PDF(Pubmed)
Abstract：
Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors\' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.
摘要：
各种方案已被证明在小鼠和人多能干细胞定向分化为骨骼肌中有效，并用于研究肌肉发生。当前的2D肌源性分化方案可以模拟肌肉发育及其在病理条件例如肌营养不良下的改变。3D骨骼肌分化方法可以，此外,对发育中的类器官内各种细胞类型之间的相互作用进行建模。我们的方案确保人类胚胎/诱导多能干细胞（hESC/hiPSC）分化为骨骼肌类器官（SMO），通过具有近轴中胚层和神经中胚层祖细胞身份的细胞，并进一步产生神经板缘和皮肌细胞组的有组织结构。连续培养省略神经谱系分化和促进胎儿肌生成,包括纤维脂肪原细胞和PAX7阳性肌原细胞的成熟。PAX7祖细胞类似于人类发育的晚期胎儿阶段，基于单细胞转录组分析，聚集在初级肌肉的成年卫星细胞附近。为了克服肌肉萎缩症患者在疾病进展过程中肌肉活检的局限性，我们建议使用SMO系统，它从患者特异性iPSCs中提供稳定的骨骼肌祖细胞群体，以研究健康和疾病条件下的人类肌肉生成。主要特征•从人类多能干细胞分化骨骼肌器官的发展，概括了肌肉发生。•早期胚胎和胎儿肌生成的分析。•提供骨骼肌祖细胞用于长达14周的类器官培养的体外和体内分析。•来自患者特异性iPSC的体外肌生成允许克服具有病理状况的患者的肌肉活检的瓶颈。
