Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen
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Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1–6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21Cip1 gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells.
KeywordsHuman primary myoblasts Oxygen Proliferation p21Cip1 Differentiation Cell cycle Species differences
Myosin heavy chain
Skeletal muscle growth medium
Skeletal muscle differentiation medium
We thank Assoc Prof David Cameron-Smith for his expertise with primer design; Prof Geoff C Nicholson for his assistance with the human ethics applications; the Orthopedic Surgeons—Drs David Bainbridge, Rick Angliss, and Rob Wood—for collection of the muscle biopsy samples; and Lisa Coleman, the Orthopedic Liaison Nurse, for her participation in subject recruitment. We gratefully acknowledge all of their contributions towards the completion of this project. The work presented in this report was supported by a NHMRC Postdoctoral Fellowship to NS and by the Deakin University Central Research Grants Scheme.
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