Single Cell RNA-Sequencing of Human Limb Skeletal Muscle across Development and Myogenic Culture from Pluripotent Stem Cells

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. To address this, we employed single cell RNA-sequencing to profile human skeletal muscle tissues from embryonic, fetal and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a “roadmap” of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols, and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. Additionally, we found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. In summary, this work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for better characterization and understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle based regenerative medicine. Overall design: Whole hindlimbs or hindlimb skeletal muscle tissues were obtained from human prenatal development up to 18 weeks as well as juveniles and adults. Dissociated cells were subjected to single cell RNA-sequencing (scRNA-seq) via the Drop-seq method (Macosko et al., Cell, 2015) without or with minimal enrichment. Similarly, myogenic cultures from three human pluripotent stem cell (hPSC) differentiation protocols (Xi et al., Cell Rep, 2017; Chal et al., Nat Biotechnol, 2015; Shelton et al., Stem Cell Rep, 2014) were also subjected to scRNA-seq/Drop-seq. Single cells from the whole tissues/cultures were analyzed using the Seurat package in R to explore the broad cell types present. The skeletal myogenic populations were subset and reanalyzed to identify myogenic subpopulations. Skeletal muscle stem and progenitor cells from in vivo tissues were computationally purified and assembled to construct a trajectory of human myogenesis, and progenitor cells derived from hPSCs in vitro were aligned to the trajectory to determine their developmental identities.