MyoD Over-expression Reprograms Elephant Seal Fibroblasts into Muscle Cells

Abstract

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 theoretically be reprogrammed into muscle cells, which are often challenging to obtain from wild organisms but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myoD. We derived primary dermal fibroblasts 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 also expressed downstream myogenenic markers (myogenin, myosin heavy chain 1 and myosin heavy chain 8). MyoD-overexpressing cells differentiate into myotubes, become multinucleated and stain positively for myosin heavy chain 1. RNAseq analysis confirmed a transcriptional profile of myoD expressing cells, which is unique and different from the naive dermal elephant seal fibroblasts. 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. The Department of Defense National Defense Science and Engineering Graduate (NDSEG) Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.