Nanopatterned Surfaces to Control Cell Fate

项目摘要/摘要： 人类胚胎干细胞(HES)正在被研究为治疗多发性硬化症的潜在细胞来源 疾病(如糖尿病、脊髓损伤、帕金森氏症、白血病、充血性心力衰竭等)。这些是相同的 细胞也被吹捧为体外组织工程或原位再生医学的理想细胞来源。 成功将HES细胞整合到这种疗法中将取决于三个关键步骤：1)干细胞 无分化的数量扩张(即自我更新)；2)定向分化成特定的细胞 细胞类型或细胞类型的集合；以及，3)细胞存活和促进其功能整合到现有 组织。精确控制这些步骤中的每一个对于最大限度地提高HES细胞的治疗效果至关重要 功效。然而，很难精确控制HES细胞的行为，因为环境条件 人们对自我更新和差异化知之甚少。我们建议开发一种可调谐的完全合成的 表面和化学定义的介质，以控制HES细胞的自我更新/扩张。如果HES细胞可以 在完全合成的环境中派生和维护，那么就有可能消除 与动物源性材料相关的病原体传播，为大规模推广提供了基础 HES细胞的产生，并为进一步开发控制HES细胞分化提供了精确的基础。 这一应用将开发材料来解决这样的假设：HES细胞的收缩状态， 表现为整合素参与的细胞核形态，调节HES细胞的自我更新。我们的 假说的中心是一种共同的机制，即细胞对两种材料中的任何一种做出不同的反应 可变的模数或材料，通过粘连部位的分布在空间上限制细胞的形状。我们建议 控制HES细胞自我更新和决定细胞命运的一个常见机制是 表现为细胞核形态、整合素结合和聚集。因此，我们希望探索 细胞粘附域的空间排列(即它们的大小、数量/细胞体和空间排列) 并评估它们对HES细胞自我更新的影响。我们建议改变一个人的身体状态 多能的HES细胞，通过其与表面的粘连的空间聚集，将影响自我更新和 分化成特定的表型。 提出了以下具体目标。 具体目标1：开发和表征纳米级细胞培养底物，其中大小、多肽 整合素结合结构域的配基密度、数目/细胞体和空间排列将改变为 控制细胞和集落形态。 具体目标2：评估支持长期生长(5-10代)的纳米颗粒基质 人类胚胎干细胞在化学定义的培养液中。

Project Summary/Abstract: Human embryonic stem (hES) cells are being studied as potential source of cells for the treatment of many diseases (e.g. diabetes, spinal cord injury, Parkinson's, leukemia, congestive heart failure, etc.). These same cells are also being touted an ideal cell source for ex vivo tissue engineering or in situ regenerative medicine. The successful integration of hES cell into such therapies will hinge upon three critical steps: 1) stem cell expansion in number without differentiation (i.e., self-renewal); 2) directed differentiation into a specific cell type or collection of cell types; and, 3) cell survival and promotion of their functional integration into existing tissue. Precisely controlling each of these steps will be essential to maximize the hES cell's therapeutic efficacy. However, it is difficult to precisely control the behavior of hES cells, since environmental conditions for self-renewal and differentiation are poorly understood. We propose to develop a tunable completely synthetic surface and chemically defined media to control the self-renewal/expansion of hES cells. If hES cells can be derived and maintained within a completely synthetic environment, then it will be possible to eliminate pathogen transmission associated with animal-derived materials, provide a scalable basis for large-scale production of hES cells, and provide a precise base for further development to control hES cell differentiation. This application will develop materials to address the hypothesis that the contractile state of a hES cell, manifested by nuclear shape morphology via integrin engagement, regulates hES cell self-renewal. Our hypothesis is centered on a common mechanism by which cells respond differentially to either materials with variable moduli or materials that spatially confine a cell's shape via adhesion site distribution. We propose that a common mechanism that controls hES cell self-renewal and cell fate determination is the contractile state of the cell manifested by nuclear morphology, and integrin engagement and clustering. Thus, we wish to explore the spatial arrangement of cell adhesion domains (i.e., their size, number/cell body, and spatial arrangement) and assess their effect on the self-renewal of hES cells. We propose that altering the physical state of a pluripotent hES cell, via spatial clustering of its adhesions with a surface, will influence self-renewal and differentiation to a specific phenotype. The following specific aims are proposed. Specific Aim 1: To develop and characterize nanopatterned cell culture substrata where the size, peptide ligand density, number/cell body, and spatial arrangement of integrin-engaging domains will be varied to control cell and colony morphology. Specific Aim 2: To evaluate the nanopatterned substrata to support the long-term growth (5-10 passages) of human ES cells in chemically-defined media.