Validating engineered hiPSC-derived cardiomyocytes as model cells

 描述：心血管疾病仍然是发达国家的头号杀手。心肌梗死或慢性细胞凋亡导致的心肌细胞丢失可导致心力衰竭，全球有2300多万人受到影响。我们目前缺乏对心脏病功能障碍的机制如何在蛋白质、肌节和细胞水平上与收缩表型相关联的细微差别的理解。遗憾的是，人类心脏组织的稀缺和在体外无法维持成熟的原代心肌细胞，阻碍了心脏毒性和人类心脏病的基本机制的研究和心肌细胞的生理学研究。相反，人类诱导的多能干细胞来源的心肌细胞很容易获得(甚至在商业上也是如此)，可以在培养中保持数月，并冷冻以备将来使用。这项工作的一个重要动机是人类心肌细胞和动物模型心肌细胞在生理、结构组成和基础生物学方面的差异。在筛选用于人类的药物或治疗的心脏毒性时，这些差异尤其明显。在这方面，人类诱导的多能干细胞来源的心肌细胞似乎有很大的希望，但目前的细胞来源方案在 结构、功能、伸缩性和其他关键参数。在这项应用中，我们寻求建立一种高风险、高回报范式转变：对具有生理形状和肌节组织的工程单个心肌细胞中的肌原纤维组织的定量分析将使研究人员能够克服在人类诱导的多能干细胞来源的心肌细胞群体中观察到的异质性，将人类诱导的多能干细胞来源的心肌细胞的收缩行为和肌原纤维组织定位为心脏毒性和心肌疾病(心肌病)的模型。我们将通过其与机械调节的细胞培养环境的接口来设计人类诱导的多能干细胞来源的心肌细胞的形态和亚细胞肌原纤维排列。通过提供原位非破坏性功能评估，同时促进单个IPSC-心肌细胞的成熟，我们将能够对终末分化的心肌细胞的收缩能力、功和功率进行定量研究。这些成熟的干细胞来源的心肌细胞模型也有可能避免原代心肌细胞模型在体外长期存活不佳的问题，减少我们对动物模型的依赖，并避免它们与人类细胞的已知差异。我们建议的项目提供了在仿生培养条件下维持干细胞来源的心肌细胞的方法和系统，以及评估干预措施挽救健康表型前后这些细胞功能所需的非破坏性收缩能力分析。我们的进一步目标是部署这些模型系统和方法来表征导致遗传性心肌病的突变的生物物理学。我们试图展示适合未来转化为高通量的治疗方法的模型，以适应患者的特异性。

 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.