A novel protocol for the direct isolation of a highly pure and regenerative population of satellite stem cells

The utility of a pure population of highly regenerative satellite stem cells (SSCs) is a prerequisite for successful cell-based muscle therapies. Previous works have reported several methods for the SSC isolation. However, the majority of cells isolated using previous methods are fibroblasts and other nonmyogenic cell types, necessitating further expensive and time-consuming purification steps often affecting the regenerative quality of the isolated SSCs. Here, we describe a simple, time-effective, and robust protocol for the isolation of a pure population of SSCs in a single direct step, eliminating the need for further purification steps. By separating the muscle fascicles from the adjacent connective tissues (i.e., epimysium and perimysium) and utilizing a defined dissociation medium, a cell pool enriched in SSCs was successfully obtained. Immunofluorescent staining confirmed the stemness and the myogenic purity of the isolated cells (~97%). Upon myogenic induction, SSCs gave rise to multinucleated myofibers that exhibited spontaneous contraction in the culture dish for up to 21 d. Efforts to optimize the culture conditions revealed that tissue culture plates (TCPs) coated with a tissue-specific extract significantly enhanced SSCs’ attachment, growth, and differentiation compared to collagen I, Matrigel-coated TCPs, or noncoated TCPs. Further studies confirmed the robust myogenic regenerative capacity of the isolated cells, as evidenced by their ability to display key regenerative characteristics, demonstrating the mild effects of our isolation protocol on their regenerative capacity. The isolation protocol presented herein can potentially be used to obtain SSCs with high myogenic purity for skeletal muscle regenerative engineering and clinical indications.