Engineered Stem Cells for Cardiac Repair

抽象的。这个项目建立在默里博士和雷格尼耶博士多年的合作基础上。 人胚胎干细胞来源心肌细胞(hESC-CMS)作为潜在细胞替代细胞的研究 心肌梗塞(MI)后的心脏修复策略。我们的小组已经证明了hESC-CMS和 人可诱导多能干细胞-CMS(HIPS-CMS)的生产规模和纯度允许在 啮齿动物模型和最有可能预测人类反应的动物：非人灵长类(NHP； 猕猴)。我们已经证明了这些细胞在啮齿动物模型中植入的能力，包括 整个疤痕，并与宿主组织电结合，以改善左心功能。 这个项目基于两个基本发现：1)2-脱氧三磷酸腺苷(DATP)是一种有效的天然 核苷酸刺激心肌收缩能力(通过改善肌球蛋白与肌动蛋白的结合和更快的脱离后 2)高表达dATP合成限速酶的HiPSC-CMS，即核糖核苷酸 还原酶(RNR)既可以增加心脏的收缩能力，又可以通过缝隙连接将dATP输送到心脏的其余部分。 因此，我们将检验工程hiPSC-CMS以提高RNR(RNR-hiPSC-CMS)将会改善的假设 心肌梗死细胞替代治疗的结果(与对照HIPSC-CMS相比)，改善心肌收缩功能 移植心肌和自体心肌均有。我们的方法有几个非常新奇的方面。1)这是第一次 建议使用细胞核苷酸操作来改善体内的心功能。2)方法不是 仅限于用功能更好的移植物替换丢失的组织(用HiPSC-CMS)，但也可以基本上 有利于改善心肌梗死后自主心肌的抑制功能。3)首次使用工程HIPSC-CMS来 提供一种有效的小分子疗法(DATP)，一种改善心肌的天然化合物 收缩。这有效地使HiPSC-CMS成为具有心脏特异性递送和 效果。目标1将开发和测试RNR的工程突变，以增加其稳定性和活性 心肌细胞及其滴定在HiPSC-CMS中产生的dATP水平增加的能力。AIM 2将使用 AAV载体用于RNR突变体，选自Aim 1，用于研究其改善心脏功能的能力。 心肌梗死和心力衰竭的小鼠模型。AIM 3将生产工程HIPS细胞系，将 作为分化后的dATP“供体细胞”，用于移植到急性心肌梗塞和更具挑战性的地方 慢性心肌梗死无菌大鼠模型的建立及其对移植后功能改善的影响 工程HIPSC-CMS。我们将评估这些影响的持续性，并确定长期影响 这些细胞系的稳定性和存活率。我们预计移植物和移植物的收缩性能都会有显著改善。 RNR-hiPSC-CMS与HiPSC-CMS的天然心肌及其dATP的调节作用 移植细胞的生产能力。这些研究的结果将阐明这一潜力 结合细胞和小分子疗法来改善甚至改善衰竭心脏的泵功能。

ABSTRACT. This project is built around years of collaborative work between Drs. Murry and Regnier studying human embryonic stem cell derived cardiomyocytes (hESC-CMs) as a potential cell replacement strategy for cardiac repair following myocardial infarction (MI). Our group has shown that hESC-CMs and human inducible pluripotent SC-CMs (hiPS-CMs) can be produced at a scale and purity that permit testing in rodent models and the animal most likely to predict the human response: the non-human primate (NHP; Macaca nemestrina). We have demonstrated the ability of these cells to engraft in rodent models, covering the entire scar, and electrically integrate with host tissue to improve left ventricular performance. This project is based on two fundamental discoveries: 1) 2-deoxy ATP (dATP) is a potent natural nucleotide stimulant of cardiac contractility (via improved myosin binding to actin & faster detachment after the power stroke), and 2) hiPSC-CMs that overexpress the rate-limiting enzyme for dATP synthesis, ribonucleotide reductase (RNR), have both increased contractility and deliver dATP to the rest of the heart via gap junctions. Thus we will test the hypothesis that engineering hiPSC-CMs to elevate RNR (RNR-hiPSC-CMs) will improve outcomes in cell replacement therapy for MI (compared with control hiPSC-CMs), improving contractility of both graft and native myocardium. There are several highly novel aspects to our approach. 1) It is the first proposed use of cellular nucleotide manipulation to improve in vivo cardiac function. 2) The approach is not limited to replacement of lost tissue (with hiPSC-CMs) with a better functioning graft, but may also substantially benefit the post-MI depressed function of native myocardium. 3) The first use of engineered hiPSC-CMs to deliver what is effectively a small molecule therapy (dATP), a natural compound that improves heart muscle contraction. This effectively makes hiPSC-CMs a drug delivery device with cardiac specific delivery and effects. Aim 1 will develop and test engineered mutations in RNR that increase it's stability and activity in cardiomyocytes and their ability to titrate increasing levels of dATP produced in hiPSC-CMs. Aim 2 will use AAV vectors for RNR variants, selected from Aim 1, to investigate their capacity to improve cardiac function in a mouse model of myocardial infarct and heart failure. Aim 3 will produce engineered hiPS cell lines that will act as dATP `donor cells' following differentiation, for transplantation into acute MI and more challenging chronic MI athymic rat models to determine their capacity to improve function beyond transplantation of non- engineered hiPSC-CMs. We will evaluate the persistence of these effects and determine the long-term stability and viability of these cell lines. We expect significant contractile improvement of both the graft and native myocardium with RNR-hiPSC-CMs vs. hiPSC-CMs and this effect will be modulated by the dATP producing capacity of the transplanted cells. Results from these studies will elucidate the potential of this combination cell- and small molecule therapy to ameliorate or even improve pump function in failing hearts.