were influencing One of us (Gardiner) —working with Tetsuya regeneration by altering the behavior of resident Endo of U.C. Irvine a few years ago—took a fibroblasts. minimalist approach to answering the basic These induced blastemas never progressed to question of how to make a blastema. Instead of the later stages of regeneration to form a new studying amputation sites on the salamander, limb, however. One more ingredient was needed. where a blastema would naturally form, we The key to inducing a blastema that produced a looked at simple wounds on the side of a sala- new limb was to graft a piece of skin from the mander limb, which would normally heal just opposite side of the limb to the wound site, which ) by regenerating the skin. Our idea was that such allowed fibroblasts from opposite regions of the wounds are similar to the site of an amputated limb to participate in the healing response. The mammalian limb that fails to generate a new resulting accessory limb was, of course, growing salamander CHER ( limb. If we could get an entire limb to grow out at an abnormal location, but it was anatomi- A where a simple wound-healing response would cally normal [see box on pages 62 and 63]. So the PFELB A typically occur, then we could further dissect basic recipe for making a blastema seemed rela- A the regeneration process. tively simple: you need a wound epidermis, nerves ); LIS After we made a small incision in the sala- and fibroblasts from opposite sides of the limb. mander leg, epidermal cells migrated to cover With this minimal view of limb regeneration in and seal the wound, as they would at an ampu- mind, we began to focus on understanding the illustrationslimb tation site, and fibroblasts from the dermis layer roles of the individual ingredients. of the skin also moved in to replace the missing We knew that the epidermis is derived from LICE Y. CHEN ( A skin. But if we carefully deviated a nerve to the one of three layers of primitive cells within an ); wound site, we could induce those fibroblasts to early developing embryo, the ectoderm, which form a blastema instead. Marcus Singer of Case is also well known to provide signals that con- photographs Western Reserve University had already demon- trol the outgrowth of limbs from limb buds on RDINER ( strated more than half a century ago that inner- the embryo. Ectoderm cells gather in the bud to A vation was required for a regeneration response, form an apical ectodermal ridge (AER), which VID M. G A but our experiments clarified that unknown transiently produces chemical signals that guide D 58 SCIENTIFIC AMERICAN © 2008 SCIENTIFIC AMERICAN, INC. April 2008 Perfect REGeneration Developing digits Original amputation site TAKING SHAPE FLESHING OUT As the blastema grows, it begins to form the outline of the As its internal anatomy and outline become more new limb, including the tip that will become the foot. The mature, the limb lengthens to fill in the missing embryonic cells give rise to new tissues by proliferating and segment between the original amputation plane differentiating into bone, muscle, fibroblasts, and so on. and the toes. the migration and proliferation of the underly- primary cellular players, the fibroblasts, carry ing limb bud cells. out this function. Although some of the critical signals from the epidermis have not yet been identified, members Location, Location, Location of the family of fibroblast growth factors (FGFs) Recall from the minimalist accessory-limb are involved.