Holothurian primary muscle cell culture: Optimization of cell culture techniques and elucidation of molecular pathways involved in muscle cell dedifferentiation

Abstract

Members of the phylum Echinodermata possess an impressive regenerative potential that could further develop the field of regenerative medicine. Holothurians have one of the most developed musculatures in terms of the myogenesis that leads to regeneration of muscle tissue. We aimed to develop an <em>in vitro</em> model for dissociated muscle tissue that could be used for regenerative studies. We focus on molecular candidates that have been previously associated with regeneration, to define their roles in the dedifferentiation of muscle cells. To do this, we optimized protocols for intestinal tissue cultures by changing parameters for ones that best suited our muscle tissue. Then, we applied pharmacological agents and analyzed the presence of SLS's and dedifferentiating fibers, structures that have been associated with the process of dedifferentiation, to elucidate which molecular pathways could induce the dedifferentiation of muscle cells. We found specific parameters such as changes in the enzymatic dissociation media, the adherence protocol and the number of cells cultured, that yielded healthy muscle fibers. Our cultures also showed a cellular component that was added to structures that indicated that dedifferentiation was occurring. For pharmacological assays, we found an increase in dedifferentiating fibers when we treated cultures with LiCl and an increase in SLS's when treated with DPI, EGTA and UK-383,367. Our results indicate that our <em>in vitro</em> model serves as an effective way to determine the effects that pharmacological agents have on dedifferentiation. We conclude that pathways involving Wnt/β- catenin and GSK-3, reactive oxygen species, Ca²⁺ and BMP-1 modulate <em>in vitro</em> dedifferentiation of muscle fibers.