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.

抽象的当前的工程人骨骼肌模型主要由肌源性细胞和成纤维细胞组成在各种类型的天然或合成生物材料支架中生长。简化的细胞构成这些组织可以限制其在疾病建模和再生疗法中的效用，可以通过结合其他肌肉居民细胞类型，例如血管细胞。但是，迄今为止，还没有研究证明了成熟功能性工程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人类骨骼肌肉组织的人（“ myobundles”）由原代人肌细胞或诱导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％内皮祖细胞混合组织形成时的细胞（EPC）产生具有收缩功能可比的血管化时间仅肌肉对照。重要的是，随着文化的时间，整个过程中形成了强大的血管网络 MyObundle体积发生早期的管腔形成。在这些承诺结果的基础上，我将测试假设高度功能的人体肌肉中的工程密集毛细管网络将：1）支持维持肌肉干细胞（卫星细胞，SC）生态位并增强体外再生能力的肌收发板和2）加速肌动物的血管化和灌注，以提高其生存和体内体积肌肉损失（VML）损伤模型的治疗效率。具体来说，我将表征效果 Myobundle-EPC共培养在居民SC的转录组概况上使用单细胞RNA-sequing和将研究血管化引起的SC激活和肌肉再生的变化，以响应毒素在体内免疫功能低下的小鼠中，我将分析血液流动通过前的建立血管化的myobundle Imprans会影响SC表型，并将进一步评估植入的潜力肌收入诱导胫骨前肌的VML损伤修复。如果成功，这项工作将确定高度功能，血管化的人骨骼肌组织的第一个仿生模型，将提供为VML的工程肌肉疗法的未来追求基础。