Engineered Cardiac Niche Arrays for Exploring and Optimizing Stem Cell Therapies

描述（由申请人提供）：由于成年心肌细胞无法增殖，心肌的自发修复或再生通常是不可能的。因此，诸如心肌梗塞的病理事件导致永久性损伤，其通常导致心力衰竭和死亡。作为一种潜在的治疗策略，干细胞提供了巨大的潜力，因为它们能够在局部小生境微环境内的线索引导下分化成组织特异性细胞类型，可能为心脏修复提供细胞来源。然而，包括低细胞保留和活力的并发症，加上苛刻和不断变化的梗死后环境，阻碍了对基于干细胞的心肌梗死治疗机制的鉴定，导致临床试验失败。工程化心脏组织提供了替代实验模型系统，其结合了比标准培养皿更生理的3D环境，具有天然心肌制剂不可能实现的长期存活力和改进的实验控制。然而，工程化心脏组织主要是为外科修复应用而开发的，而不是为研究心肌损伤、修复和再生而设计的活的体外系统。该提案旨在开发创新的新工具和方法，结合软光刻和组织工程，总体目标是提高对基于干细胞的心脏修复方法的理解和疗效。一个指导性假设是，3D微环境的力学是控制人间充质干细胞（MSC）和心肌细胞（CM）之间相互作用的关键因素。两个具体的目标是建立这个新的模型系统，测试这一假设，并提供了一个跳板，在这一领域的未来研究。目标1：开发高通量工程化心脏组织（ECT）阵列系统，以评估和优化干细胞共培养策略，从而在受控的3D微环境中增强心肌细胞收缩功能。这个目标将集中研究被动拉伸，机械刚度，和居民的心脏细胞类型的MSC的能力，以提高工程心脏组织的收缩功能，使用一个独特的模块化软光刻力传感器阵列的影响。目标二：建立组织工程三维冷冻梗死模型，研究局灶性细胞损伤对干细胞迁移、分化和心脏修复的影响。该目标将联合收割机将上述工程化心脏组织阵列系统与用于在受控的3D模型损伤环境中检查MSC功能的新型冷冻梗塞方法相结合，有助于将目标1的发现转化为心肌梗塞或ECT植入的实验动物模型。这种高风险、高产量的提议的成功应该会导致对干细胞机械生物学的理解取得新的进展，促进转化为更复杂的实验动物和临床环境。 公共卫生相关性：由于心肌的自发修复或再生通常是不可能的，因此心肌梗死等病理事件会导致永久性损伤，通常导致心力衰竭和死亡。虽然干细胞为心脏修复提供了巨大的潜力，但我们对指导干细胞分化的机制的理解受到缺乏良好控制的心肌梗死实验模型的阻碍，该模型允许长期研究损伤和修复过程。该提案旨在开发创新的新工具和方法，将软光刻和组织工程相结合，总体目标是了解和指导干细胞分化用于心脏修复应用，这将有望在理解干细胞机械生物学方面取得新进展，并促进翻译到更复杂的实验动物和人类患者。

DESCRIPTION (provided by applicant): Due to the inability of adult cardiac myocytes to proliferate, spontaneous repair or regeneration of heart muscle is not normally possible. Consequently, pathological events such as myocardial infarction result in permanent damage that often leads to heart failure and death. As a potential therapeutic strategy, stem cells offer great potential due to their ability to differentiate into tissue-specific cell types guided by cues within the local niche microenvironment, possibly providing a cell source for cardiac repair. However, complications including low cell retention and viability, combined with a demanding and evolving post-infarction environment, have impeded the identification of mechanisms governing stem cell based treatments for myocardial infarction, resulting in failed clinical trials. Engineered cardiac tissues offer alternative experimental model systems combining a more physiologic 3D environment than the standard Petri dish, with long-term viability and improved experimental control not possible with natural heart muscle preparations. However, engineered cardiac tissues have been developed primarily for surgical repair applications, rather than as living in vitro systems designed for investigating myocardial injury, repair, and regeneration. This proposal aims to develop innovative new tools and approaches combining soft lithography and tissue engineering, with the overall objective of improving the understanding and efficacy of stem cell based approaches for cardiac repair. A guiding hypothesis is that mechanics of the 3D microenvironment is a key factor governing the interaction between human mesenchymal stem cells (MSC) and cardiac myocytes (CM). Two specific aims are designed to establish this new model system, test this hypothesis, and provide a springboard for future studies in this area. Aim 1: To develop a high-throughput engineered cardiac tissue (ECT) array system to evaluate and optimize stem cell co-culture strategies for enhancing cardiomyocyte contractile function in a controlled 3D microenvironment. This aim will focus on studying the effects of passive stretch, mechanical stiffness, and resident cardiac cell types on the ability of MSC to improve the contractile function of engineered cardiac tissues using a unique modular soft lithography based force sensor array. Aim 2: To establish a tissue engineered 3D cryo-infarct model to examine the effects of focal cell injury on stem cell migration, differentiation, and cardiac repair. This aim will combine the above engineered cardiac tissue array system with a novel cryo-infarct approach for examining MSC function in a controlled 3D model injury environment, helping to translate the findings of Aim 1 to experimental animal models of myocardial infarction or ECT implantation. Success of this high- risk, high-yield proposal should lead to new advances in understanding stem cell mechanobiology, facilitating translation to more complex experimental animal and clinical settings. PUBLIC HEALTH RELEVANCE: Because spontaneous repair or regeneration of heart muscle is not normally possible, pathological events such as myocardial infarction result in permanent damage that often leads to heart failure and death. Although stem cells offer great potential for cardiac repair, our understanding of the mechanisms guiding differentiation of stem cells is hampered by a lack of well-controlled experimental models of myocardial infarction that allow long term study of injury and repair processes. This proposal aims to develop innovative new tools and approaches combining soft lithography and tissue engineering, with the overall objective of understanding and directing stem cell differentiation for cardiac repair applications, which will hopefully lead to new advances in understanding stem cell mechanobiology and facilitate translation to more complex experimental animals and human patients.