Transcriptional landscape of human myogenesis reavels a key role of TWIST1 in maintenance of skeletal muscle progenitors

Derivation of human skeletal muscle in vitro with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in myogenic specification and relevance to rare genetic diseases. We characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ PSCs, MSGN1::EGFP+ presomites, PAX7::EGFP+ skeletal muscle progenitors, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitors, providing an explanation for the musculoskeletal symptoms of a rare genetic disease, Saethre-Chotzen syndrome. We have established a foundation for future studies to identify regulators of human myogenic ontogeny. To systematically investigate the transcriptional blueprint of developing human skeletal muscle cells and to gain comprehensive insights into the molecular signatures of putative skeletal muscle stem/progenitors, we conducted step-wise isolations of stage-specific cellular subtypes during muscle differentiation in vitro and performed global gene expression analysis. Using our human genetic reporter PSC lines and a newly devised method for myotube enrichment, we isolated five distinct cell types in human embryonic myogenesis, including OCT4::EGFP+ embryonic stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ putative skeletal muscle stem/precursor cells, MYOG::EGFP+ myoblast cells, and multinucleated myotubes.