Mechanisms for Stem Cell Differentiation into Cardiac Myocytes

描述（由申请人提供）：美国有500万患者患有心力衰竭。由于这一问题的严重性而开始的干细胞治疗的临床试验取得了适度的结果。了解诱导幼稚成体干细胞分化为心肌细胞的表观遗传程序是开发干细胞治疗的重要一步。我们的应用程序测试了人类间充质干细胞（hMSC细胞）中这种表观遗传过程的组成部分，因为它们在临床试验中的潜在用途以及从患者中产生它们的能力。拟议的研究是基于我们在杜克大学、北卡罗来纳州查佩尔山大学和东卡罗莱纳大学的研究人员之间正在进行的成功合作。这种合作导致了我们的一般假设：核Ca 2+振荡诱导hMSC细胞分化为心肌细胞。更具体地说，成人来源的干细胞和相邻的收缩心肌细胞之间共享的胞质管道，间隙连接，提供了来自心肌细胞的信号进入干细胞的途径。该信号被翻译成干细胞中的从头核Ca 2+振荡和心脏基因程序的激活。我们提出以下顺序事件驱动幼稚干细胞分化为心肌细胞：干细胞和邻近的收缩心肌细胞之间形成共享的功能性连接蛋白43（Cx43）衍生的间隙连接（Cx43是心肌细胞和hMSC细胞中的主要亚型）;在干细胞的细胞质（[Ca 2 +]c）和细胞核（[Ca 2 +]n）中产生了与心肌细胞胞质钙（[Ca 2 +]i）瞬变同步的从头Ca 2+振荡。[Ca 2 +]n振荡由三磷酸肌醇受体1介导（hMSC细胞核膜IP 3R为IP 3R 1）; Ca 2+信号传导依赖性效应物CaMKIV和新描述的心脏基因表达调节因子钙调蛋白结合转录因子（CAMTA 1）和心脏基因的表达上调，干细胞获得心肌细胞表型。我们将使用体外和体内研究来验证我们的假设。公共卫生相关性：在该提案中，我们试图了解诱导幼稚成体干细胞分化为心肌细胞的过程的表观遗传基础。我们的一般假设是核内Ca ~（2+）振荡诱导hMSC细胞分化为心肌细胞。我们在体外和体内小鼠心脏中与新生心肌细胞共培养研究了这一点。

DESCRIPTION (provided by applicant): Five million patients suffer from heart failure in the United States. Clinical trials of stem cell therapy, initiated because of the severity of this problem, have yielded modest results. Understanding the epigenetic programs that induce differentiation of a naive somatic adult-derived stem cell into a cardiomyocyte is an important step in developing stem cell therapy. Our application tests components of this epigenetic process in human mesenchymal stem cells (hMSCells), because of their potential use in clinical trials and the ability to generate them from the patient. The proposed studies are based on our ongoing successful collaboration between investigators at Duke University, University of North Carolina at Chapel Hill, and East Carolina University. This collaboration has led to our General Hypothesis: Nuclear Ca2+ oscillations induce hMSCells to differentiate into cardiomyocytes. More specifically, shared cytosolic conduits, gap junctions, between adult-derived stem cells and adjacent contracting cardiomyocytes provide the route by which a signal from the cardiomyocyte enters the stem cell. This signal is translated into de novo nuclear Ca2+ oscillations in the stem cell and activation of a cardiac gene program. We propose that the following sequential events drive naive stem cell differentiation into a cardiomyocyte: shared functional connexin 43 (Cx43)-derived gap junctions develop between the stem cell and an adjacent contracting cardiomyocyte (Cx43 is the dominant isoform in cardiomyocytes and hMSCells); de novo Ca2+ oscillations develop in the stem cell cytoplasm ([Ca2+]c) and nucleus ([Ca2+]n) that are synchronous with the cardiomyocyte cytosolic calcium ([Ca2+]i) transients. The [Ca2+]n oscillations are mediated by inositol trisphosphate receptor 1 (the hMSCell nuclear envelope IP3R is IP3R1); the expression of Ca2+- signaling dependent effectors, CaMKIV and the newly described regulator of cardiac gene expression, calmodulin binding transcription factor (CAMTA1), and cardiac genes become up-regulated and the stem cell acquires a cardiomyocyte phenotype. We will use in vitro and in vivo studies to test our hypothesis. PUBLIC HEALTH RELEVANCE: In the proposal, we attempt to understand the epigenetic basis underlying the process that induces a naive somatic adult-derived stem cell to differentiate into a cardiomyocyte. Our general hypothesis is nuclear Ca2+ oscillations induce hMSCells to differentiate into cardiomyocytes. We study this in co-cultures with neonatal cardiomyocytes in vitro and in vivo in the mouse heart.