From human keratinocytes to biological pacemakers

描述（由申请人提供）：本申请的长期目标是从一种容易获得的自体细胞类型——人毛囊角质细胞（HFKT-pacemakers）——中创建一个纯的心脏起搏器细胞群，以表征其起搏器机制、其整合到心脏合胞体中的能力以及其作为体内生物起搏器的潜力。如果长期成功，这种方法的健康益处将是取代美国每年35万多名患者植入或重新植入的电子起搏器。该项目有四个具体目标：(1)扩大由HFKTs产生的诱导多能干细胞的数量，增强其向心脏谱系的分化，并选择起搏器肌细胞；(2)表征HFKT-起搏器的膜电流、起搏器功能和基因表达谱，并在体外将HFKT-起搏器与天然心脏原发性和继发性起搏器进行比较；(3)确定(a) HFKT起搏器在体外与特定位置（心房或心室）的成人心脏细胞偶联的能力，以及所产生的起搏速率是否与靶标相关(b) HFKT起搏器表达哪些连接蛋白，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（一种潜在的心律失常情况）；(4)确定在犬心房或心室放置hfkt起搏器所产生的体内生物起搏器功能。我们的方法将采用1)新颖的方法来增强起搏器细胞的选择，2)膜片箝位来表征动作电位

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Each year 350,000 pacemakers are implanted in American citizens. Although enormously successful they also have drawbacks which limit their effectiveness. The successful development of an autologous biological pacemaker will increase the quality of life for those patients who suffer disorders of cardiac rhythm.