Kruppel-like Factor and Maturation of hESC/hiPSC Derived Cardiomyoctyes

描述(申请人提供)：本R21提案旨在确定Kr？ppel样因子(KLF)是否促进干细胞来源的心肌细胞的功能成熟，并建立成熟效应的生理和分子遗传学基础。实现hESC和hPSC来源的心肌细胞用于药物发现、研究和心肌再生的一个重要障碍是干细胞来源的心肌细胞表现出胎儿细胞的电学和机械特性，而不是成人细胞。关键的是，未成熟的心肌细胞不会产生成熟心肌细胞的力量，而且它们的电学特性在移植环境中可能会导致心律失常。此外，不成熟的离子通道和生理特性降低了体外分析的预测价值，例如用于评估候选药物的不良心脏效应。尽管现在使用有效的方案从干细胞中提取心肌细胞，但即使是最好的方法也能产生未成熟的胎儿样细胞，因为很少有 已经知道了引导成熟的信号。数据显示了高通量电生理成熟度评估仪器和软件的发展。利用这项技术，我们提供了初步证据，证明KLFS可以促进hESC来源的和新生大鼠心室肌细胞的电和离子通道谱成熟。这一建议的具体目的是：1)通过离子通道和泵的功能表达、药物敏感性分析、动作电位和钙处理指标、收缩装置结构和力产生来确定KLF诱导的成熟；以及2)通过质谱学和下一代ChIPSeq分析确定KLF的结合伙伴和基因靶标，建立生理效应的分子遗传学基础。对促进心肌细胞成熟的信号的识别应该导致改进获得功能性心肌细胞的方法，这将使干细胞在基础和应用研究中的许多应用成为可能。此外，引导外源性或内源性干细胞来源的能力将是实现心脏功能再生的关键。 与公共卫生相关：该项目旨在识别和表征直接从干细胞转化为心肌细胞的生理性成熟的蛋白质和基因。干细胞来源的心肌细胞通常缺乏成年心肌细胞的电学和机械特性，而这些特性对许多研究和临床应用都很重要。这些发现将是一个概念上的进步，因为人们对生理成熟是如何控制的知之甚少，并将增加干细胞来源的心肌细胞在研究和医疗应用中的价值。

DESCRIPTION (provided by applicant): This R21 proposal is to determine if Kr¿ppel-like factors (KLFs) promote functional maturation of stem cell- derived cardiomyocytes, and to establish the physiological and molecular genetic basis for the maturational effects. An important impediment to realizing the promise of hESC and hiPSC-derived cardiomyocytes for drug discovery, research and myocardial regeneration is that stem cell-derived cardiomyocytes exhibit electrical and mechanical properties of fetal, rather than adult, cells. Critically, immatue cardiomyocytes do not generate the force of mature myocytes, and their electrical properties are potentially arrhythmogenic in a transplant setting. Moreover, immature ion channel and physiological properties diminish the predictive value of in vitro analyses such as for assessing adverse cardiac effects of drug candidates. Although efficient protocols are now used to derive cardiomyocytes from stem cells, even the best yield immature, fetal-like cells since very little is known about the signals that direct maturation. Data are presented showing the development of instrumentation and software for high throughput assessment of electrophysiological maturation. Using this technology, we provide preliminary evidence that KLFs can promote electrical and ion channel profile maturation of hESC-derived and neonatal rat ventricular cardiomyocytes. The specific aims of this proposal are to: 1) define the KLF-induced maturation by functional expression of ion channels and pumps, drug sensitivity profiling, action potential and calcium handling metrics, contractile apparatus structure, and force generation; and 2) establish the molecular genetic basis for the physiological effects, in part by determining the binding partners and gene targets of KLFs by mass spectroscopic and next generation ChIPSeq analyses. The identification of signals that promote cardiomyocyte maturation should lead to improved methods for deriving functional cardiomyocytes that will be enabling for many stem cell applications in basic and applied research. Moreover, the ability to direct exogenous or endogenous stem cell sources will be essential to achieve functional regeneration of the heart. PUBLIC HEALTH RELEVANCE: This project is to identify and characterize proteins and genes that direct physiological maturation of heart muscle cells from stem cells. Heart muscle cells derived from stem cells typically lack electrical and mechanical properties of adult cardiomyocytes that are important for many research and clinical applications. The findings will constitute a conceptual advance since little is known about how physiological maturation is controlled, and will increase the value of stem cell-derived cardiomyocytes for research and medical applications.