Electrophysiology and Cell Biology of Cardiac Stem Cells

描述(由申请人提供)：最近发现心脏是干细胞的来源，为自体细胞性心肌成形术开辟了新的前景。这样的心脏干细胞(CSCs)已经从啮齿动物的心脏和人类手术标本中分离出来。我们在从经皮心内膜心肌活检中分离和扩增这些细胞方面已经取得了实质性的进展。当在悬浮培养中生长时，CSCs形成自组织的球形簇，显示出分化心肌细胞的几个特征。我们现在建议继续在体外和体内对CSCs和心球进行功能表征。了解CSCs和心球的电生理学可能有助于深入了解心脏发育和修复过程中的事件，并对评估细胞移植的安全性和有效性至关重要。这一应用试图阐明不同分化阶段的分子和电生理表型。以前使用间充质干细胞和骨髓细胞的研究表明，在注射的细胞没有明显植入或分化为功能性心肌细胞的情况下，心功能得到了改善。自体心肌干细胞具有最高的植入和分化为功能性心肌细胞的潜力。我们建议在心肌梗死动物模型中比较注射CSCs或心球后的植入和分化。心球凭借其三维结构，可以改善细胞的植入、增殖、存活和分化。成肌细胞(一种目前正在进行临床试验的自体细胞类型)的临床试验报告称，室性心律失常的发生率很高，可能是由于成肌细胞缺乏电整合所致。我们的初步数据显示，心肌干细胞表达Cx43，并且在共培养中能够与心室肌细胞电偶联。在这里，我们计划使用光学标测、微电极记录和双光子显微镜来评估体外和体内细胞的电耦合和致心律失常的潜力。干细胞的另一个重要特征是它们能够分泌对心脏功能有有益影响的因子。通过干细胞识别分泌因子，可以在损伤后立即直接输送生长因子，而不需要细胞扩增。我们将使用蛋白质组学技术来鉴定分泌因子。鉴于临床翻译的潜力，这项工作为心力衰竭的治疗带来了革命性的可能性。

DESCRIPTION (provided by applicant): Recent discoveries identifying the heart as a source of stem cells have opened up new prospects for autologous cellular cardiomyoplasty. Such cardiac stem cells (CSCs) have been isolated from rodent hearts and from human surgical specimens. We have made substantial progress in isolating and expanding these cells from percutaneously obtained endomyocardial biopsies. When grown in suspension culture, CSCs form self-organizing spherical clusters that display several features of differentiating cardiomyocytes. We now propose to continue functional characterization of CSCs and cardiospheres in vitro and in vivo. Understanding the electrophysiology of CSCs and cardiospheres may provide insights into events in cardiac development and repair and is vital to assessing the safety and efficacy of cell transplantation. This application seeks to elucidate the molecular and electrophysiologic phenotype at different stages of differentiation. Previous work using mesenchymal stem cells and bone marrow cells has demonstrated improvement of cardiac function without significant engraftment or differentiation of the injected cells into functional cardiac myocytes. Autologous cardiac stem cells have the highest potential for engraftment and differentiation into functional myocytes. We propose to compare engraftment and differentiation after injection of CSCs or cardiospheres in an animal model of myocardial infarction. Cardiospheres may, by virtue of their 3-dimensional architecture, improve cell engraftment, multiplication, survival and differentiation. Clinical trials of myoblasts, (an autologous cell type currently in clinical trials) have reported a high incidence of ventricular arrhythmias probably secondary to lack of electrical integration of myoblasts. Our preliminary data reveals that cardiac stem cells express Cx43 and are capable of electrical coupling with ventricular myocytes in co-culture. Here, we plan to evaluate electrical coupling and arrhythmogenic potential of cells in vitro and in vivo, using optical mapping, microelectrode recordings and 2-photon microscopy. Another important feature of stem cells is their ability to secrete factors that have a beneficial effect on cardiac function. Identification of secreted factors by stem cells may permit direct delivery of growth factors immediately after injury obviating the need for cell expansion. We will use proteomic techniques to identify secreted factors. Given the potential of clinical translation, this work opens up the possibility of revolutionizing the treatment of heart failure.