| Summary: | Ex vivo tissue culture allows the study of complex cellular mechanisms that are relevant to physiological responses while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can be reprogrammed into muscle cells, which are often challenging to obtain but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myod. We isolated cells from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES cells respire, stain positive for fibroblast markers (vimentin and PDGFR), and are amenable to electroporation. We overexpressed GFP‐myod in NES fibroblasts and conducted antibiotic selection and purification by FACS. As expected, expression of myod was higher in transfected cells than in controls according to qPCR analysis (t‐test p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression. Furthermore, fibroblasts overexpressing myod expressed downstream markers of myogenesis (myogenin, myosin heavy chain 1 and myosin heavy chain 8) and the effect was enhanced when myod‐overexpressing cells were supplemented with small molecules. We are currently evaluating the capacity of myod‐overexpressing cells to differentiate into myotubes and comparing metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.
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