Abstract
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 G1 phase and decreased S phase proportions in all donors (p < 0.005). The proportions of cells in G1 and G2 phases differed in proliferating myoblasts when comparing donors (p < 0.05 to p < 0.005), but following KOSR treatment, the proportion of cells in G1 (p = 0.558), S (p = 0.606) and G2 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.
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Acknowledgements
The authors wish to thank Mrs Lize Engelbrecht and Ms Rozanne Adams from the Central Analytical Facility at Stellenbosch University for their technical assistance with imaging and flow cytometry.
Funding
This study was funded by the National Research Foundation: South African Research Chairs Initiative (SARChI). Grant number: SARCI150212114075
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10616_2019_365_MOESM1_ESM.docx
Supplementary material 1. Supplementary Figure 1: Representative images (A, B) of cell cycle analysis by flow cytometry of PHMs. Cell cycle analysis of proliferation (A) and quiescence (B) of S6.3 N (1) is represented to illustrate the method by which the data was acquired. Supplementary Table 1: qPCR instrument settings used to perform analysis on acquired samples. Supplementary Table 2: Details of the antibodies used in the flow cytometric assessment of quiescent PHMs including nuclear and cell surface markers. Supplementary Table 3 (A, B, C): Raw data Ct values of qPCR of PHMs from 3 subjects (3A - S6.3; 3B - KH1; 3C - KH3). (DOCX 58 kb)
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Gudagudi, K.B., d’Entrèves, N.P., Woudberg, N.J. et al. In vitro induction of quiescence in isolated primary human myoblasts. Cytotechnology 72, 189–202 (2020). https://doi.org/10.1007/s10616-019-00365-8
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DOI: https://doi.org/10.1007/s10616-019-00365-8