Limbs regrow without pluripotency

by Jef Akst

The cells responsible for the salamander's famed ability to regenerate amputated limbs aren't pluripotent, as scientists have thought, a study published online in Nature today reports. That's good news for regenerative medicine: If the mechanism salamander cells use for regrowing body parts doesn't depend on pluripotent stem cells, it may be easier than researchers have assumed to mimic that organism's regenerative strategy in potential therapies. Regenerated limb with GFP-labeled Schwann cells Image: Dunja Knapp and Elly Tanaka "This is a very important finding for this field and also for regenerative medicine in general," said regeneration biologist Andras Simon of the Karolinska Institutet in Sweden, who was not involved in the research. "The data very strongly suggest that during regeneration cells don't really shift lineage. I think many people expected more flexibility than what this study shows." Salamanders' regenerative abilities were thought to rely on the dedifferentiation of cells near the damaged limb to a pluripotent state -- a feat that mammalian cells are normally incapable of. Development and cell biologist Elly Tanaka of the Center for Regenerative Therapy at Dresden University of Technology in Germany and colleagues examined the lineage of these regenerative cells more closely. They created green fluorescent axolotls -- a Mexican salamander frequently used as a model system for limb regeneration -- by linking green fluorescent protein (GFP) to a promoter of cytoplamsic actin, a protein expressed in every cell of the body. By grafting cells from green axolotl embryos to normal animals before amputation, the researchers could track the GFP to examine the fate of specific cell types in a regenerating limb after amputation in juveniles. Using these techniques, the researchers looked at four different tissue types: dermis, cartilage, muscle, and Schwann cells -- neural tissue that insulates the nerves of the limbs. With the exception of dermal cells, they found that the grafted green cells showed up only in those same tissue types in the regrown limb. "What's surprising about this finding is that it shows clearly that those cells in the blastema" -- the ball of cells that forms at the site of the amputation to build the regenerated limb -- "are not homogenous," said developmental biologist Kenneth Poss of the Duke University Medical Center, who was not involved in the research. "They're retaining their memory of the tissues they came from, and they go on to form cells of that same type. That's not what most people thought was going on." Interestingly, dermal cells contributed to cartilage, connective tissue, and tendons, in addition to the dermis. This may be a result of the common origin of dermal and cartilage cells in the embryo, Tanaka said. The formation of a blastema thus either activates a stem cell that is a common progenitor of cartilage and dermis, or causes dermal cells to be dedifferentiated into one of these progenitors. Image: Laura Muzinic "It is likely that [the cells] are dedifferentiating [and somehow] retain a memory of what they need to differentiate back into," explained Alejandro Sánchez Alvarado, a developmental biologist at the University of Utah School of Medicine and author of a review article accompanying the Tanaka study. "They probably do go back to an embryonic state," Tanaka agreed, "but it's not to a pluripotent state." The results "really shift the focus" of regenerative research, Simon said. Instead of trying to generate multipotent or pluripotent cells, "one should try to understand how these cells get the appropriate signals to make a new limb in terms of organizing the different tissue types." The cell lineage specificity of the blastema cells "is probably a closer situation to what we see in other vertebrates," agreed developmental biologist Randal Voss of University of Kentucky, who did not participate in the research. Applying an understanding of that mechanism to future therapies thus becomes "something that's more achievable in regenerative medicine." However, the current study examined regeneration in juvenile axolotls, and to fully understand the implications for regenerative medicine involving adult tissue, it is important to extend the current findings to limb regeneration in adult axolotls, Sánchez Alvarado said. Also, it's still unclear whether this same loyalty of tissue type applies to other appendages and other regenerating species, Tanaka said. Regenerating cells in the axolotl tail, for example, do seem to differentiate into multiple tissue types. This may be because the tail blastema contains cells from the spinal cord, which may be more plastic than other types of tissue, or it could be an artifact of the labeling technique those studies used. "So now we have to see if the previous result is really true or whether the previous technology was unreliable," Tanaka said.