Stem cell-based tissue engineering for myotendinous junction modeling and repair

项目摘要/摘要 肌腱连接是肌肉和肌腱与受累部位之间的直接界面。 创伤性的 肌肉损伤和肌腱断裂。考虑到现有治疗方法的效果不一致 肌腱损伤，我们建议开发一种替代方法，使用人类诱导的多能干细胞 细胞(IPSCs)。这样的细胞有能力产生祖细胞，可以促进肌肉和肌腱的形成 再生。我们这项研究的具体目标是设计IPSC衍生的肌腱单位并评估 它们作为研究肌腱连接形成和修复肌肉损伤的体外模型的效用， 肌腱和肌腱连接。指导这一提议的基本假设是IPSC派生的 肌肉骨骼祖细胞(骨骼肌祖细胞和肌腱祖细胞)将与 肌腱之间相互连接，形成肌腱单位，肌腱连接具有功能。这一假设得到了支持 根据我们发表的研究和初步数据，证明了生产人类肌肉骨骼的可行性 来自iPSCs的组织。在这项计划中，我们将从IPSCs中制备人肌腱祖细胞系(HTPC)。 建立的细胞将与IPSC来源的骨骼肌祖细胞(HSMPC)共同培养，使用 新推出的IPSCs细胞培养系统、二维微图案化培养平台(目标1)。 微图案的地形和分子引导可以模拟细胞和分子的复杂性 在肌肉骨骼发育和病理学方面。接下来，我们将创建三维肌肉-肌腱组织 IPSC来源的肌肉骨骼祖细胞的培养及其解剖和生理特性分析 扩展IPSCs用于模拟肌腱连接形成的用途的特性(目标2)。在整个过程中 开发这些3D培养模型，我们希望确定外部刺激的作用，如 肌肉-肌腱分化和肌腱连接的信号分子和机械负荷 队形。最后，我们将测试IPSC来源的肌腱组织再生受损肌肉的能力， 肌腱和肌腱连接在大鼠完全肌腱连接模型中的植入研究 破裂(目标3)。这些目标将为在体外使用基于IPSC的有效方法提供高度新颖的见解 模特和治疗。由于IPSCs现在可以从人类成年躯体组织中分离出来，这种方法可以 用于开发针对患者的、基于细胞的体外模型和治疗人类疾病的方法。结果是 该项目将加快对肌肉骨骼疾病患者进行有效治疗的进展。 鉴于缺乏对肌腱损伤的有效治疗以及由此带来的负担 对于社会来说，这项研究既紧迫又及时。

PROJECT SUMMARY/ABSTRACT Myotendinous junctions are the direct interface between muscle and tendon and the affected site in traumatic muscle injury and myotendinous rupture. Given the inconsistent effectiveness of existing treatments for myotendinous injuries, we propose developing an alternative approach using human induced pluripotent stem cells (iPSCs). Such cells have the capacity to create progenitor cells that can contribute to muscle and tendon regeneration. Our specific goals of this study are to engineer iPSC-derived muscle-tendon units and evaluate their utility as an in vitro model to study myotendinous junction formation, and to repair damages in muscle, tendon, and myotendinous junctions. The fundamental hypothesis guiding this proposal is that iPSC-derived musculoskeletal progenitor cells (skeletal muscle progenitor cells and tendon progenitor cells) will interact with each other and form a muscle-tendon unit with functional myotendinous junctions. This hypothesis is supported by our published studies and preliminary data demonstrating the feasibility of producing human musculoskeletal tissues from iPSCs. In this proposal, we will prepare lines of human tendon progenitor cells (hTPCs) from iPSCs. The established cells will be co-cultured with iPSC-derived skeletal muscle progenitor cells (hSMPCs) using newly featured cell culture systems for iPSCs, two-dimensional micropatterned culture platforms (Aim 1). Topographical and molecular guidance from the micropatterns can simulate cellular and molecular complexity in musculoskeletal development and pathology. Next, we will create three-dimensional muscle-tendon tissue cultures using iPSC-derived musculoskeletal progenitor cells and analyze their anatomical and physiological properties to extend the utility of iPSCs for modeling myotendinous junction formation (Aim 2). Throughout the development of these 3D culture models, we hope to identify the roles of exogenous stimulations such as signaling molecules and mechanical loads for muscle-tendon differentiation and myotendinous junction formation. Lastly, we will test the capacity of iPSC-derived muscle-tendon tissues to regenerate injured muscle, tendon, and myotendinous junctions by studying implantation in a rat model of complete myotendinous junction rupture (Aim 3). These aims will provide highly novel insights into effective approaches using iPSC-based in vitro modeling and treatments. As iPSCs can now be derived from human adult somatic tissues, this approach can be used to develop patient-specific, cell-based in vitro models and therapy for human disease. The results of this project will accelerate progress towards effective treatments for patients with musculoskeletal disorders. Given the lack of effective treatments for myotendinous injuries and the consequential burden it places on society, this study is both urgent and timely.