Direct reprogramming of dermal fibroblasts derived from Northern elephant seals into muscle cells

Abstract

Ex vivo tissue culture is a powerful technique that allows the study of complex cellular mechanisms that are relevant to physiological responses in animals 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 progenitor cells (myoblasts), 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 dermal fibroblasts from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES skin fibroblasts stained positive for the Platelet‐derived growth factor receptor. Primary NES fibroblasts were amenable to eGFP transfection by both, lipofection and electroporation. Maximal transfection efficiency was achieved using the Neon Electroporation system and corresponded to 55% of living cells. We overexpressed myod in NES fibroblasts and conducted antibiotic selection with 2 ug/mL puromycin for 3 days. As expected, expression of myod was significantly higher in transfected cells 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 dermal fibroblasts to differentiate into myotubes and comparing differentiation and metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types such as fibroblasts could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.