Engineered Cardiac Niche Arrays for Exploring and Optimizing Stem Cell Therapies

描述（由申请人提供）：由于成体心肌细胞无法增殖，心肌的自发修复或再生通常是不可能的。因此，心肌梗塞等病理事件会导致永久性损伤，常常导致心力衰竭和死亡。作为一种潜在的治疗策略，干细胞具有巨大的潜力，因为它们能够在局部利基微环境的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在内的并发症，加上苛刻且不断变化的梗塞后环境，阻碍了基于干细胞治疗心肌梗塞的机制的识别，导致临床试验失败。工程心脏组织提供了替代的实验模型系统，它结合了比标准培养皿更符合生理学的 3D 环境，具有天然心肌制剂不可能实现的长期生存能力和改进的实验控制。然而，工程心脏组织主要是为了外科修复应用而开发的，而不是作为设计用于研究心肌损伤、修复和再生的体外活体系统。该提案旨在开发结合软光刻和组织工程的创新工具和方法，总体目标是提高对基于干细胞的心脏修复方法的理解和功效。一个指导性假设是，3D 微环境的力学是控制人类间充质干细胞 (MSC) 和心肌细胞 (CM) 之间相互作用的关键因素。设计了两个具体目标来建立这个新的模型系统，检验这个假设，并为该领域的未来研究提供一个跳板。目标 1：开发高通量工程心脏组织 (ECT) 阵列系统，以评估和优化干细胞共培养策略，以在受控 3D 微环境中增强心肌细胞收缩功能。该目标将重点研究被动拉伸、机械刚度和驻留心肌细胞类型对 MSC 使用基于力传感器阵列的独特模块化软光刻改善工程心脏组织收缩功能的能力的影响。目标 2：建立组织工程 3D 冷冻梗死模型，以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。该目标将上述工程心脏组织阵列系统与新型冷冻梗塞方法相结合，用于在受控 3D 模型损伤环境中检查 MSC 功能，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。这一高风险、高收益提案的成功应该会导致干细胞力学生物学的理解取得新进展，促进转化为更复杂的实验动物和临床环境。 公众健康相关性：由于心肌的自发修复或再生通常是不可能的，因此心肌梗塞等病理事件会导致永久性损伤，常常导致心力衰竭和死亡。尽管干细胞在心脏修复方面具有巨大的潜力，但由于缺乏控制良好的心肌梗死实验模型（无法对损伤和修复过程进行长期研究），我们对引导干细胞分化的机制的理解受到阻碍。该提案旨在开发结合软光刻和组织工程的创新工具和方法，总体目标是理解和指导心脏修复应用中的干细胞分化，这将有望在理解干细胞力学生物学方面带来新的进展，并促进转化为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.