From human keratinocytes to biological pacemakers

项目总结：本申请的长期目标是创建一个纯粹的心脏起搏器人群 来自容易获得的自体细胞类型的细胞，人毛囊角质形成细胞（HFKT-起搏器）， 以表征它们的起搏机制、它们整合到心脏合胞体中的能力以及它们的 作为体内生物起搏器发挥作用的潜力。如果长期成功， 这种方法将取代超过350，000个植入的电子起搏器， 在美国每年都有病人被重新植入。该项目有四个具体目标：（1）扩大 由HFKT产生的诱导多能干细胞群增强了它们向心脏干细胞的分化， 谱系和选择的起搏心肌细胞;（2）表征膜电流在HFKT- 心脏起搏器及其起搏器功能和基因表达谱，并比较HFKT- 起搏器与体外天然心脏初级和次级起搏器的比较;（3）确定（a） HFKT起搏器在体外与特定位置（心房或心室）的成年心脏细胞偶联， 所产生的起搏速率是否是靶依赖性的（B）HFKT-起搏器表达的连接蛋白，以及 HFKT-起搏器与表达成纤维细胞连接蛋白的细胞偶联的能力（一种潜在的 （4）确定植入起搏器后产生的体内生物起搏器功能。 HFKT-犬心房或心室中的起搏器。我们的方法将采用1）新的方法来提高 选择起搏细胞，2）膜片钳以表征动作电位形态和膜 3）基因芯片，以确定起搏细胞的 表达谱，4）双全细胞膜片钳，以及生物化学和分子技术来确定 连接蛋白表达和功能性细胞与细胞偶联5）将HFKT-起搏器注射到犬中 心房或心室以确定体内起搏器功能。这些实验将由一个团队进行， 他是斯托尼布鲁克大学和以色列理工学院的长期合作者。该团队拥有广泛的 在干细胞生物学、诱导多能干细胞、心脏起搏、膜片钳和体内 生物起搏器功能的研究。研究计划的成功执行将提高选择 从IPSC心脏细胞谱系的技术，表征了HFKT中起搏器活性的基础， 起搏器，并确定其作为体内生物起搏器的有效性。如果HFKT起搏器 在犬心脏中功能良好，未来的目标将是推进这种新的自体细胞方法 临床部署。

Project Summary: The long term goal of this application is to create a pure population of cardiac pacemaker cells from an easily accessible autologous cell type, the human hair follicle keratinocyte (HFKT-pacemakers), to characterize their pacemaker mechanism, their ability to integrate into the cardiac syncytium and their potential to function as an in vivo biological pacemaker. If successful in the long term, the health benefit of such an approach will be to substitute for the more than 350,000 electronic pacemakers implanted or reimplanted in patients in the United States each year. The project has four specific aims: (1) to expand the population of induced pluripotent stem cells created from the HFKTs, enhance their differentiation to a cardiac lineage and select for pacemaker myocytes; (2) to characterize the membrane currents in the HFKT- pacemakers as well as their pacemaker function and gene expression profile, and to compare the HFKT- pacemaker to native cardiac primary and secondary pacemakers in vitro; (3) to determine (a) the ability of HFKT pacemakers to couple to adult heart cells from specific locations (atrium or ventricle) in vitro and whether the pacing rate generated is target dependent (b) which connexins HFKT-pacemakers express and the ability of the HFKT-pacemakers to couple to cells expressing fibroblast connexins (a potentially arrhythmogenic situation); (4) to determine in vivo biological pacemaker function generated by placement of HFKT-pacemakers in the canine atrium or ventricle. Our approach will employ 1) novel methods to enhance selection of pacemaker cells, 2) patch clamping to characterize action potential morphology and membrane currents in the isolated pacemaker cells generated, 3) gene chips to determine the pacemaker cells' expression profile, 4) dual whole cell patch clamp, and biochemical and molecular techniques to determine connexin expression and functional cell to cell coupling 5) Injection of the HFKT- pacemakers into the canine atrium or ventricle to determine in vivo pacemaker function. The experiments will be carried out by a team of long term collaborators at Stony Brook University and at the Technion in Israel. The team has extensive expertise in stem cell biology, induced pluripotent stem cells, cardiac pacemaking, patch clamp,, and in vivo studies of biological pacemaker function. Successful execution of the research plan will enhance selection techniques for cardiac cell lineages from IPSCs, characterize the basis of pacemaker activity in the HFKT- pacemakers and determine their effectiveness as an in vivo biological pacemaker. If the HFKT-pacemaker functions well in the canine heart, a future goal would be to advance this novel autologous, cellular approach towards clinical deployment.