From human keratinocytes to biological pacemakers

从人类角质形成细胞到生物起搏器

基本信息

批准号：
8219449
负责人：
IRA S COHEN
金额：
$ 68.12万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8219449
关键词：
Action Potentials Adult American Animal Experiments Anterior Antigens Autologous Automobile Driving Biochemical Biological Biological Pacemakers Calcium Canis familiaris Cardiac Cardiac Myocytes Cell Lineage Cells Characteristics Clinical Complement Connexins Coupling Development Disease Dreams Effectiveness Excision Exhibits Fibroblasts Future Gap Junctions Gene Chips Gene Expression Gene Expression Profile Genetic Giant Cells Goals Hair follicle structure Health Benefit Heart Heart Atrium Heart Block Human Immunohistochemistry Immunosuppression Implant In Vitro Individual Injection of therapeutic agent Ion Channel Israel Label Left Location Mechanics Membrane Methods Microelectrodes Molecular Molecular Profiling Monitor Morphology Muscle Cells Pacemakers Patients Physiological Population Property Protocols documentation Quality of life Research Reverse Transcriptase Polymerase Chain Reaction Sinus Site Source Staining method Stains Techniques Testing Therapeutic United States Universities Ventricular Western Blotting base cell type connexin 40 electronic pacemaker heart cell heart rate variability heart rhythm human GJB2 protein implantation in vivo induced pluripotent stem cell keratinocyte nodal myocyte novel novel strategies patch clamp performance site repaired research study response stem cell biology

项目摘要

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.

描述（由申请人提供）：本申请的长期目标是从易于获得的自体细胞类型（人毛囊角质形成细胞（HFKT-起搏器））创建纯心脏起搏器细胞群，以表征其起搏器机制、其融入心脏合胞体的能力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取代美国每年植入或重新植入患者体内的超过 350,000 个电子起搏器。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体，增强其向心脏谱系的分化并选择起搏器肌细胞； (2) 表征 HFKT 起搏器中的膜电流及其起搏器功能和基因表达谱，并将 HFKT 起搏器与体外心脏主起搏器和次级起搏器进行比较； (3) 确定 (a) HFKT 起搏器在体外与来自特定位置（心房或心室）的成人心脏细胞耦合的能力，以及产生的起搏速率是否依赖于目标 (b) HFKT 起搏器表达的连接蛋白和能力HFKT 起搏器与表达成纤维细胞连接蛋白的细胞耦合（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）新方法来增强起搏细胞的选择，2）膜片钳来表征动作电位产生的分离起搏细胞的形态和膜电流，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化和分子技术以确定连接蛋白表达和功能性细胞与细胞偶联 5) 注射将 HFKT 起搏器放入犬心房或心室以确定体内起搏器功能。这些实验将由石溪大学和以色列理工学院的长期合作者团队进行。该团队在干细胞生物学、诱导多能干细胞、心脏起搏、膜片钳和生物起搏器功能的体内研究方面拥有广泛的专业知识。该研究计划的成功执行将增强IPSC心脏细胞谱系的选择技术，表征HFKT起搏器中起搏器活性的基础，并确定其作为体内生物起搏器的有效性。如果 HFKT 起搏器在犬心脏中发挥良好作用，未来的目标将是将这种新型自体细胞方法推向临床部署。公共健康相关性：每年有 350,000 个起搏器被植入美国公民体内。尽管它们取得了巨大成功，但也有限制其有效性的缺点。自体生物起搏器的成功开发将提高心律失常患者的生活质量。