Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. A loss of dystrophin leads to severe and progressive muscle wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) and their derivatives offer important opportunities to treat a number of diseases. Here, we investigated whether givinostat (Givi), a histone deacetylase inhibitor, with muscle differentiation properties could reprogram hiPSCs into muscle progenitor cells (MPC) for DMD treatment. MPC were generated from hiPSCs by treatment with CHIR99021 and givinostat called Givi-MPC or with CHIR99021 and fibroblast growth factor as control-MPC. The proliferation and migration capacity were investigated by CCK-8, colony, and migration assays. Engraftment, pathological changes, and restoration of dystrophin were evaluated by in vivo transplantation of MPC. Conditioned medium from cultured MPC was collected and analyzed for extracellular vesicles (EVs). Givi-MPC exhibited superior proliferation and migration capacity compared to control-MPC. Givi-MPC produced less reactive oxygen species (ROS) after oxidative stress and insignificant expression of IL6 after TNF-α stimulation. Upon transplantation in cardiotoxin (CTX)-injured hind limb of Mdx/SCID mice, the Givi-MPC showed robust engraftment and restored dystrophin in the treated muscle than in those treated with control-MPC or human myoblasts. Givi-MPC significantly limited infiltration of inflammatory cells and reduced muscle necrosis and fibrosis. Additionally, Givi-MPC seeded the stem cell pool in the treated muscle. Moreover, EVs released from Givi-MPC were enriched in several miRNAs related to myoangiogenesis including miR-181a, miR-17, miR-210 and miR-107, and miR-19b compared with EVs from human myoblasts. It is concluded that hiPSCs reprogrammed into MPC by givinostat possessing anti-oxidative, anti-inflammatory, and muscle gene-promoting properties effectively repaired injured muscle and restored dystrophin in the injured muscle.

中文翻译：

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多能干细胞诱导的骨骼肌祖细胞与 givinostat 促进肌血管生成并恢复受损杜兴营养不良肌中的肌营养不良蛋白

杜氏肌营养不良症 (DMD) 是由编码肌营养不良蛋白的基因突变引起的。肌营养不良蛋白的丧失会导致骨骼肌和心肌严重的进行性肌肉萎缩。人类诱导多能干细胞（hiPSC）及其衍生物为治疗多种疾病提供了重要机会。在这里，我们研究了具有肌肉分化特性的组蛋白脱乙酰酶抑制剂 givinostat (Givi) 是否可以将 hiPSC 重编程为肌肉祖细胞 (MPC) 以进行 DMD 治疗。MPC 是通过用 CHIR99021 和 givinostat（称为 Givi-MPC）处理或用 CHIR99021 和成纤维细胞生长因子（作为对照-MPC）处理从 hiPSC 生成的。通过CCK-8、集落和迁移测定来研究增殖和迁移能力。通过MPC体内移植来评估抗肌营养不良蛋白的植入、病理变化和恢复。收集来自培养的 MPC 的条件培养基并分析细胞外囊泡 (EV)。与对照-MPC 相比，Givi-MPC 表现出优异的增殖和迁移能力。Givi-MPC 在氧化应激后产生较少的活性氧 (ROS)，在 TNF-α 刺激后产生微不足道的 IL6 表达。移植到心脏毒素 (CTX) 损伤的 Mdx/SCID 小鼠后肢后，与对照 MPC 或人成肌细胞治疗的肌肉相比，Givi-MPC 在治疗的肌肉中表现出强大的植入和恢复的肌营养不良蛋白。Givi-MPC 显着限制了炎症细胞的浸润并减少了肌肉坏死和纤维化。此外，Givi-MPC 将干细胞库接种到接受治疗的肌肉中。此外，与人类成肌细胞的 EV 相比，Givi-MPC 释放的 EV 富含多种与肌血管生成相关的 miRNA，包括 miR-181a、miR-17、miR-210 和 miR-107 以及 miR-19b。结论是，具有抗氧化、抗炎和肌肉基因促进特性的givinostat将hiPSC重编程为MPC，可有效修复受损肌肉并恢复受损肌肉中的肌营养不良蛋白。