MyoD over-expression reprograms marine mammal fibroblasts into muscle cells

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 by overexpression of the myogenic transcription factor myod. Muscle cells are often challenging to obtain from wild organisms but offer a unique system to study metabolic responses. 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, myod expression was higher in transfected cells than in non-transfected cells according to RT-qPCR analysis (p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression in transfected cells. 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 analyses confirmed that the transcriptional profile of Myod-expressing cells is different from the naive dermal elephant seal fibroblasts. Reprogrammed cells share 67% of the differentially expressed genes found in primary muscle cells and primary differentiated myotubes. Exposure to the synthetic glucocorticoid, dexamethasone (0.1, 1, and 100mM) for 48h resulted in reduced immunofluorescence expression of myosin heavy chain 1 with increasing dose. Furthermore, we successfully reprogrammed fibroblasts into muscle cells derived from the common dolphin ( Delphinus delphis ) providing a unique and comparative platform for mechanistic studies in species where muscle tissue samples cannot be obtained from live animals. NDSEG This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.