Abstract: Adult skeletal muscle harbors a population of muscle stem cells (MuSCs) that are required to repair or reform multinucleated myofibers after a tissue injury. In youth, a portion of MuSCs return to a reversible state of cell cycle arrest, termed quiescence, after injury resolution. By contrast, a proportion of aged MuSCs exist in a semi-activated state under homeostatic conditions, and prematurely respond to subsequent injury cues, thereby failing to return the tissue to its pre-injury state. The heterogeneity of MuSC function is linked to quiescence depth, but regulation of the balance between MuSC quiescence and activation in youth and in age is incompletely understood. This is due in part to the paucity of scalable methods that support MuSC quiescence in culture, and in turn necessitates reliance on low-throughput in vivo studies. To fill this gap, we developed a simple, 96-well format method to inactivate MuSCs isolated from skeletal muscle tissue, and return them to a quiescent-like state for at least one-week by culturing them within a three-dimensional engineered sheet of myotubes. Seeding the myotube sheets with different numbers of MuSCs elicited population-level adaptation activities that converged on a common steady-state niche repopulation density. By evaluating MuSC engraftment over time in culture, we observed reversible cell cycle exit that required both myotubes and a 3D culture environment. Additional quiescence-associated hallmarks were identified including a Pax7+CalcR+MyoD-c-FOS- molecular signature, quiescent-like morphology including oval-shaped nuclei and long cytoplasmic projections with N-cadherin+ tips, as well as the acquisition of polarized niche markers. We further demonstrate a relationship between morphology and cell fate signature using high-content imaging and CellProfilerTM-based image analysis pipelines. MuSC functional heterogeneity during engraftment was observed across all metrics tested, suggesting in vivo-like subpopulation activities are reflected in the assay. Notably, aged MuSCs introduced into young 3D myotube cultures displayed aberrant proliferative activities, delayed inactivation kinetics, and activation-associated morphologies that we show are rescued by wortmannin treatment. Thus, this miniaturized, biomimetic culture assay offers an unprecedented opportunity to uncover regulators of quiescence in youth and in age.

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