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

描述（由申请人提供）：本R21提案旨在确定Kr¿pel样因子（KLFs）是否促进干细胞源性心肌细胞的功能成熟，并为其成熟效应建立生理和分子遗传学基础。实现hESC和hipsc衍生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，干细胞衍生的心肌细胞表现出胎儿细胞而不是成人细胞的电学和力学特性。关键是，未成熟的心肌细胞不能产生成熟心肌细胞的力量，并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，例如评估候选药物对心脏的不良影响。尽管从干细胞中提取心肌细胞的有效方法现已被采用，但即使是最好的方法也只能产生未成熟的、胎儿样的细胞，因为这种方法很少

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.