From human keratinocytes to biological pacemakers

从人类角质形成细胞到生物起搏器

• 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
• 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

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））中产生纯心脏起搏器细胞群，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植入或重新植入患者的35万多个电子起搏器。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，增强其向心脏谱系的分化并选择起搏肌细胞;（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，并在体外将HFKT起搏器与天然心脏初级和二级起搏器进行比较;（3）确定（a）HFKT起搏器与来自特定位置的成体心脏细胞偶联的能力b）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和HFKT-起搏器与表达成纤维细胞连接蛋白的细胞偶联的能力（潜在的致心律失常情况）;（4）确定通过将HFKT起搏器放置在犬心房或心室中产生的体内生物起搏器功能。我们的方法将采用1）新的方法来提高起搏细胞的选择，2）膜片钳来表征动作电位 产生的分离的起搏细胞中的形态和膜电流，3）基因芯片以确定起搏细胞的表达谱，4）双全细胞膜片钳，以及生物化学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）将HFKT-起搏器注射到犬心房或心室中以确定体内起搏器功能。这些实验将由斯托尼布鲁克大学和以色列理工学院的一个长期合作小组进行。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。研究计划的成功实施将增强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起搏器在犬心脏中功能良好，未来的目标将是将这种新型自体细胞方法推向临床部署。 公共卫生相关性：每年有35万个心脏起搏器植入美国公民体内。虽然它们非常成功，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。