Richardson Louise, Wang Dapeng, Hughes Ruth, Johnson Colin A, Peckham Michelle
School of Molecular and Cellular Biology, University of Leeds, Leeds, UK.
LeedsOmics, University of Leeds, Leeds, LS2 9JT, UK.
Sci Rep. 2022 Feb 18;12(1):2841. 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-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.
在柔软表面（12千帕）上培养的骨骼肌卫星细胞比在坚硬表面（约100千帕）上培养的细胞表现出更好的分化能力。为了更好地理解其原因，我们对源自H2k-tsA58永生小鼠的单个卫星细胞克隆（C1F）进行了RNA测序分析，该克隆在严格调控的条件下（撤除γ干扰素，37℃）分化为肌管。随着细胞从增殖状态转变为分化状态，整体基因表达的最大变化发生在第1天。令人惊讶的是，进一步分析表明，增殖的C1F细胞表达Pax3而非Pax7，免疫染色证实了这一点，然而它们随后分化为肌管的过程是正常的，并且在较软的表面上得到增强，这表现为成肌调节因子、肌节基因的表达水平显著更高，融合增强以及肌原纤维形成改善。在坚硬表面上，编码细胞外基质结构成分和相关基因的mRNA水平持续上调，这表明在坚硬表面上分化的卫星细胞的一个结果是它们在分化之前试图操纵其微环境。这个全面的RNA测序数据集将成为理解表达Pax3的细胞的有用资源。
