Engineering iPSC-Derived Skeletal Muscle and Cells for Transplantation

摘要 慢性肌肉疾病，包括Duchenne肌营养不良症(DMD)和与衰老相关的骨量减少 肌肉无力，丧失独立性，死亡风险增加。此外，创伤性肌肉损伤 由于事故、手术和战时受伤造成的损失需要长期恢复。骨骼肌是一种高度的 再生组织，其中卫星细胞，骨骼肌的干细胞群，在 创造和修复骨骼肌。然而，这种潜力最终会随着疾病和衰老而失效。 自体卫星细胞移植是创造和修复骨骼肌纤维的一种潜在方法，但 卫星细胞很少见(只占所有肌肉细胞核的几%)，而且通常很难分离。患者派生的诱导 多能干细胞是获得无限数量的肌源性细胞的理想细胞来源 避免移植后的免疫排斥反应。然而，高效的成肌分化和放大的 肌源性分化仍然难以捉摸，必须进一步发展才能产生有效的细胞 治疗。在宿主动物体内产生人类肌源性细胞和骨骼肌的关键是 选择性敲除胚泡中对器官发育至关重要的基因。因此，在这份提案中， (1)我们将确定注射后小鼠IPSC衍生肢体骨骼肌的生成程度 将小鼠IPSCs移植到Pax3突变的小鼠囊胚中，创造了一个干细胞可以占据和 在四肢中形成骨骼肌。这种方法将为创造完整的骨骼肌提供证据 通过小鼠体内的ipscs。此外，(2)我们将使用Pax3突变体生成人源化骨骼肌 小鼠胚胎宫内注射人ipscs联合Pax3突变胚胎和ipscs。 这种协调一致的方法将帮助我们在体内创造IPSC来源的骨骼肌和肌源性细胞， 可以移植到患者体内，最终治愈肌肉疾病和肌肉损伤。此外, 人源化的小鼠骨骼肌将作为研究其特性和功能的动物模型。 人类骨骼肌病的再生特点及对药物的反应 并为为自体肌肉生成患者来源的细胞和组织资源提供概念证明 移植。 。

Abstract Chronic muscle diseases including Duchenne muscular dystrophy (DMD) and aging-related sarcopenia result in muscle weakness, loss of independence, and increased risk of death. In addition, traumatic muscle injury and loss due to accidents, surgery, and wartime injuries needs prolonged recovery. Skeletal muscle is a highly regenerative tissue in which satellite cells, a stem cell population for skeletal muscle, play essential roles in creating and repairing skeletal muscle. However, this potential ultimately fails with disease and aging. Autologous satellite cell transplantation is a potential approach to create and repair skeletal muscle fibers, but satellite cells are rare (a few % of all muscle nuclei) and often difficult to isolate. Patient-derived induced pluripotent stem cells (iPSCs) are the ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. However, efficient myogenic differentiation and the scale-up of myogenic differentiation remain elusive and must be developed further in order to generate effective cellular therapies. A key to the generation of human myogenic cells and skeletal muscle in a host animal is the selective knockout of genes in the blastocyst that are critical for organ development. Therefore, in this proposal, (1) we will determine to which extent mouse iPSC-derived limb skeletal muscle will be generated after injection of mouse iPSCs into Pax3 mutant mouse blastocysts, creating a niche in which stem cells can occupy and form skeletal muscle in the limb. This approach will provide evidence for the creation of entire skeletal muscle by mouse iPSCs in vivo. In addition, (2) we will generate a humanized skeletal muscle using Pax3 mutant mouse embryos via in utero injection of human iPSCs in combination with Pax3 mutant embryos and iPSCs. This concerted approach will help us to create iPSC-derived skeletal muscle and myogenic cells in vivo that can be transplanted into patients for a definitive cure of myopathic diseases and muscle injuries. In addition, the humanized skeletal muscle in mice will serve as an animal model to study the characteristics and regeneration characteristics of the human skeletal muscle diseases and responses to pharmacological agents and to provide a proof of concept for generating patient-derived cell and tissue sources for autologous muscle transplantation. .