GSE202308
An Engineered Multicellular Stem Cell Niche for the 3D Derivation of Human Myogenic Progenitors from iPSCs
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 identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of consecutive Wnt and FGF pathway induction, three-component embryoids (iTCEs) containing these cell types yielded between 40-50% human Pax7 positive embryonic-like myogenic progenitors (eMPs). In a secondary screen we identified cell surface markers allowing for flow cytometric isolation of >99% of the Pax7 positive eMP population from iTCEs. Myogenic differentiation of hiPSCs in iTCEs relies on a specialized structural microenvironment and activates the MAPK, PI3K/AKT, and Notch pathways. After transplantation in a mouse model Duchenne muscular dystrophy, eMPs repopulate the stem cell niche, reactivate after repeated injury and, compared to adult human myoblasts, display enhanced fusion and lead to stronger muscles. Altogether, we provide a highly efficient and scalable suspension-based protocol for 3D derivation of Pax7 positive myogenic progenitors from hiPSCs within a two-week time window. Overall design: 2D cultured human induced pluripotent stem cells (hiPSCs) (day 0) and of 7 and 13 day 3D-hiPSC organoids analyzed using scRNAseq.