Mechanisms enhancing functional coupling between native and embryonic stem cell d

描述（由申请人提供）：本申请的目标是通过“受飓风桑迪不利影响的新研究者试点项目的恢复”机会获得资金。2012年10月，我收集了大量的初步数据，准备在2013年6月截止日期前提交R01申请。我的研究受到超级风暴桑迪的严重影响。我的损失包括一群connnexin43（缝隙连接）突变小鼠，作为细胞移植研究的受体，一群繁殖的大鼠，以及珍贵的冷冻细胞裂解液和等待分子和组织学分析的组织样本。本申请中提出的实验将恢复试点项目数据，以提交竞争性拨款，预计将于2014年夏季/秋季提交。我的长期研究兴趣是改善多能干细胞（PSC）在心脏病治疗中的应用。PSC允许基因操作，可以在培养皿中分化成功能性心肌细胞。PSC已经被用于个性化医疗方法，并且PSC将成为未来自体细胞替代治疗的合适来源。然而，要认真考虑治疗应用，仍然存在一些挑战，包括移植细胞的长期存活和功能植入。细胞的植入通过细胞间的连接或细胞间的接触得到加强，这种联系是由特殊的蛋白质形成的。细胞接触提供结构组织支持（机械连接：粘附连接；桥粒），并允许快速脉冲传播和心肌的同步收缩（电连接：间隙连接通道）。事实上，缺乏间隙连接蛋白connexin43的PSC来源的心肌细胞移植会导致小鼠心律失常的增加。我们的初步数据表明，干细胞衍生的心肌细胞与天然心肌细胞形成很少的功能性间隙连接接触。该建议的总体假设是，间隙连接通道形成和功能的增加将提高干细胞来源的心肌细胞的治疗效果。我将采用三种不同的方法进行测试

DESCRIPTION (provided by applicant): The goal of this application is to obtain funding through the "Restoration of New Investigator Pilot Projects Adversely Affected by Hurricane Sandy" opportunity. In October 2012, I had collected strong preliminary data and was preparing an R01 application for the June 2013 deadline. My research was seriously impacted by the after-effects of Super-storm Sandy. My losses include a colony of connexin43 (gap junction) mutant mice, generated as recipients for cell-transplantation studies, a breeding colony of rats, as well as precious frozen cell lysates and tissue samples awaiting molecular and histological analyses. Experiments proposed in this application will restore pilot project data towards a competitive grant submission, anticipated for summer/fall 2014. My long-term research interest is improving the therapeutic use of pluripotent stem cells (PSC) for heart diseases. PSC allow gene manipulation and can be differentiated into functional heart muscle cells in the dish. Already, PSC are being used for personalized medicine approaches, and PSC will be a suitable source for autologous cell replacement therapy in the future. To be seriously contemplated for therapeutic applications, however, several challenges remain, including long-term survival and functional engraftment of transplanted cells. Engraftment of cells is enhanced by intercellular connections, or cell-cell contacts, formed by specialized proteins. Cell contacts provide structural tissue support (mechanical junctions: adherens junctions; desmosomes) and allow fast impulse propagation and the synchronous contraction of the heart muscle (electrical junctions: gap junction channels). Indeed, transplantation of cardiomyocytes derived from PSC lacking the gap junction protein connexin43, lead to increased arrhythmogenesis in mice. Our preliminary data demonstrate that stem cell derived cardiomyocytes form very few functional gap junction contacts with native cardiomyocytes. The overarching hypothesis of this proposal is that an increase in gap junction channel formation and function will improve the therapeutic efficacy of stem cell derived cardiomyocytes. I will employ three different approaches to test this hypothesis. Based on preliminary data, I will investigate how the formin protein Daam1 enhances gap junction formation. Further, I will analyze the influence of forced gap junction channel formation for cell engraftment using PSC expressing a mutant gap junction channel, K258stop. Additionally I will use a high throughput screen to identify small molecules regulating gap junction expression in stem cell derived cardiomyocytes. While experiments proposed in this study will elucidate ways to enhance the formation of gap junctions in stem cell derived cardiomyocytes, the mechanisms identified might very well also be applicable and therapeutically relevant to the treatment of cardiac diseases related to changes in gap junction formation.