Mechanisms enhancing functional coupling between native and embryonic stem cell d

描述(申请人提供)：本申请的目的是通过“修复受飓风桑迪影响的新调查员试点项目”的机会获得资助。2012年10月，我收集了强大的初步数据，正在准备2013年6月截止日期的R01申请。我的研究受到超级风暴桑迪的后遗症的严重影响。我的损失包括一群作为细胞移植研究接受者而产生的连接蛋白43(缝隙连接)突变小鼠，一群繁殖的大鼠，以及等待分子和组织分析的珍贵的冷冻细胞裂解物和组织样本。本申请中提议的实验将恢复试点项目数据，以提交预计于2014年夏/秋提交的竞争性赠款。我的长期研究兴趣是改善多能干细胞(PSC)在心脏疾病中的治疗用途。PSC可以进行基因操作，并可以在培养皿中分化为具有功能的心肌细胞。PSC已经被用于个性化的医学方法，PSC将是未来自体细胞替代治疗的合适来源。然而，要认真考虑将其应用于治疗，仍然存在一些挑战，包括移植细胞的长期存活和功能植入。细胞的植入通过特殊蛋白质形成的细胞间连接或细胞-细胞接触而增强。细胞接触提供结构性组织支持(机械连接：粘连连接；桥粒)，并允许快速脉冲传播和心肌的同步收缩(电子连接：缝隙连接通道)。事实上，移植缺乏缝隙连接蛋白43的PSC来源的心肌细胞，会导致小鼠心律失常的增加。我们的初步数据表明，干细胞来源的心肌细胞与天然心肌细胞形成的功能性缝隙连接接触很少。这一建议的主要假设是，缝隙连接通道的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。我将使用三种不同的方法来测试它 假设。基于初步数据，我将研究Forin蛋白Daam1如何促进缝隙连接的形成。此外，我将利用表达突变的缝隙连接通道K258停止的PSC来分析强制缝隙连接通道的形成对细胞植入的影响。此外，我将使用高通量筛选来确定干细胞来源的心肌细胞中调节缝隙连接表达的小分子。虽然这项研究中提出的实验将阐明促进干细胞来源的心肌细胞缝隙连接形成的方法，但所确定的机制也很可能适用于与缝隙连接形成变化相关的心脏病的治疗。

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.