Engineering iPSC-Derived Skeletal Muscle and Cells for Transplantation

摘要 慢性肌肉疾病，包括杜氏肌营养不良症（DMD）和衰老相关的肌肉减少症 肌肉无力，丧失独立性，死亡风险增加。此外，创伤性肌肉损伤 因事故，手术，战伤等造成的损失，需要长期恢复。骨骼肌是一种高度 再生组织，其中卫星细胞，骨骼肌的干细胞群，在以下方面发挥重要作用： 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消失。 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， 卫星细胞是罕见的（所有肌肉细胞核的百分之几），并且通常难以分离。患者源性诱导 多能干细胞（iPSC）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，有效的肌源性分化和肌源性分化的规模扩大， 肌源性分化仍然难以捉摸，必须进一步发展以产生有效的细胞分化， 治疗在宿主动物中产生人肌源性细胞和骨骼肌的关键是 选择性敲除囊胚中对器官发育至关重要的基因。因此，在本提案中， (1)我们将确定在注射后小鼠iPSC衍生的肢体骨骼肌将在多大程度上产生 将小鼠iPSC转化为Pax3突变小鼠胚泡，创造了干细胞可以占据的小生境， 在肢体中形成骨骼肌。这种方法将为整个骨骼肌的产生提供证据 通过小鼠iPSC在体内。此外，（2）我们将使用Pax3突变体产生人源化骨骼肌。 通过子宫内注射人iPSC与Pax3突变体胚胎和iPSC的组合来制备小鼠胚胎。 这种协调一致的方法将帮助我们在体内创造iPSC衍生的骨骼肌和肌源性细胞， 可以移植到患者体内，用于彻底治愈肌病和肌肉损伤。此外，本发明还提供了一种方法， 小鼠中的人源化骨骼肌将作为研究其特性的动物模型， 人骨骼肌疾病的再生特点及对药物的反应 并提供用于产生自体肌肉的患者来源的细胞和组织来源的概念验证 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. .