Characterisation of stemness and multipotency of ovine muscle-derived stem cells from various muscle sources

Abstract

Muscle stem cells (MSCs) are a promising tool for cell-based therapy and tissue regeneration in veterinary medicine. Evaluation of MSCs from muscles of different origins improves our understanding of their regenerative potential. The present study compared the stemness, cell proliferation, migration potential, myogenic differentiation (MD), and multipotency of MSCs for four developmentally different muscles of ovine origin. MSCs were isolated from the hind limb (HL), diaphragm (DI), extraocular (EO), and masseter (MS) muscles. Cell proliferation, migration, and stemness were examined using sulforhodamine B, and colony formation assays. Evaluation of multipotency was examined using histological and morphometric analyses, alkaline phosphatase (ALP) activity, and the expression of myogenic, adipogenic, and osteogenic markers using RT-qPCR. Data were statistically analysed using analysis of variance. The results revealed that all experimental groups expressed stem cell markers paired box transcription factor Pax7, α7-integrin, CD90, and platelet-derived growth factor receptor alpha. DI and HL muscle cells displayed higher proliferation, migration, and colony formation capacities compared to the EO and MS muscle cells. HL and DI muscle cells showed increased MD, as indicated by myotube formation and relative expression of MyoD at day 7 and Myogenin at day 14. Although MS and EO muscle cells displayed impaired MD, these cells were more prone to adipogenic differentiation, as indicated by Oil Red O staining and upregulated fatty acid-binding protein 4 and peroxisome proliferator-activated receptor gamma expression. DI muscle cells demonstrated a higher osteogenic differentiation capability, as shown by the upregulation of osteopontin expression and an elevated ALP activity. Our data indicate that ovine HL and DI MSCs have a higher regenerative and multipotent potential than the EO and MS muscle cells. These results could be valuable for regional muscle biopsies and cell-based therapies.