Making Embryonic Stem Cells Infarct Avid

描述（由申请人提供）：本提案的总体目标是使胚胎干细胞（ESCs）能够特异性识别并结合梗死组织，它们有望取代和再生梗死组织。干细胞移植修复心脏有望成为梗死后的治疗选择。与心肌内注射相比，急性心肌梗死（MI）患者血管内输注干细胞的侵入性更小，耐受性更好。然而，经血管输注后ESC在梗死部位的保留率相对较低，这是阻碍充分植入和再生的限制因素。为了解决这一挑战，我们将修改干细胞表面，使输血的细胞能够通过识别一种定义明确的细胞死亡分子标记——阴离子磷脂（APLDs）与梗死组织结合。在细胞凋亡和坏死过程中，APLDs暴露于细胞外环境，从而为同源部分识别梗死组织提供了一个特异性和充足的结合靶标。这种同源片段是synaptotagmin I的C2A结构域，synaptotagmin I是一种特性良好的aplds结合蛋白，具有高特异性和亲和力，可识别死亡和垂死细胞。最近，我们证明静脉注射的C2A在梗死心肌中大量积聚。为了从逻辑上扩展这一有趣的发现，我们将应用这种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附着在ESC表面来赋予ESCs梗死亲和性。作为初步研究的一部分，我们已经证明ESCs可以用C2A表面标记，而不会影响细胞生理学或多能性。而且，作为可行性的证明，我们观察到c2a偶联的ESCs可以结合死亡的心肌细胞，但不能结合活的心肌细胞。根据这些初步数据，我们假设配备了aplds结合活性的ESCs将显著改善体内梗死部位的植入。为了加强心脏修复治疗并验证这一假设，我们提出了以下具体目标：1)改进和优化C2A粘附于ESCs表面的偶联方法；2)评估c2a共轭ESCs在梗死心肌组织中的移植率，并评估急性心肌梗死小鼠模型的长期心功能。一旦建立，赋予梗死亲和性的概念应适用于其他类型的移植细胞，包括ESCs衍生的分化细胞类型。总的来说，这项工作探索了一种新的移植策略，将有助于心脏修复与转化潜力。公共卫生相关性：干细胞治疗在梗死心肌再生方面有希望。虽然血管内移植干细胞具有微创性，且患者耐受性较好，但其疗效受到灌注细胞在梗死部位的低保留率的限制。这项建议的目标是使干细胞能够识别和结合梗死组织。这将通过在不改变细胞生理的情况下将梗死蛋白附着在干细胞表面来实现。使干细胞对死亡组织和濒死组织具有结合亲和力，不仅有助于将移植的干细胞靶向到梗死部位，而且还能提高它们在梗死组织内的保留和植入率。

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.