Gudagudi Kirankumar B, d'Entrèves Niccolò Passerin, Woudberg Nicholas J, Steyn Paul J, Myburgh Kathryn H
Department of Physiological Sciences, Stellenbosch University, Matieland, Private Bag X1, Stellenbosch, 7602, South Africa.
Department of Human Biology, University of Cape Town, Anzio Road, Observatory, South Africa.
Cytotechnology. 2020 Apr;72(2):189-202. doi: 10.1007/s10616-019-00365-8. Epub 2020 Jan 28.
Adult skeletal muscle stem cells, satellite cells, remain in an inactive or quiescent state in vivo under physiological conditions. Progression through the cell cycle, including activation of quiescent cells, is a tightly regulated process. Studies employing in vitro culture of satellite cells, primary human myoblasts (PHMs), necessitate isolation myoblasts from muscle biopsies. Further studies utilizing these cells should endeavour to represent their native in vivo characteristics as closely as possible, also considering variability between individual donors. This study demonstrates the approach of utilizing KnockOut™ Serum Replacement (KOSR)-supplemented culture media as a quiescence-induction media for 10 days in PHMs isolated and expanded from three different donors. Cell cycle analysis demonstrated that treatment resulted in an increase in G phase and decreased S phase proportions in all donors (p < 0.005). The proportions of cells in G and G phases differed in proliferating myoblasts when comparing donors (p < 0.05 to p < 0.005), but following KOSR treatment, the proportion of cells in G (p = 0.558), S (p = 0.606) and G phases (p = 0.884) were equivalent between donors. When cultured in the quiescence-induction media, expression of CD34 and Myf5 remained constant above > 98% over time from day 0 to day 10. In contrast activation (CD56), proliferation (Ki67) and myogenic marker MyoD decreased, indicated de-differentiation. Induction of quiescence was accompanied in all three clones by fold change in p21 mRNA greater than 3.5 and up to tenfold. After induction of quiescence, differentiation into myotubes was not affected. In conclusion, we describe a method to induce quiescence in PHMs from different donors.
成年骨骼肌干细胞，即卫星细胞，在生理条件下于体内处于不活跃或静止状态。细胞周期进程，包括静止细胞的激活，是一个受到严格调控的过程。使用卫星细胞、原代人成肌细胞（PHM）进行体外培养的研究，需要从肌肉活检中分离成肌细胞。利用这些细胞的进一步研究应尽可能努力呈现其天然的体内特征，同时也要考虑个体供体之间的差异。本研究展示了一种方法，即利用添加了KnockOut™血清替代物（KOSR）的培养基作为静止诱导培养基，对从三个不同供体分离并扩增的PHM进行为期10天的培养。细胞周期分析表明，该处理导致所有供体的G期增加，S期比例降低（p < 0.005）。在比较供体时，增殖的成肌细胞中G期和G期细胞的比例存在差异（p < 0.05至p < 0.005），但经过KOSR处理后，供体之间G期（p = 0.558）、S期（p = 0.606）和G期（p = 0.884）细胞的比例相当。当在静止诱导培养基中培养时，从第0天到第10天，CD34和Myf5的表达随时间保持在> 98%的恒定水平。相比之下，激活（CD56）、增殖（Ki67）和成肌标记物MyoD减少，表明去分化。在所有三个克隆中，静止诱导伴随着p21 mRNA的倍数变化大于3.5且高达十倍。静止诱导后，向肌管的分化不受影响。总之，我们描述了一种在来自不同供体的PHM中诱导静止的方法。
