An Engineered Multicellular Stem Cell Niche to Study Skeletal Muscle Physiology and Function

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human induced pluripotent stem cell (hiPSC) differentiation, we screened cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identify the combination of embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. Combined with Wnt and FGF pathway mediated lineage-priming, embryoid differentiation allows for the isolation of >99% pure Pax7 positive skeletal muscle progenitors within a two-week time-window. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and activates the MAPK, PI3K/AKT and Notch pathways. Embryoid derived myogenic cells reactivate after repeated injury and, compared to adult human myoblasts, display enhanced fusion to muscle fibers and lead to stronger muscles when transplanted into a mouse model of Duchenne muscular dystrophy. Altogether, we provide a highly efficient and scalable suspension-based protocol for 3D derivation of Pax7 positive myogenic progenitors from hiPSCs.