MEndR: An In Vitro Functional Assay to Predict In Vivo Muscle Stem Cell‐Mediated Repair

Functional assessment of stem cell‐mediated endogenous repair relies on animal studies. Here an in vitro assay is described that recapitulates important early steps of the in vivo skeletal muscle endogenous repair (MEndR) process. The assay is integrated with a custom semi‐automated image analysis pipeline to enable high‐content data analysis of donor‐derived muscle fiber content and morphology. Myotube sheets, generated by infiltrating a cellulose scaffold with myoblasts, are engrafted with muscle stem cells (MuSCs), injured to induce a regenerative microenvironment, and muscle repair is assessed. Significantly, the spatiotemporal dynamics of in vitro repair closely matched those observed in vivo, when both stem cells and injury are present. By exploiting the easy imaging geometry of the engineered tissue, cellular mechanisms of action driving the MuSC response to the regenerative template are explored. In vivo outcomes of two known modulators of MuSC‐mediated repair, measured by donor fiber production, MuSC niche repopulation, and response to a secondary injury, are phenocopied in the platform only when both the stem cells and injured 3D template are present. The MEndR platform represents a powerful opportunity to explore MuSC‐mediated repair and potentially compress the discovery pipeline by combining drug screening and validation in one step. The in vitro MEndR assay recapitulates key early phases of in vivo skeletal muscle regeneration and can be used to assess the capability of drugs to modulate muscle stem cell‐mediated repair to prioritize hits for in vivo transplantation experiments. This modular assay provides a practical tool to study the dynamics of muscle regeneration in response to injury, as well as niche repopulation, in a highly controllable format.