A Limb Regeneration Mystery Solved

Salamanders have an enviable ability to regrow appendages that are amputated or injured; they re-create all the bones, muscle, skin, blood vessels, and nerves of the new body part so adeptly that it’s hard to tell that it was ever missing. Because of this ability, salamanders have been popular subjects for scientists studying regeneration–and trying to learn how human cells might be coaxed to perform the same feat.

In salamanders, new tissues come from a tumorlike mass of cells that forms at the site of the injury, called the blastema. Until now, most scientists thought that the blastema contained a population of stem cells that had become pluripotent–capable of giving rise to all the needed tissues. But a new paper in the journal Nature provides evidence that this is not the case. Instead, stem cells involved in regeneration only create cells of the tissue that they came from. The finding suggests that regeneration does not require cells to reprogram themselves as dramatically as scientists had assumed.

Elly Tanaka, lead scientist of the study at the Center for Regenerative Therapies, in Dresden, Germany, says that “a lot of people had the impression that these blastema cells were all the same.” Tanaka’s lab had even shown previously that a single muscle fiber could give rise to several types of cells in a regenerated limb. But previous studies, she says, relied on imperfect methods of tracking cells, such as using fluorescent dyes that may have leaked out to other cells.

In the latest study, Tanaka’s team employed a novel method for tracking the fate of cells from different tissues in a type of salamander called the axolotl. The researchers first created transgenic axolotls that carried green fluorescent protein (GFP) in their entire bodies. When the animals were still embryos, the researchers removed a piece of tissue from the limb region of the transgenic animals and transplanted the tissue into the same location in nontransgenic axolotls. The transplants were incorporated into the growing body as normal cells, and when the limb of the transplant recipients were then severed, the researchers could track the fate of the fluorescent cells as the limb regrew.

The researchers used this method to track the fate of cells of the inner and outer skin, muscles, and cartilage, as well as Schwann cells, which insulate nerve fibers. They found that, contrary to previous evidence, muscle cells at the amputation site only become muscle cells in the new limb. Other cell types also stuck to their previous identities; the only exception, Tanaka says, is that cells of the inner layers of skin and cartilage seem to be able to transform into one another. But for the most part, she says, the blastema is not a homogeneous mass of cells but “a mix of stem or progenitor cells from different tissues that stay separate during the whole process.”

The researchers also found that some cells remember not only their identities but also their position in the body. Cartilage cells, for instance, remember if they are supposed to form an upper arm, lower arm, or hand, while Schwann cells simply migrate anyplace that they are needed.

Tanaka says that the finding will provoke a major shift in thinking about the requirements of regeneration. In explaining why salamanders can regrow limbs and humans can’t, she says, “the hypothesis was that it’s because salamanders can powerfully alter the identity of cells.” But in fact, their cells never really lose their identities; instead, they seem to use tissue-specific stem cells capable of generating a certain part of the new limb. Tanaka points out that humans also have tissue-specific stem cells that replace different kinds of tissue. Perhaps salamanders “are not doing something much more complicated than what human stem cells would do,” she says. Coaxing human cells to regenerate might not require steps as drastic as making cells pluripotent.

Alejandro Sánchez Alvarado, a scientist who studies regeneration at the University of Utah School of Medicine, says that this method of “tattooing” the transplanted cells genetically is “a novel technique for the field of regeneration.” Tanaka believes that previous studies may have misled researchers by using imperfect tracking methods such as dyes by culturing cells before transplanting them and possibly altering them, or by allowing different cell types to contaminate samples.

Sánchezalso says that the idea that blastemas held several different cell types was a “minority hypothesis” and that this study “shows that this hypothesis turns out to be correct.” He cautions that scientists now need to determine whether this phenomenon is the same in adult axolotls and in newts, which are a primary model organism for regeneration studies. But if the same mechanism turns out to underlie other cases of regeneration, it would change what scientists believe is required to regrow body parts, Sánchezsays. But it leaves a major question unanswered: if humans already have tissue-specific stem cells, what exactly is the difference between our cells and those of salamanders?