Nanopatterned Surfaces to Control Cell Fate

描述(申请人提供)：人类胚胎干细胞(HES)正在被研究为治疗许多疾病(如糖尿病、脊髓损伤、帕金森病、白血病、充血性心力衰竭等)的潜在细胞来源。这些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。将HES细胞成功地整合到这种疗法中将取决于三个关键步骤：1)干细胞在数量上的扩张而不分化(即自我更新)；2)定向分化为特定的细胞类型或细胞类型的集合；以及3)细胞存活和促进其与现有组织的功能整合。精确控制这些步骤中的每一步对于最大化HES细胞的治疗效果至关重要。然而，很难精确控制HES细胞的行为，因为人们对自我更新和分化的环境条件知之甚少。我们建议开发一种可调的、完全合成的表面和化学定义的介质来控制HES细胞的自我更新/扩张。如果HES细胞能够在完全人工合成的环境中获得和维持，那么就有可能消除与动物来源材料相关的病原体传播，为HES细胞的大规模生产提供可扩展的基础，并为进一步研究控制HES细胞分化提供精确的基础。这一应用将开发材料来解决这一假设，即HES细胞的收缩状态，通过整合素参与的核形状表现出来，调节HES细胞的自我更新。我们的假设集中在一个共同的机制上，即细胞对具有可变模数的材料或通过黏附位置分布在空间上限制细胞形状的材料做出不同的反应。我们认为，控制HES细胞自我更新和决定细胞命运的一个共同机制是细胞的收缩状态，表现为核形态、整合素参与和聚集。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。我们认为，改变多能HES细胞的物理状态，通过其与表面的粘连的空间聚集，将影响自我更新和向特定表型的分化。提出了以下具体目标。具体目标1：开发和表征纳米修饰的细胞培养底物，其中整合素结合结构域的大小、肽配体密度、数量/细胞体和空间排列将发生变化，以控制细胞和克隆的形态。具体目标2：评估纳米基质在化学定义的培养基中支持人ES细胞长期生长(5-10代)的能力。与公共健康相关：这项应用将特别专注于设计一个可调和定义明确的环境，为HES细胞提供一个完全合成的细胞培养表面和化学定义的介质，以促进自我更新。其结果将是一个合成的微环境，既可以作为HES细胞大规模扩增的再生医学技术平台，又可以提供一个新颖的高度模块化的系统，用于剖析HES细胞自我更新的基本信号机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This application will focus specifically on engineering a tunable and well-defined environment presenting hES cells with a completely synthetic cell culture surface and chemically-defined media to promote self-renewal. The result will be a synthetic microenvironment that can both serve as a regenerative medicine technology platform for large scale hES cell expansion, as well as provide a novel and highly modular system for dissecting basic signaling mechanisms underlying hES cell self-renewal.