Gene Expression profile at the single cell level of quiescent and activated muscle stem cells (MuSC) from Extraocular muscle (EOM) and Tibialis Anterior (TA) muscle

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and scRNA-seq to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogenintdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as the driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells. MuSCs from Tg:Pax7-nGFP mice were isolated by Fluorescence-based active cell sorting (FACS) based on GFP fluorescence. Quiescent MuSCs were immediatly processed for single-cell RNA sequencing and activated MuSCs were put in culture for 4 days before sequencing.

骨骼肌干细胞（Skeletal muscle stem cells, MuSCs）被学界普遍认为具有功能异质性。据报道，与肢体来源的MuSCs相比，颅面来源的MuSCs具有更强的增殖与再生能力。目前学界对这一功能异质性背后的调控机制仍缺乏全面认知。本研究通过克隆分析、活细胞成像及单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，鉴定出区分眼外肌（extraocular muscle, EOM）来源与肢体来源骨骼肌干细胞群的关键特征。借助MyogenintdTom报告基因的实验结果显示，EOM来源MuSCs的增殖能力增强与其分化延迟以及肌源性定向基因MyoD（Myod）的低表达密切相关。出乎意料的是，体外激活的EOM来源MuSCs会表达大量间充质非肌细胞特有的细胞外基质成分。生物信息学分析表明，一套肢体来源MuSCs所不具备的独特共调控模块，是介导上述特征的核心驱动因素。以叉头框C1（Foxc1）为核心调控因子的EOM来源MuSCs转录因子网络，似乎被固有编程为眼外肌干细胞身份，该网络在细胞生长、疾病状态以及体外传代多次后仍可维持稳定。本研究结果揭示了高性能MuSCs如何通过重塑局部微环境并获得非肌源性细胞典型特性，来调控肌源性定向过程。本研究从Tg:Pax7-nGFP转基因小鼠中分离MuSCs，通过基于GFP荧光的荧光激活细胞分选术（Fluorescence-activated cell sorting, FACS）完成纯化。静息状态的MuSCs被立即用于单细胞RNA测序，而激活状态的MuSCs则先在体外培养4天后再进行测序。