Metabolic and Transcriptional Reprogramming of Cardiac Maturation

项目摘要 干细胞科学的最新进展导致了心脏再生的加速进展。我们的团队 成功地实现了猕猴梗死心脏的大规模肌化 多能干细胞来源的人心肌细胞移植(hPSC-CMS)。这些心肌细胞 射血从~40%恢复到~62%，这是我们所知的最大程度的心功能恢复。这 治疗因出现持续数周的一过性室性心律失常而变得复杂。 在消失之前。我们的电生理研究表明，心律失常是起搏引起的。 这反过来又是由于hPSC-CMS在移植时不成熟所致。主 这项建议的目标是促进hPSC-CM的成熟，使其非致心律失常，使用 代谢和转录重新编程。在目标1中，我们建立在我们的观察基础上，调制 新陈代谢对成熟有广泛的影响，包括降低自律性和增加 生理性肥大，力排量大，钙循环更像成人。最强大的新陈代谢 干预、底物转换、代谢激素和能量感应将得到系统优化，以 提高体外电成熟度。优化后，我们将探索底层机制，然后测试 这种成熟是否通过将它们移植到猪心来减少心律失常。Aim 2利用 在最近生成的资源中，我们对一系列定时的人类心肌移植物进行了RNA-SEQ 当它们在体内成熟到成体水平时，在老鼠的心脏中。通过将这些数据与未成熟的心肌细胞进行比较， 我们确定了一组转录调控因子，它们是成熟的候选驱动因素。我们将表演 基于CRISPR的体外激活这些因子的功能增益研究。利用基因表达和 电生理分析，然后我们将确定最佳组合，以促进成熟。如果成功， 这些研究将解决干细胞心脏再生的最大障碍，并使我们更接近 临床试验。

Project Summary Recent advances in stem cell science have led to accelerated progress in cardiac regeneration. Our group successfully achieved large-scale re-muscularization of the infarcted hearts of macaque monkeys by transplanting human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). These cardiomyocytes restored ejection from ~40% to ~62%, the largest restoration of cardiac function of which we are aware. This therapy is complicated by the appearance of transient ventricular arrhythmias, which last for several weeks before disappearing. Our electrophysiological studies indicate that the arrhythmias result from pacemaking activity, which in turn results from the immaturity of the hPSC-CMs at the time of transplantation. The main goal of this proposal is to enhance the maturation of hPSC-CM to make them non-arrhythmogenic, using metabolic and transcriptional reprogramming. In Aim 1 we build on our observations that modulating metabolism has wide-ranging effects on maturation, including reducing automaticity, and increasing physiological hypertrophy, force production, and more adult-like calcium cycling. The most powerful metabolic interventions, substrate switching, metabolic hormones, and energy sensing, will be systematically optimized to enhance electrical maturity in vitro. Once optimized, we will explore the underlying mechanisms, and then test whether this maturation reduces arrhythmias by transplanting them into porcine hearts. Aim 2 takes advantage of a recently generated resource, where we performed RNA-seq on a timed series of human myocardial grafts in the rat heart as they matured to adult levels in vivo. By comparing these data to immature cardiomyocytes, we identified a set of transcriptional regulators that are candidate drivers of maturation. We will perform CRISPR-based gain-of-function studies to activate these factors in vitro. Using gene expression and electrophysiology analyses, we will then identify optimal combinations to enhance maturation. If successful, these studies will solve the greatest barrier to stem cell-based heart regeneration and bring us much closer to clinical trials.