Expression of MyoD and M-Cadherin in C2C12 cells: differentiation induced by cell-to-cell contact vs reduction in growth factor

C2C12 murine cells differentiate through the fusion of myoblasts to form multinucleated myotubes as a cell model used to study myogenesis in vitro. Differentiation can be induced through serum starvation, which is achieved with adult serum that has reduced growth factors (GF), or cell-to-cell contact which induces growth arrest. Fetal Bovine Serum (FBS) is commonly used as a source of GF for cell proliferation, whilst Horse Serum (HS) lacks GF, allowing for induction of cell differentiation. Common methodologies in literature utilized 2% HS as differentiation media (DM) while ATCC recommends 10% HS as DM; DM switch also varied at 80% or 100% cell confluency. However, effects of such variations have yet to be studied. Hence, we aim to study the effects of C2C12 cell differentiation due to cell-to-cell contact versus GF reduction using MyoD and m-Cadherin as biomarkers. MyoD, a master regulator of muscle-specific genes, is activated in the nucleus to initiate myogenesis. m-Cadherin is a transmembrane protein mediating cell fusion prior to myotube formation. Samples were switched to 2% HS at 80% or 100% cell confluency and 10% HS at 100% confluency, compared to control samples maintained in 10% FBS. Localization and relative expression of biomarkers between the condition groups were studied via immunocytochemistry (ICC) and qPCR analysis, respectively, at myoblast stage (MB), days 0, 4, 7, and 11. Varying culture conditions across the literature poses obstacles for data reproducibility and comparability.The outcomes of this study may contribute in establishing a standardized cell culture protocol within the discipline.