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

皮埃尔，布伦达提案编号：1445650机构：明尼苏达大学双子城分校标题：急切：ECM拓扑图中发育特定的变化影响心脏特性干细胞在治疗急慢性疾病方面具有巨大潜力，包括心脏疾病；然而，将这些细胞输送给患者的最佳方法尚不清楚。大部分困难在于通常生长细胞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，组织被包裹在由纳米和微米级3D纤维组成的网络中，该网络由胶原、层粘连蛋白和弹性蛋白等蛋白质组成，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，蓝图将从不同发育阶段的心脏组织的显微镜数据中得出。确定干细胞对复制的组织结构的反应将对干细胞如何被操纵和输送用于研究和治疗具有重大而根本的影响。此外，这一建议的成功将对研究其他类型的健康或疾病组织中的细胞-基质相互作用具有广泛的实用价值。更好地了解干细胞-细胞外基质(ECM)相互作用可以完善干细胞培养体系，改进诱导心脏细胞类型功能分化的方案。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向心脏的治疗性输送提供信息。据推测，发育中心脏的三维地形图特征可以引导多能干细胞或心脏前体细胞分化为功能心肌细胞类型。为了验证这一假设，将通过严格概括体内ECM结构来开发促进干细胞或其后代分化和交付的方法。具体地说，将使用多光子激发(MPE)光化学来创建3D矩阵，每次一个平面，具有亚微米特征尺寸，模板来自定义明确的时间点的高分辨率心脏显微图像数据。为此，将使用牛血清白蛋白或甲基丙烯酸甲酯制造基质，以有目的地消除与细胞外基质蛋白质相关的生化信号(尽管应该注意，该技术能够用细胞外基质蛋白质制造)。能够完全分化为成熟心肌细胞类型的室祖细胞(VPC)将被种植在基质中。在之后的每周时间点，将对基质中的细胞进行心脏细胞类型的表型和功能属性测试。与发育时间点相关的基质之间的主要比较指标将是具有心肌细胞表型和功能的细胞的百分比。二级比较器将是每种心肌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，相对于对应于较早和较晚时间点的基质，从对应于胚胎后期心脏发育的模板衍生的功能心肌细胞的百分比将在统计上显著增加。干细胞对细胞外基质结构的成功响应将对人们如何操纵和运送用于研究和治疗的干细胞产生重大和根本性的影响，并将对更广泛地了解细胞与细胞外基质在健康和疾病中的相互作用的研究战略产生重大影响。

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.