No abstract

Muscle cell fusion is a multistep process involving cell migration, adhesion, membrane remodeling and actin-nucleation pathways to generate multinucleated myotubes. However, molecular brakes restraining cell–cell fusion events have remained elusive. Here we show that transforming growth factor beta (TGFβ) pathway is active in adult muscle cells throughout fusion. We find TGFβ signaling reduces cell fusion, regardless of the cells’ ability to move and establish cell-cell contacts. In contrast, inhibition of TGFβ signaling enhances cell fusion and promotes branching between myotubes in mouse and human. Exogenous addition of TGFβ protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFβR2 induces the formation of giant myofibers. Transcriptome analyses and functional assays reveal that TGFβ controls the expression of actin-related genes to reduce cell spreading. TGFβ signaling is therefore requisite to limit mammalian myoblast fusion, determining myonuclei numbers and myofiber size.

No abstract

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.