Mechanisms for Stem Cell Differentiation into Cardiac Myocytes

描述(申请人提供)：美国有500万心力衰竭患者。由于这一问题的严重性而启动的干细胞治疗临床试验取得了不大的成果。了解诱导幼稚的体细胞成体干细胞分化为心肌细胞的表观遗传学程序是发展干细胞疗法的重要一步。我们的应用程序在人类间充质干细胞(HMSCells)中测试这种表观遗传过程的组件，因为它们可能在临床试验中使用，并且能够从患者身上产生它们。拟议的研究是基于我们在杜克大学、北卡罗来纳大学教堂山分校和东卡罗来纳大学的研究人员正在进行的成功合作。这一合作导致了我们的一般假设：核钙振荡诱导hMSCcell分化为心肌细胞。更具体地说，成体干细胞和相邻收缩心肌细胞之间共享的胞浆管道，即缝隙连接，提供了来自心肌细胞的信号进入干细胞的途径。这一信号被转化为干细胞中从头开始的核钙振荡和心脏基因程序的激活。我们认为以下一系列事件促使干细胞分化为心肌细胞：在干细胞和相邻收缩的心肌细胞之间发展共享的功能性连接蛋白43(Cx43)衍生的缝隙连接(Cx43是心肌细胞和hMSC细胞中的主要异构体)；在干细胞细胞质([Ca2+]c)和细胞核([Ca2+]n)中产生与心肌细胞胞浆钙([Ca2+]i)瞬时同步的从头钙振荡。[Ca~(2+)]_n振荡由三磷酸肌醇受体1(hMSCell核膜IP3R为IP3R)介导；钙信号依赖效应物CaMKIV和新近发现的心脏基因表达调节因子钙调素结合转录因子(CAMTA1)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的体细胞成人来源的干细胞分化为心肌细胞的过程中潜在的表观遗传学基础。我们的一般假设是核钙振荡诱导hMSC细胞分化为心肌细胞。我们在体外和活体小鼠心脏中与新生心肌细胞共培养来研究这一点。

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.