Effects of hypoxia on stemness, survival and angiogenic capacity of muscle-derived stem/progenitor cells

Abstract

Muscle-derived stem/progenitor cells (MDSPCs) have been demonstrated as a promising source of cellular therapy for the regeneration of peripheral nerves and how to improve the therapeutic potential of these cells is the focus of future research. This study aims to investigate the role of hypoxia on functional effects in MDSPCs. In this study, MDSPCs were isolated from skeletal muscles of limbs; meanwhile, the flow cytometry analysis, immunocytochemistry and multilineage differentiation were performed to characterize MDSPCs. We set cultured MDSPCs in a hypoxic condition with 3% oxygen. The quantitative RT–PCR results showed that hypoxia inhibits myogenic differentiation genes expression, while it is beneficial for the stemness and pro-angiogenic genes. The CCK-8, flow cytometry, Hoechst 33342 and trypan blue dye assays revealed that hypoxia promotes survival and reduces apoptotic cells from oxidative stress damage. The western blot analysis indicated increased expression of HIF-1α and its downstream proteins by hypoxia. Moreover, the angiogenesis assay showed that condition media extracted from hypoxia-treated MDSPCs support endothelial cells’ survival, migration and tube formation in vitro. The cultured cells under hypoxia appear as a salutary strategy to improve the survival and stemness of MDSPCs and enhance angiogenesis capacity in tissue injury repair and reconstruction applications. Abbreviations: MDSPCs, Muscle-derived stem/progenitor cells; ESCs, embryonic stem cells; PPSCs, induced pluripotent stem cells; ACSs, adult stem cells; PNI, Peripheral nerve injuries; BMSCs, bone marrow mesenchymal stem cells; NSCs, neural stem cells; ADSCs, adipose-derived stem cells; HIF-1α; Hypoxia-inducible factor-1α ; VEGF, vascular endothelial growth factor; SDF-1, stromal-derived factor 1; PM, proliferation media; DMEM, Dulbecco’s modified eagle medium; FBS, fetal bovine serum; MYHC, Myosin heavy chain; CCK-8, cell counting kit-8; H2O2, hydrogen peroxide; CM, conditioned media; CON, control group; qPCR, Quantitative real-time polymerase chain reaction; MRL/MpJ, Murphy Roths Large