Making Embryonic Stem Cells Infarct Avid

描述（由申请人提供）：该提案的广泛目标是使胚胎干细胞（ESC）能够特异性识别并结合梗死组织，预期它们将取代和再生。通过干细胞移植进行心脏修复有望成为梗死后的治疗选择。与心肌内注射相比，血管内输注干细胞的侵入性更小，并且急性心肌梗死（MI）患者的耐受性更好。然而，经血管输注后ESC在梗死部位的保留率相对较低，这是阻止充分植入和再生的限制因素。为了应对这一挑战，我们将修改干细胞表面，使输注的细胞能够通过识别细胞死亡的明确分子标志物阴离子磷脂（APLD）与梗死组织结合。APLD在细胞凋亡和坏死期间暴露于细胞外环境，从而为同源部分识别梗死组织提供特异性和充足的结合靶标。这种同源部分是突触结合蛋白I的C2 A结构域，突触结合蛋白I是一种充分表征的APLD结合蛋白，其以高特异性和亲和力识别死亡和垂死细胞。最近，我们证明了静脉注射的C2 A在梗死心肌中大量积聚。为了在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递送的ESC植入到梗死心肌中。主要目标是通过将C2 A附着到ESC表面来赋予ESC梗死亲合力。作为初步研究的一部分，我们已经证明了ESC可以用C2 A进行表面标记，而不会影响细胞生理学或多能性。而且，作为可行性的证据，我们已经观察到C2 A缀合的ESC结合死亡的心肌细胞，但不结合存活的心肌细胞。根据这些初步的数据，我们假设，ESCs配备APLD结合活性将导致一个显着改善体内梗死部位内的植入。为了增强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C2 A连接到ESC表面的缀合方法; 2）评估C2 A缀合的ESC在梗死心脏组织中的植入率并评估急性MI小鼠模型中的长期心脏功能。一旦建立，赋予梗死亲合力的概念应该适用于其他类型的移植细胞，包括来自ESC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翻译潜力。公共卫生相关性：干细胞疗法有望再生梗死心肌。虽然干细胞的血管内递送是微创的并且患者更好地耐受，但是其功效受到梗塞部位中输注细胞的低留存率的限制。这项提议的目标是使干细胞能够识别并结合梗死组织。这将通过在不改变细胞生理学的情况下将梗塞亲和蛋白附着到干细胞表面来实现。使干细胞对死亡和垂死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死部位，而且还可以提高它们在梗死组织中的保留和植入率。

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.