Efficient In Vitro Generation and Isolation of Quiescent Pax7⁺ Human Muscle Reserve Cells

Human muscle reserve cells (MuRCs) represent a quiescent myogenic population generated in vitro and an attractive model for studying muscle stem cell biology. This protocol outlines a robust and reproducible method to generate in vitro, quiescent human MuRCs from primary myoblasts (MBs) obtained from muscle biopsies of healthy donors undergoing orthopedic surgery. Muscle tissue is enzymatically dissociated and cultured in a defined growth medium (GM) to establish pure MB populations, which are isolated by flow cytometry sorting using CD56⁺/CD146⁺/CD82⁺ markers. Human MBs can be expanded for up to seven passages (approximately 30 cell divisions). Upon reaching 80% confluency, human MBs are induced to differentiate by exposure to a serum-free differentiation medium (DM) for 48-72 h. Following brief trypsinization, multinucleated myotubes are removed and mononucleated MuRCs are isolated through a 20 µm filtration step. This approach yields a high number of quiescent human MuRCs, suitable for investigating muscle stem cell quiescence, activation, and function. The protocol provides a valuable platform to study the mechanisms underlying muscle regeneration and to develop therapeutic strategies that target muscle repair and disease.