Geometric Cues in the Stem Cell Microenvironment

描述（由申请人提供）：该项目的目的是探索生态位中的几何线索如何引导间充质干细胞（MSCs）走向特定的命运。人间充质干细胞是多能干细胞，可分化为脂肪、骨、肌腱、肌肉和神经元样细胞。多能性和易于收集（即脂肪抽吸，骨髓）使间充质干细胞成为再生治疗和组织工程中更广泛研究的干细胞类型之一。在确定指导MSC命运决定的化学和生物信号方面取得了重大进展。然而，目前对生态位几何在指导MSC自我更新和谱系承诺中的作用知之甚少。大多数试图模拟干细胞微环境的系统不能充分控制细胞形状、空间分布和增殖的变化。提出的研究旨在使用模型表面来研究一系列单一MSCs的分化，这些MSCs被限制在精确的形状中。细胞粘附和细胞骨架特征在组织形态发生的许多方面都很重要。因此，这项工作的形状选择将受到先前显示的直接粘合，收缩和突出元素的特定几何线索的影响。作为第一个具体目标，MSCs将在生长和分化条件下的混合形状模式中培养，以评估几何线索本身以及与谱系促进可溶性因子结合的影响。组织化学，免疫荧光和RT-PCR将用于比较不同条件和形状的细胞。第二个具体目标将探索细胞骨架元件和谱系调节因子的抑制作用，以分离形状促进命运决定的重要生化途径。此外，焦点粘附密度在生态位中的作用将通过改变不同形状的表面化学来平行探索。随着目标1中开发的技术，DNA微阵列分析，蛋白质电泳，western blotting和酶活性分析将用于分离重要的网络，以彻底了解MSC生态位内的信号事件。来自成人骨髓的间充质干细胞（MSCs）有望成为再生医学中多能细胞的丰富来源。不适当的间充质干细胞命运调节涉及许多病理包括：骨质疏松症，动脉粥样硬化和癌细胞转移。因此，破解贴壁干细胞形状如何影响其命运对于理解再生和病理过程以及设计基于干细胞的治疗材料都具有重要意义。

DESCRIPTION (provided by applicant): The objective of this project is to explore how geometric cues in the niche guide mesenchymal stem cells (MSCs) towards specific fates. Human MSCs are multipotent stem cells that differentiate into fat, bone, tendon, muscle and neuron-like cells. The multipotency and ease of collection (i.e. fat aspirates, bone marrow) makes MSCs one of the more widely studied stem cell types for regenerative therapy and tissue engineering. Significant strides have been made in determining the chemical and biological signals that direct MSC fate decisions. However, there is currently very little understanding of the role of the niche geometry in directing MSC self-renewal and lineage commitment. The majority of systems that attempt to model the microenvironment of stem cells in their niche do not adequately control for variations in cell shape, spatial distribution and proliferation. The proposed research aims to use model surfaces to study the differentiation of an array of single MSCs confined to precise shapes. Cell adhesion and cytoskeletal features are important in many aspects of tissue morphogenesis. Thus, the choice of shapes for this work will be influenced by specific geometric cues previously shown to direct adhesive, contractile and protrusive elements. As the first specific aim, MSCs will be cultured on mixed patterns of shapes under growth and differentiation conditions to evaluate the effect of geometric cues by themselves and in combination with lineage promoting soluble factors. Histochemistry, immunofluorescence and RT-PCR will be used to compare cells across conditions and shapes. The second specific aim will explore inhibition of cytoskeletal elements and lineage regulators to isolate important biochemical pathways in shape-promoted fate decisions. Furthermore, the role of focal adhesion density within the niche will be explored in parallel by varying the surface chemistry across shapes. Along with techniques developed in aim 1, DNA microarray analysis, protein electrophoresis, western blotting and enzyme activity assays will be used to isolate important networks towards a thorough understanding of signaling events within the MSC niche. Mesenchymal stem cells (MSCs) from adult bone marrow hold promise as an abundant source of multipotent cells for regenerative medicine. Improper MSC fate regulation has been implicated in numerous pathologies including: osteoporosis, atherosclerosis and cancer cell metastasis. Therefore, deciphering how an adherent stem cells shape influences its fate is of vast importance for both understanding regenerative and pathological processes as well as for designing materials for stem cell based therapies.