EAGER: Development-specific changes in ECM topography influence cardiac specification

项目负责人：Ogle， Brenda提案编号：1445650机构：明尼苏达大学双子城分校（University of Minnesota-Twin cities）标题：急切：发育特异性ECM地形变化影响心脏规格干细胞在治疗急性和慢性疾病（包括心脏疾病）方面具有巨大潜力；然而，将这些细胞输送给患者的最佳方法还有待研究。大部分困难在于细胞通常生长的组织培养环境与体内组织的复杂结构之间的脱节。在后者中，组织被包裹在由胶原蛋白、层粘连蛋白和弹性蛋白等蛋白质组成的纳米和微型3D纤维网络中，这些蛋白质促进细胞生长和新组织的合成。该项目旨在复制体内组织结构，其中蓝图将来自心脏组织在不同发育阶段的显微镜数据。确定干细胞对复制组织结构的反应将对干细胞如何用于研究和治疗产生重大而根本的影响。此外，这一建议的成功将对研究与健康或疾病有关的其他组织类型的细胞-基质相互作用具有广泛的效用。更好地了解干细胞-细胞外基质（ECM）的相互作用可以改进干细胞培养制度和改进方案，以诱导心脏细胞类型的功能分化。由此产生的分化细胞类型可用于体外毒性测试，并为干细胞或其后代的心脏治疗递送提供信息。据推测，心脏发育的三维地形特征可以引导多能干细胞或心脏前体向功能性心脏细胞类型的分化。为了验证这一假设，将通过严格再现体内ECM结构来开发促进干细胞或其后代分化和传递的方法。具体来说，多光子激发（MPE）光化学将用于创建三维矩阵，每次一个平面，具有亚微米特征尺寸，模板来源于在明确定义的时间点发育中的心脏的高分辨率显微镜图像数据。为此，将使用BSA或甲基丙烯酸甲酯制造基质，以有目的地消除与ECM蛋白相关的生化信号（尽管应该注意的是，该技术能够用ECM蛋白制造）。心室祖细胞（VPCs），能够完全分化为成熟的心脏细胞类型，将被播种在基质中。在此后的每周时间点，将对基质中的细胞进行检测，以了解心肌细胞类型的表型和功能属性。与发育时间点相关的基质之间的主要比较物将是具有心肌细胞表型和功能的细胞的百分比。次级比较指标将是每种心脏细胞类型的相对百分比。预计在BSA或基于甲基丙烯酸酯的结构中检测到的功能性心肌细胞的百分比将在统计上显着增加，这些结构来自于对应于胚胎心脏发育晚期的模板，相对于对应于较早和较晚时间点的基质。干细胞对ECM结构的成功反应将对如何操纵和提供用于研究和治疗的干细胞产生重大而根本的影响，并将对研究策略产生重大影响，从而更广泛地了解细胞-ECM在健康和疾病中的相互作用。

PI: Ogle, Brenda Proposal Number: 1445650Institution: University of Minnesota-Twin CitiesTitle: EAGER: Development-specific changes in ECM topography influence cardiac specificationStem cells have great potential to treat acute and chronic conditions, including cardiac disorders; however, the optimal methods of delivering these cells to patients have yet to be learned. Much of the difficulty lies in the disconnect between the tissue culture environments in which cells are typically grown, versus the complicated architecture of in vivo tissues. In the latter, tissues are encased in a network of nano and micoscale 3D fibers comprised of proteins such as collagen, laminin, and elastin that promote cell growth and synthesis of new tissue. This project aims to replicate the in vivo tissue structure, where the blueprint will be derived from microscopy data of cardiac tissues at various stages of development. Determining the stem cell response to the replicated tissue structure would have a significant and fundamental impact on how stem cells could be manipulated and delivered for both research and therapy. In addition, success of this proposal would have broad reaching utility for the study of cell-matrix interactions in other tissue types with health or disease.A better understanding of stem cell-extracellular matrix (ECM) interactions could refine stem cell culture regimes and improve protocols to induce functional differentiation of cardiac cell types. Resultant differentiated cell types could be used for in vitro toxicity testing and to inform therapeutic delivery of stem cells or their progeny to the heart. It is hypothesized that 3D topographical features of the developing heart can guide differentiation of pluripotent stem cells or cardiac precursors into functional cardiac cell types. To test this hypothesis, methods to advance the differentiation and delivery of stem cells or their progeny will be developed by rigorous recapitulation of in vivo ECM architecture. Specifically multiphoton-excited (MPE) photochemistry will be used to create 3D matrices, one plane at a time, with sub-micron feature sizes, from templates derived from high resolution microscopy image data of the developing heart at well-defined timepoints. For this objective, matrices will be fabricated using BSA or methyl methacrylate to purposefully eliminate biochemical signals associated with ECM proteins (though it should be noted that the technology is capable of fabricating with ECM proteins). Ventricular progenitor cells (VPCs), capable of differentiating exclusively to mature cardiac cell types, will be seeded in the matrices. At weekly time points thereafter, cells in matrices will be tested for phenotypic and functional attributes of cardiac cell types. The primary comparator between matrices associated with developmental time points will be the percentage of cells with cardiomyocyte phenotype and function. The secondary comparator will be the relative percentage of each cardiac cell type. It is anticipated that a statistically significant increase in the percentage of functional cardiomyocytes will be detected in BSA or methacrylate-based structures derived from templates corresponding to late embryonic cardiac development relative to matrices corresponding to earlier and later time points. A successful stem cell response to ECM structure would have a significant and fundamental impact on how one can manipulate and deliver stem cells for both research and therapy, and would substantially impact research strategies for more generally understanding cell-ECM interactions in health and disease.