RNA-Seq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation

• Published in: Scientific reports Vol. 12; no. 1; pp. 2841 - 16
• Main Authors: Richardson, Louise, Wang, Dapeng, Hughes, Ruth, Johnson, Colin A., Peckham, Michelle
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.02.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/514/1949; 631/532/2439; Animals; Cell culture; Cell Culture Techniques; Cell differentiation; Cell Differentiation - genetics; Cell proliferation; Cell Proliferation - drug effects; Cloning; Extracellular matrix; Gene expression; Gene Expression Regulation, Developmental - drug effects; Humanities and Social Sciences; Interferon; Mice; multidisciplinary; Muscle Fibers, Skeletal - cytology; Muscle Fibers, Skeletal - drug effects; Myoblasts; Myoblasts - cytology; Myoblasts - metabolism; Myotubes; Pax3 protein; PAX3 Transcription Factor - genetics; RNA-Seq; Satellite cells; Satellite Cells, Skeletal Muscle - cytology; Satellite Cells, Skeletal Muscle - drug effects; Science; Science (multidisciplinary); Single-Cell Analysis; Skeletal muscle; Surface Properties
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-06795-3

Skeletal muscle satellite cells cultured on soft surfaces (12 kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100 kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2k b -tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of ɣ-interferon, 37 °C). The largest change in overall gene expression occurred at day 1, as cells switched from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of mRNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNA-Seq dataset will be a useful resource for understanding Pax3 expressing cells.