Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.

中文翻译：

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用于从 iPSC 中 3D 衍生人肌源性祖细胞的工程化多细胞干细胞生态位

胚胎中的命运决定受三维生态位中过多的微环境相互作用控制。为了研究是否可以设计这种微环境复杂性的各个方面来指导肌源性人类诱导的多能干细胞 (hiPSC) 分化，我们在这里筛选了存在于异型悬浮胚胎中发育或成体干细胞生态位中的鼠类细胞类型。我们发现胚胎内皮细胞和成纤维细胞高度允许 hiPSC 的肌源性规范。经过两周的连续 Wnt 和 FGF 通路诱导，这些三组分胚状体富含 Pax7 阳性胚胎样肌源性祖细胞，可以通过流式细胞术分离。异型胚状体中 hiPSC 的成肌分化依赖于专门的结构微环境，并依赖于 MAPK、PI3K/AKT 和 Notch 信号传导。在杜氏肌营养不良症小鼠模型中移植后，胚胎样肌原性祖细胞重新填充干细胞生态位，在反复损伤后重新激活，并且与成人成肌细胞相比，显示出增强的融合并导致肌肉功能增强。总而言之，我们提供了一个为期两周的协议，用于从 hiPSC 中高效且可扩展地基于悬浮液的 3D 推导 Pax7 阳性肌源性祖细胞。显示增强的融合并导致肌肉功能增加。总而言之，我们提供了一个为期两周的协议，用于从 hiPSC 中高效且可扩展地基于悬浮液的 3D 推导 Pax7 阳性肌源性祖细胞。显示增强的融合并导致肌肉功能增加。总而言之，我们提供了一个为期两周的协议，用于从 hiPSC 中高效且可扩展地基于悬浮液的 3D 推导 Pax7 阳性肌源性祖细胞。