Preparation and Culture of Limb Blastema Stem Cells from Regenerating Larval and Adult Salamanders

INTRODUCTION Adult salamanders show extensive ability to regenerate their body parts, and this can be attributed at least in part to their cellular plasticity. Cell reprogramming, dedifferentiation, proliferation and redifferentiation occur naturally in adult salamanders in response to tissue damage or removal. Limb regeneration proceeds by mesenchymal dedifferentiation and cell migration at the end of the stump. This generates a cohort of mesenchymal stem cells called blastema cells, which accumulate under the wound epithelium. The “limb blastema” is an autonomous structure that retains the positional identity of its location of origin as well as morphogenetic cues for reconstructing a new limb. At the cellular level, blastema stem cells maintain characteristics such as positional memory derived from their location, while expressing new markers relevant to the process of limb regeneration. In this protocol, a nonenzymatic method is described for the dissociation of blastema stem cells from newts ( Notophthalmus viridescens ) and axolotls ( Ambystoma mexicanum ).