Methods to Mimic In Vivo Muscle Cell Biology in Primary Human Myoblasts Using Quiescence as a Guidepost in Regenerative Medicine Research.

Abstract

Regenerative medicine research and testing of new therapeutics for muscle-related human diseases call for a deeper understanding of how human myoblasts gain and maintain quiescence in vitro versus in vivo. The more closely we can experimentally simulate the in vivo environment, the more relevance in vitro research on myoblasts will have. In this context, isolation of satellite cells from muscle tissue causes activation while myoblasts remain activated in culture, thus not simulating quiescence as in their in vivo niche. Cells synchronized for cell cycle present a good starting point for experimental intervention. In the past, myoblast quiescence has been induced using suspension culture (SuCu) and, recently, by knockout serum replacement (KOSR)-supplemented culture media. We assessed the proportion of cells in G0 and molecular regulators after combining the two quiescence-inducing approaches. Quiescence was induced in primary human myoblasts (PHMs) in vitro using KOSR-treatment for 10 days or suspension in viscous media for 2 days (SuCu), or suspension combined with KOSR-treatment for 2 days (blended method, SuCu-KOSR). Quiescence and synchronization were achieved with all three protocols (G0/G1 cell cycle arrest >90% cells). Fold-change of cell cycle controller p21 mRNA for KOSR and SuCu was 3.23 ± 0.30 and 2.86 ± 0.15, respectively. Since this was already a significant change (p < 0.05), no further change was gained with the blended method. But SuCu-KOSR significantly decreased Ki67 (p = 0.0019). Myogenic regulatory factors, Myf5 and MyoD gene expression in PHMs were much more suppressed (p = 0.0004 and p = 0.0034, respectively) in SuCu-KOSR, compared to SuCu alone. In conclusion, a homogenous pool of quiescent primary myoblasts synchronized in the G0 cell cycle phase was achieved with cells from three different donors regardless of the experimental protocol. Myogenic dedifferentiation at the level of Myogenic Regulatory Factors was greater when exposed to the blend of suspension and serum-free culture. We suggest that this blended new protocol can be considered in future biomedical research if differentiation is detected too early during myoblast expansion. This shall also inform new ways to bridge the in vitro and in vivo divides in regenerative medicine research.