Engineered Stem Cells for Cardiac Repair

用于心脏修复的工程干细胞

• 批准号: 10588153
• 负责人: MICHAEL REGNIER
• 金额: $ 78.83万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10588153
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Actins; Affect; Award; Behavior; Binding; Binding Sites; Canis familiaris; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cell Line; Cell Survival; Cell Transplantation; Cells; Cellular Metabolic Process; Chronic; Computer Models; Congestive Heart Failure; Contractile Proteins; Contracts; Data; Dependence; Depressed mood; Development; Disease; Dose; Electrostatics; Engineering; Engraftment; Enzymes; Equilibrium; Family suidae; Funding; Gap Junctions; Genes; Genetic Transcription; Goals; Heart; Heart failure; Human; Human Engineering; In Vitro; Infarction; Ischemia; Kinetics; Left; Left Ventricular Function; Macaca nemestrina; Mechanics; Mediating; Microscopy; Modeling; Molecular; Movement Disorders; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscle relaxation phase; Myocardial; Myocardial Infarction; Myocardium; Myosin ATPase; Nucleotides; Nude Rats; Pathology; Pathway interactions; Performance; Perfusion; Phosphocreatine; Phosphorylation; Physiologic intraventricular pressure; Positioning Attribute; Proteins; Publishing; Rattus; Recovery; Relaxation; Reperfusion Injury; Reporting; Rest; Ribonucleotide Reductase; Rodent; Rodent Model; Roentgen Rays; Sarcomeres; Stimulant; Structure; Testing; Thick Filament; Thin Filament; Tissues; Transgenic Mice; Transgenic Organisms; Transplantation; Ventricular; Ventricular Function; Vertebral column; Viral Vector; Work; X ray diffraction analysis; cardiac repair; cell replacement therapy; engineered stem cells; experimental study; functional improvement; heart function; human stem cells; imaging approach; improved; improved outcome; induced pluripotent stem cell; inhibitor; molecular dynamics; multi-scale modeling; nonhuman primate; novel; overexpression; pressure; promoter; recruit; single molecule; small molecule; small molecule therapeutics; stem cells; targeted treatment; time use; ultra high resolution

ABSTRACT. Aim 1 of this project is built around years of collaborative work between Drs. Regnier and Murry studying human stem cell derived cardiomyocytes as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). We have shown that human stem cells can be differentiated into cardiomyocytes (CMs), produced at a scale and purity that permit testing in rodent models and non-human primates (NHP; Macaca nemestrina) and that these cells engraft and integrate with host tissue to improve left ventricular performance. The premise for the proposed experiments is based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of contractility when used by cardiac myosin, and 2) hiPSC-CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have increased contractility and also deliver dATP to the native myocardium heart via gap junctions. In our current award we made excellent progress in testing the hypothesis that engineered hiPSC-CMs with elevated RNR (hiPSC-CMRNR) improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs). For this proposal, we have generated new hiPSC-CM lines with gene-edited RNR and different transcriptional promotors. These cells have greater RNR expression and produce multi-fold greater levels of cellular dATP. Thus, we will test the dose dependence of elevated dATP for hiPSC-CMRNR engrafted into infarcted rat hearts. The novel aspect of our approach is to go beyond replacement of lost tissue (with hiPSC-CMs) by using engineered hiPSC-CMRNR to produce and deliver a small molecule therapeutic (dATP) that improves native heart muscle contraction. This has the potential to substantially recover the post-MI depressed function of native myocardium. Aim 2 will explore the mechanistic basis of how small increases in myocardial dATP result in significant increases in contractile force and kinetics of activation and relaxation of muscle, and in the magnitude of LV pressure development (LVDP) and kinetics of pressure development (+dP/dt) and decline (-dP/dt) of the heart. Our recent reports and preliminary data strongly suggest at least three mechanisms are involved: 1) disruption of the super-relaxed state (SRX) from the myosin backbone to a disordered relaxed state (DRX), 2) movement of DRX myosin towards thin filaments via greater electrostatic interactions with actin, and 3) faster crossbridge cycling. We have published multiple studies on the chemo-mechanics of faster crossbridge cycling (3), so will focus primarily on mechanisms 1 and 2 here using multiple state of the art approaches. These include low angle x-ray diffraction analysis of isolated myosin, cardiac muscle, and whole heart (Langendorff) levels, stopped-flow ATPase, super-localization single molecule microscopy of thick filament zones, structure-based computational models of myosin ± actin and multi-scale models of the heart. This project will elucidate the potential of our combination cell-small molecule therapy approach to improve function in failing hearts and provide understanding of the detailed molecular mechanisms of the myosin activator dATP.

摘要。该项目的目标1是围绕着Regnier博士和Murry博士多年的合作工作而建立的 研究人类干细胞衍生的心肌细胞作为心脏修复的潜在细胞替代策略 心肌梗死（MI）。我们已经证明人类干细胞可以分化成 心肌细胞（CM），以允许在啮齿动物模型和非人类模型中进行测试的规模和纯度生产 灵长类动物（NHP; Macaca nemestrina），这些细胞移植并与宿主组织整合，以改善左 心室性能实验的前提是基于两个基本发现： 1)2-脱氧ATP（dATP）是一种有效的天然核苷酸刺激剂，当被心肌肌球蛋白使用时， 2)hiPSC-CM过表达dATP合成的限速酶，核糖核苷酸还原酶（RNR）， 具有增加的收缩性，并且还通过间隙连接将dATP递送至天然心肌。在我们 目前的奖项，我们在测试假设，工程hiPSC-CM与升高 RNR（hiPSC-CMRNR）改善了MI细胞替代疗法的结局（与对照hiPSC-CM相比）。 对于该提议，我们已经产生了具有基因编辑的RNR和不同转录水平的新的hiPSC-CM系。 发起人这些细胞具有更高的RNR表达，并产生数倍更高水平的细胞dATP。 因此，我们将测试移植到梗塞大鼠心脏中的hiPSC-CMRNR的升高的dATP的剂量依赖性。 我们方法的新颖之处在于，通过使用hiPSC-CM， 工程化hiPSC-CMRNR以产生和递送改善天然心脏的小分子治疗剂（dATP）， 肌肉收缩。这有可能基本上恢复心肌梗死后受抑制的天然心肌细胞的功能。 心肌目的2将探讨心肌dATP的微小增加如何导致心肌缺血的机制基础。 显著增加肌肉的收缩力和激活和松弛的动力学，以及 左心室压力发展（LVDP）和压力发展（+dP/dt）和下降（-dP/dt）的动力学 心我们最近的报告和初步数据强烈表明至少有三种机制参与：1） 从肌球蛋白骨架到无序松弛状态（DRX）的超松弛状态（SRX）的破坏，2） DRX肌球蛋白通过与肌动蛋白更大的静电相互作用向细丝运动，以及3）更快 骑自行车过桥。我们已经发表了多项关于更快的过桥自行车的化学力学的研究 (3)因此，这里将主要关注使用多种现有技术方法的机制1和机制2。这些包括 分离的肌球蛋白、心肌和整个心脏（Langendorff）水平的低角X射线衍射分析， 停流ATP酶，粗丝区超定位单分子显微镜，基于结构 肌球蛋白±肌动蛋白的计算模型和心脏的多尺度模型。该项目将阐明 我们的组合细胞-小分子治疗方法改善衰竭心脏功能的潜力， 提供肌球蛋白激活剂dATP的详细分子机制的理解。