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

总结/摘要 由于成体干细胞的存在，成体骨骼肌具有显著的再生能力 这些细胞被称为卫星细胞（SC）。关于人类这些细胞的发育起源知之甚少 我们的大部分知识都来自于在小鼠身上进行的工作。SC很罕见， 它们在体外失去了再生潜力，因此被无限放大。这些问题 构成了研究它们在人类中再生特性的主要障碍， 开发翻译应用，如细胞治疗。 发现体细胞重编程可以用来产生几乎无限数量的细胞 iPSCs的发现为开发允许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协议，以区分小鼠和人类胚胎多能 干细胞（ES和iPS）体外转化为横纹肌纤维和SC [1，6]。本建议旨在 这些体外模型与小鼠体内研究相结合的优势，以了解 人类的SC谱系。 在小鼠中，SC构成异质群体，其包含少量休眠细胞， 重要的再生能力和更快地划分更大的人口。这种异质性是否 在人类中是未知的。此外，这种异质性的发展起源并不是 明白因此，拟议项目的一个重要目的是了解这一发展基础， 异质性我们将比较小鼠Pax7+肌源性前体在体内发育的异质性， 并在体外使用新引入的单细胞测序（InDrop）方法， 在一个实验中使用数千个细胞。将进行平行分析，比较人SC 在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SC中Pax7+亚群的发育轨迹和异质性。 我们还将利用我们的体外肌源性分化系统，进行高分辨率的实时成像， PAX7+谱系发育的成像研究。我们将描述记录不良的方面 SC分化，如他们进入静止和他们的贡献肌管。我们终于有 确定视黄酸（RA）及其共激活剂复合物WHHERE作为能够诱导可逆性 在体外分化的PAX7+细胞中静止。RA在分化和维持中的作用 PAX7+谱系尚未被研究，我们建议在体外和体内表征其功能 发育和成熟的SC。我们希望我们的工作能够阐明 人SC谱系，这可能对开发基于细胞的治疗方法具有重要意义， 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.