DISEASE MODELING AND PHENOTYPIC DRUG SCREENING FOR DYSTROPHIC CARDIOMYOPATHY

项目摘要 目标：我们建议利用一系列新的基于人类iPSC的心脏微生理系统， 研究营养不良心肌细胞的成熟是必要的这一假设的复杂性增加 阐明正确的疾病表型体外发育。其目的是建立一个多方面的筛选 系统使用我们小组开发的几种核心技术来评估心肌的不同方面， 机电功能我们已经开发出从病人尿液中制造多能干细胞的能力， 使得能够从人类受试者进行非侵入性细胞取样。此外，我们还收集了初步数据， 证明了将组合成熟刺激物应用于健康和肌营养不良蛋白缺失 心肌细胞有助于对疾病表型进行分层。基于这些成果，我们认为， 结合适当的成熟刺激物的靶向功能测定集将提供更好的 全面了解肌营养不良症的疾病进展。焦点/目标：我们的建议 研究重点是使用具有协同作用潜力的技术， 通过操作不同的细胞因子在干细胞衍生的心肌细胞中的表型发育 机制等具体来说，我们将研究纳米图案化基板的结构组织的影响， 甲状腺激素引起的核受体信号传导和Let-7引起的代谢信号传导途径的改变 microRNA过表达对健康心肌细胞发育及其抗肌萎缩蛋白缺失的影响 使用CRISPR-Cas9基因编辑技术创建的对应物。纳米形貌微电极阵列 将用于评估电生理功能（目标1），而纳米图案化细胞片堆叠 技术将用于创建3D心脏补丁，用于分析收缩功能（目标2）以及 有组织的3D心室结构，用于评估压力生成和每搏输出量（目标3）。这一切成功都 系统将用于评估一组药物改善营养不良表型的潜力。 本研究选择的化合物靶向一系列已知的代谢，结构和信号通路， 与肌营养不良病理学的不同方面有关。多功能分析 因此，每种化合物的终点将提供关于 当药物被用于人类患者时。从具有较高吞吐量水平的平台向 随着工作从目标1过渡到目标3，那些具有更高程度的仿生学， 药物筛选过程的“乐趣”。使用更简单的多路复用模型确定的候选人将被重新 使用能够更接近地代表天然组织并提供生理信息的系统进行评估 终点类似于患者中监测的终点。因此，所提出的研究成熟的方法 营养不良表型的发展可以为下一代筛选过程提供框架 旨在用越来越具有生理代表性的动物模型取代动物试验， 心肌

PROJECT SUMMARY Goal: We propose to utilize a series of novel human iPSC-based cardiac microphysiological systems of increasing complexity to investigate the hypothesis that maturation of dystrophic cardiomyocytes is necessary to elucidate correct disease phenotype development in vitro. The aim is to create a multifaceted screening system using several core technologies developed by our group to evaluate different aspects of myocardial electromechanical function. We have developed the ability to engineer pluripotent stem cells from patient urine, enabling non-invasive cell sampling from human subjects. Furthermore, we have collected preliminary data demonstrating that application of combinatorial maturation stimuli to healthy and dystrophin-null cardiomyocytes helps stratify the disease phenotype. Based on these achievements, we posit that the use of a targeted set of functional assays in combination with appropriate maturation stimuli will provide a more comprehensive understanding of disease progression in muscular dystrophy. Focus/Aim: Our proposed research focuses on the use of techniques with the potential to act synergistically to enhance cardiac phenotype development in stem cell-derived cardiomyocytes through manipulation of different cellular mechanisms. Specifically, we will investigate the effect of structural organization by nanopatterned substrates, nuclear receptor signaling by thyroid hormone, and alterations in metabolic signaling pathways by Let-7 microRNA over-expression on the development of healthy cardiomyocytes and their dystrophin-null counterparts created using CRISPR-Cas9 gene editing technology. Nanotopographic microelectrode arrays will be used to evaluate electrophysiological function (Aim 1), while nanopatterned cell sheet stacking technology will be used to create 3D cardiac patches for analyzing contractile function (Aim 2) as well as organized 3D ventricle structures for assessing pressure generation and stroke volume (Aim 3). Each of these systems will be used to evaluate a panel of drugs for their potential to ameliorate the dystrophic phenotype. The compounds chosen for this study target a range of metabolic, structural, and signaling pathways known to be associated with different aspects of muscular dystrophy pathology. The analysis of multiple functional endpoints for each compound will therefore provide more comprehensive information on the likely effect of drugs when administered to human patients. The movement from platforms with higher levels of throughput to those with higher degrees of biomimicry, as the work transitions from Aim 1 to Aim 3, constitutes a natural “funneling” of the drug screening process. Candidates identified using simpler multiplexed models will be re- evaluated using systems that offer closer representations of the native tissue, and provide physiological endpoints analogous to those monitored in patients. As such, the proposed method for studying maturation and dystrophic phenotype development could provide the framework for a next generation screening process geared towards replacing animal testing with increasingly physiologically representative models of the myocardium.