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

工程设计用于体外和体内应用的高功能预血管化人体骨骼肌

基本信息

批准号：
10535766
负责人：
Torie M Broer
金额：
$ 3.95万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10535766
关键词：
3-Dimensional Affect Attenuated Biomimetics Blood Vessels Blood capillaries Blood flow Cell Therapy Cell physiology Cells Coculture Techniques Complex Disease Disease model Dorsal Endothelial Cells Engineering Erythrocytes Fibroblasts Foundations Future Generations Harvest Heterogeneity Histologic Human Human Engineering Image Immunocompromised Host Implant In Situ In Vitro Injections Injury Ischemia Label Laboratories Ligands Maintenance Metabolism Methods Modeling Mus Muscle Muscle Development Muscle Fibers Muscle satellite cell Myoblasts NOTCH3 gene Neuromuscular Junction Nude Mice Pathway interactions Perfusion Phenotype Population Protocols documentation Rattus Recovery of Function Regenerative capacity Regenerative response Rodent Role Signal Transduction Site Skeletal Muscle Skeletal Myoblasts Supporting Cell Testing Therapeutic Time Tissue Engineering Tissue Survival Tissues Toxin Treatment Efficacy Vascularization Work alpha Toxin bioscaffold cell type comparative density design drug discovery endothelial stem cell experience human model implantation improved in vivo in vivo Model induced pluripotent stem cell intravital imaging muscle engineering muscle form muscle regeneration muscular structure neurovascular notch protein progenitor receptor regenerative regenerative therapy repaired response response to injury satellite cell single-cell RNA sequencing stemness tibialis anterior muscle transcriptomics volumetric muscle loss

项目摘要

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.

摘要目前的工程化人类骨骼肌模型主要由肌源性细胞和成纤维细胞组成它们生长在各种类型的天然或合成生物材料支架中。简化的蜂窝结构这些组织可能会限制它们在疾病建模和再生治疗中的用途，这可以通过以下方法来改进并入其他肌肉驻留细胞类型，如血管细胞。然而，到目前为止，还没有研究表明成熟的功能工程化肌肉在体外成功地血管化而不会丢失收缩功能。Bursac实验室是第一个设计收缩人体骨骼肌组织的实验室 (“肌束”)由原代人类成肌细胞或诱导的多能干细胞来源的肌祖细胞组成。我的初步结果显示，在优化的条件下，成肌细胞与5%的内皮祖细胞混合组织形成时的细胞(内皮祖细胞)产生血管组织，具有类似的收缩功能到只有肌肉的控制组。重要的是，随着培养时间的推移，健壮的血管网络在整个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，我将检验这些假设在高度功能的工程化人体肌肉中设计致密的毛细血管网络将：1)支持维持肌肉干细胞(卫星细胞，SC)的生态位并增强其体外再生能力肌束和2)加速肌束植入物的血管化和灌流，以提高其存活率和体内VML损伤模型的治疗效果。具体地说，我将描述这种影响应用单细胞RNA测序和免疫印迹技术研究Myobundle-EPC共培养对驻留干细胞转录谱的影响将研究血管形成引起的SC激活和肌肉再生对毒素的反应的变化受伤。在体内免疫低下的小鼠中，我将分析如何通过预先建立血流血管化肌束植入影响SC表型，并将进一步评估植入的潜力肌束诱导胫前肌VML损伤修复。如果成功，这项工作将建立第一个高功能、血管化的人类骨骼肌组织的仿生模型，将提供为VML的工程化肌肉疗法的未来追求奠定基础。