From human keratinocytes to biological pacemakers

从人类角质形成细胞到生物起搏器

• 批准号: 8423701
• 负责人: IRA S COHEN
• 金额: $ 63.59万
• 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/8423701
• 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

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.

项目摘要：该应用程序的长期目标是创建一个纯粹的心脏起搏器群体 来自一种容易获得的自体细胞类型--人毛囊角质形成细胞(HFKT起搏器)的细胞， 为了描述它们的起搏机制，它们整合到心脏合胞体的能力，以及它们的 作为体内生物起搏器的潜力。如果在长期内取得成功， 这种方法将取代植入的超过35万个电子起搏器或 每年在美国的患者身上重新植入。该项目有四个具体目标：(1)扩大 从HFKT诱导的多能干细胞群体，促进其向心脏分化 起搏器肌细胞的谱系和选择；(2)表征HFKT的膜电流。 起搏器及其起搏器功能和基因表达谱，并比较HFKT- 体外实验：(3)测定(A)心脏起搏器的起搏能力 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 产生的起搏频率是否依赖于靶点(B)哪些连接蛋白HFKT起搏器表达和 HFKT起搏器与表达成纤维细胞连接蛋白(潜在的 致心律失常的情况)；(4)测定体内生物起搏器功能 HFKT-犬心房或心室的起搏器。我们的方法将采用新的方法来增强 起搏细胞的选择，2)膜片钳技术研究动作电位的形态和膜 在分离的起搏器细胞中产生电流，3)基因芯片以确定起搏器细胞的 表达谱，4)双全细胞膜片钳，以及生化和分子技术 犬体内注射HFKT起搏器后缝隙连接蛋白的表达及功能细胞间的偶联 以确定在体心脏起搏器的功能。这些实验将由一组 石溪大学和以色列理工学院的长期合作伙伴。这支队伍拥有广泛的 擅长干细胞生物学、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加强选择 IPSCs心肌细胞谱系的技术，表征HFKT起搏器活动的基础- 并确定其作为体内生物起搏器的有效性。如果HFKT起搏器 未来的目标是推动这种新的自体细胞方法的发展 走向临床部署。