Engineering Highly Functional Pre-Vascularized Human Skeletal Muscle for In Vitro and In Vivo Applications

摘要 目前工程化人骨骼肌模型主要由成肌细胞和成纤维细胞组成 其在各种类型的天然或合成生物材料支架中生长。简化的细胞组成 这些组织会限制它们在疾病建模和再生治疗中的效用， 还包括其他的肌肉驻留细胞类型，如血管细胞。然而，到目前为止，还没有研究表明 证明了成熟的功能性工程化肌肉的成功体外血管化，而没有损失 收缩功能Bursac实验室是第一个设计收缩性人类骨骼肌组织的实验室 在一些实施方案中，所述肌束由原代人成肌细胞或诱导的多能干细胞衍生的肌肉祖细胞制成。 我的初步结果表明，在优化条件下，将成肌细胞与5%的内皮祖细胞混合， 在组织形成时，内皮祖细胞（EPCs）产生具有收缩功能的血管化组织， 到肌肉控制的程度。重要的是，随着培养时间的推移，在整个细胞中形成了强大的血管网络。 肌束体积经历早期管腔形成。在这些有希望的结果的基础上， 在高功能的工程化人体肌肉内设计密集的毛细血管网络将：1）支持 维持肌肉干细胞（卫星细胞，SC）的生态位，增强体外再生能力， 2）加速血管化和肌束植入物的灌注以改善它们的存活， 在体内体积肌肉损失（VML）损伤模型中的治疗功效。具体来说，我将描述 使用单细胞RNA测序， 将研究血管化诱导的SC激活和肌肉再生对毒素的反应变化 损伤在免疫功能低下的小鼠体内，我将分析如何建立血流通过前， 血管化肌束植入物影响SC表型，并将进一步评估植入的 肌束诱导胫骨前肌VML损伤的修复。如果成功，这项工作将建立 第一个高功能的仿生模型，血管化的人类骨骼肌组织，并将提供一个 为未来追求VML的工程肌肉疗法奠定了基础。

Abstract Current models of engineered human skeletal muscle mainly consist of myogenic cells and fibroblasts which are grown in various types of natural or synthetic biomaterial scaffolds. The simplified cellular makeup of these tissues can limit their utility in disease modeling and regenerative therapies, which can be improved by incorporating additional muscle-resident cell types such as vascular cells. However, to date, no studies have demonstrated successful in vitro vascularization of mature functional engineered muscle without loss of contractile function. The Bursac lab has been the first to engineer contractile human skeletal muscle tissues (“myobundles”) made of primary human myoblasts or induced pluripotent stem cell-derived muscle progenitors. My preliminary results show that under optimized conditions, mixing myoblasts with 5% endothelial progenitor cells (EPCs) at the time of tissue formation produces vascularized tissues with contractile function comparable to that of muscle-only controls. Importantly, with time of culture, robust vascular networks formed throughout the myobundle volume undergo early lumen formation. Building on these promising results, I will test the hypotheses that engineering dense capillary networks inside highly functional engineered human muscle will: 1) support the maintenance of the muscle stem cell (satellite cell, SC) niche and enhance in vitro regenerative capacity of myobundles and 2) accelerate vascularization and perfusion of myobundle implants to improve their survival and therapeutic efficacy in volumetric muscle loss (VML) injury model in vivo. Specifically, I will characterize the effect of myobundle-EPC coculture on the transcriptomic profile of resident SCs using single cell RNA-sequencing and will investigate vascularization-induced changes in SC activation and muscle regeneration in response to a toxin injury. In immunocompromised mice in vivo, I will analyze how establishment of blood flow through pre- vascularized myobundle implants affects SC phenotype and will further assess potential of implanted myobundles to induce repair of VML injury in the tibialis anterior muscle. If successful, this work will establish the first biomimetic model of highly functional, vascularized human skeletal muscle tissue and will provide a foundation for future pursuits of engineered muscle therapies for VML.