Validating engineered hiPSC-derived cardiomyocytes as model cells

验证工程化 hiPSC 衍生心肌细胞作为模型细胞

• 批准号: 9678119
• 负责人: Beth L Pruitt
• 金额: $ 18.46万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9678119
• 关键词: Validating; engineered; hiPSC; derived; cardiomyocytes

 DESCRIPTION: Cardiovascular disease remains the #1 killer in the developed world. Loss of cardiomyocytes due to myocardial infarction or chronic apoptosis can lead to heart failure, affecting more than 23 million people worldwide. We currently lack a nuanced understanding of how the mechanisms of dysfunction of heart disease are linked to contractile phenotypes at the protein, sarcomere and cellular levels. Unfortunately, the scarcity of human heart tissue and the inability to maintain mature, primary cardiomyocytes in vitro has hindered investigation of the basic mechanisms of cardiotoxicity and human heart disease and physiology of cardiomyocytes. Conversely, human induced pluripotent stem cell-derived cardiomyocytes are readily available (even commercially), can be maintained for months in culture, and frozen for future use. A crucial motivation for this work is the divergence between human cardiomyocytes and animal model cardiomyocytes in physiology, structural composition, and fundamental biology. These differences are particularly acute when screening cardiotoxicity of drugs or treatments for use in humans. Human induced pluripotent stem cell-derived cardiomyocytes seem to hold great promise in this regard, but current protocols for deriving cells yield heterogeneous populations in terms of structure, function, contractility, and other crucial parameters. In this application, we seek to establish a high-risk, high-reward paradigm shift: that quantitative analysis of myofibril organization in engineered single cardiomyocytes with physiological shape and sarcomere organization will empower researchers to overcome the heterogeneities observed in human induced pluripotent stem cell-derived cardiomyocyte populations, positioning the contractile behavior and myofibril organization of human induced pluripotent stem cell-derived cardiomyocyte as models of cardiotoxicity and diseases of the myocardium (cardiomyopathies). We will engineer the morphology and subcellular myofibril alignment of human induced pluripotent stem cell-derived cardiomyocytes through their interface with mechanically tuned cell culture environments. By providing in situ non-destructive functional assessment, while driving maturity in single iPSC-cardiomyocytes, we will enable quantitative studies of contractility, work and power in terminally differentiated cardiomyocytes. These models of matured stem cell-derived cardiomyocytes also have the potential to avoid the problems of poor long-term survival of primary cardiomyocyte models in vitro, to reduce our reliance on animal models and to avoid their known differences from human cells. Our proposed project provides methods and systems for sustaining stem cell-derived cardiomyocytes in biomimetic culture conditions along with non-destructive contractility assays required to assess the function of these cells before and after interventions to rescue healthy phenotypes. We further aim to deploy these model systems and methods to characterize the biophysics of mutations causing heritable cardiomyopathies. We seek to demonstrate models suitable for future translation towards high- throughput testing of therapies with patient specificity.

 描述：心血管疾病仍然是发达国家中排名第一的杀手。由于心肌梗塞或慢性细胞凋亡导致的心肌细胞丧失会导致心力衰竭，影响全球超过2300万人。目前，我们对心脏病功能障碍的机制如何与蛋白质，肉瘤和细胞水平的收缩表型有关。不幸的是，人类心脏组织的稀缺性和无法维持成熟的原发性心肌细胞体外的稀缺性阻碍了对心脏毒性和人心脏病的基本机制以及心肌细胞生理学的研究。相反，人类诱导的多能干细胞衍生的心肌细胞容易获得（甚至在商业上），可以在培养中维持几个月，并冷冻以备将来使用。这项工作的关键动机是人类心肌细胞和动物模型心肌细胞在生理，结构组成和基本生物学方面的差异。当筛选药物或治疗中的心脏毒性时，这些差异尤其严重。人类诱导的多能干细胞衍生的心肌细胞似乎在这方面持有巨大的前景，但是当前用于导致细胞的方案在 结构，功能，收缩性和其他关键参数的术语。在此应用中，我们试图建立高风险，高等的范式转变：对具有身体形状和萨马尔组织的工程单一心肌细胞中肌原纤维组织的定量分析，将使研究人员能够克服人类诱导的多能干细胞衍生的人类造成的人类固定型和肌动物群体的固定型菌群，并在人类诱导的人类诱导的人类诱导的异质性中观察到的异质性，并在心肌细胞作为心肌毒性和疾病的模型（心肌病）。我们将通过机械调谐的细胞培养环境的界面来设计人类诱导的多能干细胞衍生的心肌细胞的形态和亚细胞肌原纤维比对。通过提供原位的非破坏性功能评估，在单个IPSC核心们肌细胞中推动成熟度时，我们将启用对终末分化心肌细胞的收缩力，工作和功率的定量研究。这些成熟的干细胞衍生的心肌细胞模型也有可能避免在体外原发性心肌细胞模型长期存活不良的问题，以减少我们对动物模型的保留率，并避免其与人类细胞的已知差异。我们提出的项目提供了在仿生培养条件下维持干细胞衍生的心肌细胞的方法和系统，并在干预措施中被暗杀以评估这些细胞的功能以挽救健康表型。我们进一步旨在部署这些模型系统和方法，以表征引起遗传性心肌病的突变的生物物理学。我们试图展示适合将将来翻译成具有患者特异性疗法的高通量测试的模型。