Abstract
Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).
Highlights
Stem cell biology using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, is providing unprecedented opportunities for the study of human development by recapitulating embryogenesis (Tchieu et al, 2017; Irie et al, 2015; van de Leemput et al, 2014; Qi et al, 2017; Guo et al, 2017)
Distinct protein expression patterns were observed during our in vitro myogenic specification: OCT4 expressing cells were 96.42 ± 2.55% of undifferentiated hESCs; at day 4, 87.78 ± 4.46% of the cell population expressed TBX6; at day 20, 31.72 ± 5.78% of the cell population expressed PAX7; at day 25, 53.30 ± 6.39% of the cell population expressed MYOG; at day 40, 87.99 ± 3.64% of the cell population expressed MF20
Cardiac troponin T and smooth muscle alpha actin (SMAA)-positive cells were hardly detected, demonstrating that there is almost no contamination of cardiac muscle or smooth muscle lineage. These data demonstrated that using our skeletal muscle protocol, human pluripotent stem cells (hPSCs) can be directed to skeletal muscle lineages with the expression of key marker genes
Summary
Stem cell biology using human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), is providing unprecedented opportunities for the study of human development by recapitulating embryogenesis (Tchieu et al, 2017; Irie et al, 2015; van de Leemput et al, 2014; Qi et al, 2017; Guo et al, 2017). To systematically investigate the transcriptional blueprint for 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 (Loh et al, 2006; Fior et al, 2012; Nichols et al, 1998; Seale et al, 2000; Hasty et al, 1993). One of the transcription factors we identified is TWIST1, which has not been extensively studied in human muscle biology and relevant genetic diseases
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