Deciphering the mechanisms of c-kit+ cells in heart repair

项目摘要 c-kit阳性（c-kit+）细胞是在1998年发现的第一个推定的心脏干细胞（CSC）群体。 哺乳动物，在体外具有自我更新、克隆形成和多能活性。它还报告说，成人心脏c- Kit+细胞是心肌再生所必需和足够的。虽然这些发现令人鼓舞， 最近在小鼠中的谱系追踪研究表明，心脏驻留c-kit+细胞具有最小的潜力， 在体内分化成心肌细胞。为了确定心脏c-kit+细胞的真实身份，我们产生了一个 通过靶向报告基因（H2 B-tdTomato、核lacZ和H2 B-GFP）构建了一系列新的小鼠模型， 将MerCreMer盒插入小鼠中c-kit的起始密码子。有了他们，我们第一次发现c-kit实际上标记了一个 PECAM+心脏内皮细胞亚群（~43%）。急性心肌损伤后，常驻c-kit+细胞 仍然保持其内皮特性，并且几乎没有或没有成为心肌细胞潜力。然而，在这方面， 忽略了发育期间和心脏移植后心脏驻留c-kit+细胞的低成肌潜力， 外源性扩增的c-kit+细胞的移植一直显示出改善心脏 功能和减弱缺血性和非缺血性心肌病中不利的左心室重构。 因此，c-kit+细胞疗法的基础必须有未被认识的机制。鉴于我们的新发现c-kit 实际上标记了心脏内皮细胞的亚群，我们假设移植的c-kit+细胞修复了 通过它们的内皮性质损伤心脏。c-kit+细胞可以通过以下方式改善受体心脏的自我更新： 产生新的冠状血管，或通过释放旁分泌因子。移植的c-kit+细胞也可能 重编程并在体外扩增后获得多能性。在这项研究计划中，我们将使用国家的- art小鼠模型和人c-kit+心脏内皮细胞来检验这些假设，有三个目的：目的1 将确定c-kit+细胞的内皮性质是否有助于心脏修复;目标2将确定c-kit+细胞的内皮性质是否有助于心脏修复。 移植的c-kit+内皮细胞促进受体心脏的自我更新，如果它们作为祖细胞， 再生冠状血管和/或心肌细胞; Aim 3将确定是否有旁分泌因子从 移植的c-kit+细胞对于心脏修复是必需的。该项目将提供明确的答案，以阐明 c-kit+细胞促进心脏修复的最终机制。

PROJECT SUMMARY The c-kit-positive (c-kit+) cells are the first putative population of cardiac stem cells (CSCs) identified in mammals, with self-renewing, clonogenic and multipotent activities in vitro. It also reported that adult cardiac c- kit+ cells are necessary and sufficient for myocardial regeneration. While these findings are encouraging, a recent lineage tracing study in mice indicated that cardiac resident c-kit+ cells have minimal potential to differentiate into cardiomyocytes in vivo. To ascertain the true identity of cardiac c-kit+ cells, we generated a series of new mouse models by targeting reporter genes (H2B-tdTomato, nuclear lacZ and H2B-GFP) and MerCreMer cassette into the start codon of c-kit in mice. With them, we first uncovered that c-kit in fact labels a subpopulation (~43%) of PECAM+ cardiac endothelial cells. After acute cardiac injury, the resident c-kit+ cells still retain their endothelial identity, and have little or no potential to become cardiomyocytes. However, disregard the low myogenic potential of cardiac resident c-kit+ cells during development and after cardiac injury, transplantation of exogenously expanded c-kit+ cells has been consistently shown to improve heart function and attenuate adverse left ventricular remodeling in both ischemic and non-ischemic cardiomyopathy. Thus, there must be unrecognized mechanisms underlying c-kit+ cell therapy. Given our new finding that c-kit actually labels a subpopulation of cardiac endothelial cells, we hypothesized transplanted c-kit+ cells repair the injured heart through their endothelial nature. c-kit+ cells may improve self-renew of the recipient heart by generating new coronary vasculature, or by releasing paracrine factors. Transplanted c-kit+ cells may also be reprogramed and gain multipotency after in vitro expansion. In this research program, we will use state-of-the- art mouse models and human c-kit+ cardiac endothelial cells to test these hypotheses with three Aims: Aim 1 will determine if the endothelial nature of c-kit+ cells contributes to heart repair; Aim 2 will determine if the transplanted c-kit+ endothelial cells promote self-renew of the recipient heart, and if they serve as progenitors to regenerate coronary vessels and/or cardiomyocytes; Aim 3 will determine if the paracrine factors from transplanted c-kit+ cells are essential for heart repair. This project will provide definitive answers to elucidate ultimate mechanisms by which c-kit+ cells promote heart repair.