From human keratinocytes to biological pacemakers

从人类角质形成细胞到生物起搏器

• 批准号: 8795752
• 负责人: IRA S COHEN
• 金额: $ 66.03万
• 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 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795752
• 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：此应用程序的长期目标是创建纯粹的心脏起搏器人群 来自易于访问的自体细胞类型的细胞，人毛囊角质形成细胞（HFKT-Pacemaker）， 为了表征其起搏器机制，它们将其整合到心脏合成及其的能力及其 作为体内生物起搏器的潜力。如果从长远来看成功，那么健康的好处 这种方法将是替代350,000多个植入或 每年在美国的患者重新植入。该项目具有四个具体目标：（1）扩展 由HFKT产生的诱导多能干细胞的种群增强了其与心脏的分化 谱系并选择起搏器肌细胞； （2）表征HFKT-中的膜电流 起搏器以及其起搏器功能和基因表达谱，并比较HFKT- 在体外与本地心脏初级和次要起搏器的起搏器； （3）确定（a） HFKT起搏器在体外与特定位置（中庭或心室）的成人心脏细胞搭配 产生的起搏率是否取决于目标（b），康纳克斯素HFKT-PACEMAKERS和 HFKT-Pacemaker夫妇与表达成纤维细胞连接素的细胞的能力（潜在的 心律不齐的情况）； （4）确定通过放置产生的体内生物起搏器功能 犬室或心室中的HFKT-PACEMAKER。我们的方法将采用1）增强的新方法 选择起搏器细胞，2）贴片夹具以表征动作电势形态和膜 生成的分离的起搏器细胞中的电流，3）基因芯片，以确定起搏器细胞的细胞' 表达曲线，4）双重全细胞斑块夹以及生化和分子技术以确定 连接蛋白的表达和功能性细胞与细胞耦合5）将HFKT-起搏器注入犬类 心房或心室以确定体内起搏器功能。实验将由一个团队进行 斯托尼·布鲁克大学（Stony Brook University）的长期合作者和以色列的技术。团队有广泛的 干细胞生物学，诱导多能干细胞，心脏起搏，斑块夹和体内的专业知识 生物起搏器功能的研究。成功执行研究计划将增强选择 来自IPSC的心脏细胞谱系技术，表征了HFKT-的起搏器活动的基础 起搏器并确定其作为体内生物起搏器的有效性。如果是HFKT-Pacemaker 在犬心中很好地发挥作用，未来的目标是推进这种新颖的自体细胞方法 临床部署。

项目成果

Functional cardiomyocytes from human stem cells: a tool for determining the cardiotoxic potential of preclinical drugs.

来自人类干细胞的功能性心肌细胞：确定临床前药物心脏毒性潜力的工具。

• DOI: 10.4155/fmc.13.22
• 发表时间: 2013
• 期刊: Future medicinal chemistry
• 影响因子: 4.2
• 作者: Zeevi-Levin,Naama;Itskovitz-Eldor,Joseph;Binah,Ofer
• 通讯作者: Binah,Ofer

Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

• DOI: 10.1161/circep.116.004508
• 发表时间: 2017-05
• 期刊: Circulation. Arrhythmia and electrophysiology
• 作者: Chauveau S;Anyukhovsky EP;Ben-Ari M;Naor S;Jiang YP;Danilo P Jr;Rahim T;Burke S;Qiu X;Potapova IA;Doronin SV;Brink PR;Binah O;Cohen IS;Rosen MR
• 通讯作者: Rosen MR