Modulation of the Cellular Microenvironment by Mechanical Fluid Shear Stress and Hypoxia Alters the Differentiation Capacity of Skeletal Muscle-Derived Stem Cells

Abstract

Skeletal muscle-derived stem cells (MDSCs) are the key modulators of muscle regeneration. An inappropriate cellular microenvironment can reduce the regenerative capacity of MDSCs. This study evaluates the effect of microenvironmental alterations on the cell differentiation capacity using either mechanical fluid shear stress (FSS) or hypoxic conditions. C2C12 mouse myoblasts were differentiated under cyclic FSS (CFSS), periodic FSS (PFSS) for one hour, and hypoxia (3% O2) for up to seven days. Cell proliferation and myogenic differentiation capacities were evaluated using cell viability assays, immunohistochemical staining, and morphometric analysis. The expression of MyoD, myogenin, myosin heavy chain, nitric oxide, hypoxia-inducible factor 1 alpha (HIF1α), vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) was quantified by means of RT-qPCR. The data showed that FSS conditions altered cell morphology and increased cell viability and cell distribution compared to static conditions. MyoD and myogenin expression was upregulated under both FSS conditions. CFSS induction improved myogenic differentiation parameters including myotube number, size and fusion capacity. Although hypoxia enhanced cell viability compared to normoxia, it reduced differentiation capacity, as indicated by the downregulation of myogenin and mTOR expression, as well as reducing myotube formation. Under hypoxic conditions, increased nitric oxide production and upregulation of VEGF expression were detected for up to 72 h. The data suggest an improved myogenic differentiation capacity under mechanical FSS; in contrast, the cell differentiation capacity was impaired under hypoxic conditions. The data point out that optimizing the biomechanical and oxidative stressors in the cellular microenvironment could improve stem cell transplantation and enhance their regenerative potential in the context of cell-based therapies.