Making Embryonic Stem Cells Infarct Avid

描述(由申请人提供)：这项提议的广泛目标是使胚胎干细胞(ESCs)能够特异性地识别并结合到梗死组织上，这些组织有望被取代和再生。干细胞移植的心脏修复有望成为脑梗塞后的治疗选择。与心肌内注射相比，血管内输注干细胞侵入性更小，对急性心肌梗死(MI)患者的耐受性更好。然而，经血管输注后ESC在梗死区的滞留率相对较低，这是阻碍充分植入和再生的限制因素。为了应对这一挑战，我们将通过识别细胞死亡的明确分子标记物--阴离子磷脂(APLD)来修饰干细胞表面，使输注的细胞能够与梗塞组织结合。APLD在细胞凋亡和坏死过程中暴露在细胞外环境中，从而为同源部分识别梗死组织提供了一个特异和丰富的结合靶点。这种同源部分就是突触素I的C2a结构域，它是一种特性良好的APLDs结合蛋白，具有高度的特异性和亲和力识别死亡和濒临死亡的细胞。最近，我们证明了静脉注射的C2a在梗死心肌中大量积聚。为了合理地扩展这一有趣的发现，我们将应用这一独特的分子识别机制来介导血管内输送的ESCs植入梗死心肌。主要目的是通过将C2a附着到ESCs表面来赋予ESCs梗塞亲和力。作为初步研究的一部分，我们已经证明了ESCs可以在表面标记C2a，而不影响细胞生理学或多能性。而且，作为可行性的证据，我们观察到C2a结合的ESCs结合死亡的心肌细胞，但不结合存活的心肌细胞。根据这些初步数据，我们假设，配备了APLDs结合活性的ESCs将显著改善体内梗塞部位的植入率。为了促进治疗性心脏修复并验证这一假设，我们提出了以下具体目标：1)改进和优化将C2a连接到ESCs表面的连接方法；以及2)评估C2a连接的ESCs在梗塞心肌组织中的植入率，并评估在急性心肌梗死小鼠模型中的长期心功能。一旦确定，赋予梗塞亲和力的概念应该适用于其他类型的移植细胞，包括来源于ESCs的分化细胞类型。总体而言，这项工作探索了一种新的移植策略，将有助于具有翻译潜力的心脏修复。与公共卫生相关：干细胞疗法有望再生梗死心肌。虽然干细胞的血管内输送是微创的，患者对干细胞的耐受性更好，但其疗效受到梗死区输注细胞保留率低的限制。这项提议的目标是使干细胞能够识别和结合梗死组织。这将通过在干细胞表面附着一种抗梗死蛋白来实现，而不会改变细胞生理。使干细胞对死亡和濒临死亡的组织具有结合亲和力，不仅有助于将移植的干细胞靶向梗塞部位，而且还可以提高它们在梗塞组织中的保留率和植入率。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to enable embryonic stem cells (ESCs) to specifically recognize and bind to infarcted tissues, which they are expected to replace and regenerate. Cardiac repair by stem cell implantation holds promise as a post-infarct therapeutic option. Compared to intra- myocardial injection, intra-vascular infusion of stem cells is less invasive and better tolerated by patients with acute myocardial infarction (MI). However, the relatively low rate of ESC retention in the infarct site after transvascular infusion is a limiting factor that prevents sufficient engraftment and regeneration. To address the challenge, we will modify the stem cell surface so that transfused cells are able to bind to infarcted tissue by recognizing a well-defined molecular marker of cell death, anionic phospholipids (APLDs). APLDs become exposed to the extracellular milieu during apoptosis and necrosis, thereby providing a specific and ample binding target for the recognition of infarcted tissue by cognate moieties. Such a cognate moiety is the C2A domain of synaptotagmin I, a well- characterized APLDs-binding protein that recognizes dead and dying cells with high specificity and affinity. Recently, we demonstrated that intravenous-injected C2A avidly accumulates in infarcted myocardium. To logically extend this intriguing finding, we will apply this unique molecular recognition mechanism to mediate the engraftment of intravascularly delivered ESCs into infracted myocardium. The primary goal is to confer infarct avidity to ESCs by attaching the C2A to the ESC surface. As part of the Preliminary Studies, we have demonstrated that ESCs can be surface-labeled with C2A, without affecting cellular physiology or pluripotency. And, as a proof of feasibility, we have observed that C2A-conjugated ESCs bind dead cardiomyocytes, but not viable cardiomyocytes. In light of these preliminary data, we hypothesize that ESCs equipped with APLDs-binding activity will lead to a significantly improved engraftment within infarcted sites in vivo. To enhance therapeutic cardiac repair and to test this hypothesis, we propose the following Specific Aims: 1) to refine and optimize the conjugation methods for attaching the C2A to the surface of ESCs; and 2) to assess the engraftment rate of C2A-conjugated ESCs in infarcted cardiac tissue and evaluate the long-term cardiac function in a mouse model of acute MI. Once established, the concept of conferring infarct avidity should be applicable to other types of transplanted cells, including differentiated cell types derived from ESCs. Overall, this work explores a novel transplantation strategy that will aid in cardiac repair with translational potential. PUBLIC HEALTH RELEVANCE: Stem cell therapy holds promise in regenerating infarcted myocardium. While the intra-vascular delivery of stem cells is minimally invasive and better tolerated by patients, its efficacy is limited by a low retention rate of infused cells in the infarct site. The goal of this proposal is to make stem cells capable of recognizing and binding to infarcted tissues. This will be accomplished by attaching an infarct-avid protein to the stem cell surface without altering cellular physiology. Making stem cells with a binding affinity toward dead and dying tissues will not only help target transplanted stem cells to the infarct site, but also improve their rate of retention and engraftment within the infarcted tissues.