Regulators of Development and Quiescence in the Human Muscle Stem Cell Lineage

摘要/摘要 由于成体干细胞的存在，成年骨骼肌被赋予了显著的再生能力 细胞，称为卫星细胞(SC)。关于这些细胞在人类中的发育起源，我们知之甚少。 而我们的大部分知识都来自于在老鼠身上进行的工作。SCS是罕见的，也不可能是 当它们在体外失去再生能力时，它们会无限放大。这些问题有 构成了研究它们在人类体内的再生特性以及 翻译应用程序的开发，如细胞疗法。 体细胞重新编程可以用来产生几乎无穷无尽的细胞的发现 (IPSCs)为开发能够在人类细胞中研究这些过程的策略带来了新的希望。 我们最近开发了有效的方案来区分小鼠和人类胚胎的多能性 干细胞(ES和iPS)在体外转化为横纹肌纤维和干细胞[1，6]。目前的建议旨在采取以下措施 这些体外模型结合小鼠体内研究的优势，以了解糖尿病的发生发展 人类的干细胞血统。 在小鼠中，干细胞构成了一个异质群体，包括一小部分休眠细胞 重要的再生能力和更快地划分更多的人口。无论这种异质性是 在人类中是否保守尚不清楚。此外，这种异质性的发展根源并不是 明白了。因此，拟议项目的一个重要目标是了解该项目的发展基础 异质性。我们将比较体内发育的小鼠Pax7+肌源性前体细胞的异质性 并在体外使用一种新引入的单细胞测序方法(InDrop)，该方法允许对 在一次实验中有数千个细胞。一项平行分析将对人类干细胞进行比较 体外分化为胎儿和成人肌肉组织。这些研究有望揭示 小鼠和人类干细胞的发育轨迹和Pax7+亚群的异质性。 我们还将利用我们的体外成肌分化系统进行高分辨率的活体- PAX7+谱系发育的影像研究。我们将描述记录不充分的方面 对干细胞分化的影响，如进入静止状态和对肌管的贡献。最后，我们有 维甲酸及其共激活剂复合体作为可逆诱导途径的研究 PAX7+细胞在体外分化为静止期。类风湿因子在细胞分化和维持中的作用 PAX7+的谱系尚未被研究，我们建议在体外和体内表征其功能 在发育和成熟干细胞方面。我们期望我们的工作能让我们了解 人类干细胞谱系，这可能对基于细胞的治疗方法的发展具有重要意义 肌肉疾病。

SUMMARY/ABSTRACT Adult skeletal muscle is endowed with significant regenerative capacity due to the presence of adult stem cells, called satellite cells (SCs). Very little is known about the developmental origin of these cells in humans and most of our knowledge derives from work performed in mouse. SCs are rare and they cannot be amplified in unlimited numbers, as they lose their regenerative potential in vitro. These issues have constituted a major roadblock for the study of their regenerative properties in humans and for the development of translational applications, such as cell therapy. The discovery that somatic cell reprogramming can be used to generate virtually endless numbers of cells (iPSCs) has brought new hope for developing strategies allowing to study these processes in human cells. We have recently developed efficient protocols to differentiate mouse and human embryonic pluripotent stem cells (ES and iPS) into striated muscle fibers and SCs in vitro [1, 6]. The present proposal aims to take advantage of these in vitro models combined with in vivo studies in mice to understand the development of the SC lineage in humans. In mouse, SCs constitute a heterogeneous population comprising a minor fraction of dormant cells with important regenerative capacity and a faster dividing larger population. Whether this heterogeneity is conserved in human is not known. Moreover, the developmental origin of this heterogeneity is not understood. Thus an important aim of the proposed project is to understand the developmental basis of this heterogeneity. We will compare the heterogeneity of mouse Pax7+ myogenic precursors developing in vivo and in vitro using a newly introduced method of single cell sequencing (InDrop) which allows to sequence thousands of cells in one experiment. A parallel analysis will be performed comparing human SCs differentiated in vitro to fetal and adult human muscle tissue. These studies are expected to reveal the developmental trajectories and the heterogeneity of the Pax7+ subpopulations in mouse and human SCs. We will also take advantage of our in vitro myogenic differentiation systems to perform high-resolution live- imaging studies of the development of the PAX7+ lineage. We will characterize poorly documented aspects of SC differentiation such as their entry in quiescence and their contribution to myotubes. Finally, we have identified Retinoic Acid (RA) and its co-activator complex WHHERE as a pathway able to induce reversible quiescence in PAX7+ cells differentiated in vitro. The role of RA in the differentiation and the maintenance of the PAX7+ lineage has not been investigated and we propose to characterize its function in vitro and in vivo in developing and mature SCs. We expect our work to shed light on the development and function of the human SC lineage, which could have significant implications for the development of cell-based therapies for muscle diseases.