Metabolic and Transcriptional Reprogramming of Cardiac Maturation

项目摘要 干细胞科学的最新进展加速了心脏再生的进展。我们集团 成功地实现了猕猴梗塞心脏的大规模肌肉再生， 移植来源于多能干细胞（hPSC-CM）的人心肌细胞。这些心肌细胞 射血率从~40%恢复到~ 62%，这是我们所知道的最大的心脏功能恢复。这 治疗因出现持续数周的短暂室性心律失常而变得复杂 然后消失了我们的电生理研究表明，心律失常是由起搏引起的 活性，这又是由于移植时hPSC-CM的不成熟。主要 该提议的目标是增强hPSC-CM的成熟以使其非致瘤性， 代谢和转录重编程。在目标1中，我们建立在我们的观察基础上， 代谢对成熟有广泛的影响，包括降低自动性，增加 生理性肥大，力的产生，以及更像成人的钙循环。最强大的新陈代谢 干预，底物转换，代谢激素和能量传感，将系统地优化， 提高体外电成熟度。一旦优化，我们将探索潜在的机制，然后测试 这种成熟是否可以通过移植到猪心来减少心律失常。目标2利用 最近生成的资源，我们对一系列定时的人类心肌移植物进行了RNA测序 在体内成熟到成年水平时，通过将这些数据与未成熟的心肌细胞进行比较， 我们鉴定了一组转录调节因子，它们是成熟的候选驱动因子。我们将执行 基于CRISPR的功能获得研究在体外激活这些因子。利用基因表达和 电生理分析，然后我们将确定最佳组合，以提高成熟。如果成功， 这些研究将解决基于干细胞的心脏再生的最大障碍， 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.