工程化骨骼肌再现了人类肌肉的发育、再生和萎缩。

BACKGROUND: Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. METHODS: Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. RESULTS: The established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7 ± 0.5 mN tetanic twitch tension at 100 Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107 ± 2 to 75 ± 4 ms (P < 0.05) and from 146 ± 6 to 100 ± 6 ms (P < 0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7 cells (75 ± 6% Ki67 ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57 ± 8% of the pre-injury force 21 days post-injury (P < 0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (-35 ± 7%, P < 0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175 ± 14 ms, P < 0.05 vs. gene-edited control) and relaxation (238 ± 22 ms, P < 0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. CONCLUSIONS: We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

背景：人类多能干细胞衍生的肌肉模型在转化研究中显示出巨大的潜力。在这里，我们描述了受发育启发的方法，用于衍生骨骼肌细胞，并将其用于骨骼肌组织工程，旨在体外模拟骨骼肌再生和营养不良。

方法：关键步骤包括将人多能干细胞定向分化为胚胎肌肉祖细胞，然后进行原代和次代胎儿肌发生，形成三维肌肉。为了模拟杜氏肌营养不良症（DMD），我们比较了一个患者特异性诱导多能干细胞系和一个 CRISPR/Cas9 编辑的同基因对照系。

结果：建立的骨骼肌分化方案在二维和三维培养中可靠且忠实地再现了胚胎肌发生的关键步骤，从而产生了功能性的人类骨骼肌类器官（SMO）和工程化的骨骼肌（ESM），具有再生能力的卫星样细胞池。组织工程化的肌肉表现出器官样成熟和功能（在 ESM 中，100Hz 时的最大单收缩张力可达 5.7±0.5mN）。通过定时甲状腺激素处理可以进一步增强收缩性能，缩短单个收缩的收缩（达到收缩峰值的时间）和松弛（达到 50%松弛的时间），从 107±2 到 75±4ms（P<0.05）和从 146±6 到 100±6ms（P<0.05）。可以记录到卫星样细胞主要为静止的 PAX7 细胞（Ki67 为 75±6%），位于受层粘连蛋白包含的基底膜限制的肌纤维旁边。在心脏毒素损伤模型中，工程化的卫星样细胞龛被激活，在损伤后 21 天，收缩力恢复到损伤前的 57±8%（与损伤后第 2 天相比，P<0.05），而在前照射后完全被阻断。DMD ESM 中缺乏肌营养不良蛋白导致收缩力显著降低（-35±7%，P<0.05），并影响快肌肌球蛋白同工型的表达，导致收缩（175±14ms，P<0.05 与基因编辑对照相比）和松弛（238±22ms，P<0.05 与基因编辑对照相比）时间延长。通过基因编辑恢复肌营养不良蛋白水平可挽救 ESM 中的 DMD 表型。

结论：我们引入了具有体内真实骨骼肌典型特性的人类肌肉模型，以研究肌肉发育、成熟、疾病和修复。