Interrogating functional and molecular properties of PAX7+ putative skeletal muscle stem/progenitor cells derived from human iPSCs of healthy donors and Duchenne muscular dystrophy patients

肌肉萎缩，由衰老、基因突变、康康病相关的恶病质或创伤引起， 会导致严重的功能损害，是一个具有挑战性的临床问题，具有显著的 给我们的医疗体系带来社会经济负担。 我们已经证明，有功能的成肌细胞很容易从人类胚胎干细胞中获得。 (HESCs)和人类诱导多能干细胞(HiPSCs)，使我们能够开始研究Duchenne肌肉 营养不良(DMD)，最常见的肌肉遗传性疾病。然而，我们对i)有多早知之甚少 肌源性事件在发育过程中受基因控制，II)胚胎是否表达PAX7 肌源性干/祖细胞在出生后采用“卫星样”的命运，以及iii)DMD是如何在骨骼中发生的 肌肉干/祖细胞阶段及其与细胞替代治疗的相关性。 首先，用多个基因报告系来概括人类的生肌事件，我们将描绘一个时间-- 转录景观的过程分析，紧随其后的是“功能丧失”分析，以解决基本问题 关于哪个关键细胞内在/外在成分(S)支配骨骼肌规格的问题 过程和干细胞维护。 其次，通过对人PAX7：：GFP+假想骨骼肌进行连续移植 干细胞/祖细胞在小鼠模型中，我们将询问胚胎细胞是如何在出生后成为 卫星般的细胞命运。 第三，基于我们对人骨骼肌中肌营养不良蛋白表达的观察 干细胞/祖细胞，我们将研究Dystrophin的阶段特异性作用(S)及其漫长的非基因间隔 编码RNA(LincRNAs)，在健康和DMD条件下。此外，我们将审问体内再生 转基因DMD-hiPSC系患者特异性PAX7：：GFP+细胞的能力。 我们提议的实验有望扩展和加强我们目前对肌源性的概念。 规范事件，并加快对骨骼肌疾病的广泛研究，例如创伤性 肌肉损伤、遗传性肌营养不良、神经肌肉疾病、II型糖尿病和癌症 恶病质。

Muscle wasting, caused by aging, genetic mutations, cancan-associated cachexia, or traumatic injury, can result in significant functional impairment, and is a challenging clinical problem with a significant socioeconomic burden on our healthcare system. We have shown that functional myoblasts are readily derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), allowing us to begin to study Duchenne muscular dystrophy (DMD), the most common genetic disorder of muscle. However, we know little about i) how early myogenic events are genetically controlled during development, ii) whether embryonic PAX7 expressing myogenic stem/progenitor cells adopt postnatal `satellite-like' fate, and iii) how DMD is occurred in skeletal muscle stem/progenitor cell stage as well as their relevance for cell replacement therapy. First, using multiple genetic reporter lines to recapitulate human myogenic events, we will depict a time- course analysis of transcriptional landscape followed by `loss of function' analysis to address essential questions regarding which critical cell intrinsic/extrinsic component(s) govern the skeletal muscle specification process and stem cell maintenance. Secondly, by performing serial transplantation of human PAX7::GFP+ putative skeletal muscle stem/progenitor cells in mouse model, we will interrogate how the embryonic cells become to postnatal satellite-like cell fate. Thirdly, based on our observation on DYSTROPHIN expression in human skeletal muscle stem/progenitor cells, we will investigate stage-specific role(s) of DYSTROPHIN and its long intergenic non- coding RNAs (LincRNAs), in healthy and DMD condition. In addition, we will interrogate in vivo regeneration ability of patient-specific PAX7::GFP+ cells of genetically corrected DMD-hiPSC lines. Our proposed experiments are expected to expand and strengthen our current conception of myogenic specification events, and to accelerate a wide range of research on skeletal muscle disorders, e.g. traumatic muscle damages, genetic muscular dystrophies, neuromuscular diseases, type II diabetes and cancer-induced cachexia.