Nanopatterned Surfaces to Control Cell Fate

描述（由申请人提供）：正在研究人胚胎干（hES）细胞作为治疗许多疾病（例如糖尿病、脊髓损伤、帕金森氏病、白血病、充血性心力衰竭等）的潜在细胞来源。这些相同的细胞也被吹捧为离体组织工程或原位再生医学的理想细胞来源。将hES细胞成功整合到这些疗法中将取决于三个关键步骤：1）干细胞在数量上扩增而不分化（即，自我更新）; 2）定向分化成特定的细胞类型或细胞类型的集合;和3）细胞存活和促进它们功能整合到现有组织中。精确控制这些步骤中的每一个对于最大化hES细胞的治疗功效将是至关重要的。然而，很难精确控制hES细胞的行为，因为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成分确定的培养基来控制hES细胞的自我更新/扩增。如果hES细胞可以在完全合成的环境中衍生和维持，那么将有可能消除与动物源性材料相关的病原体传播，为大规模生产hES细胞提供可扩展的基础，并为进一步开发控制hES细胞分化提供精确的基础。本申请将开发材料以解决hES细胞的收缩状态（通过整联蛋白接合由核形状形态表现）调节hES细胞自我更新的假设。我们的假设是集中在一个共同的机制，通过该机制，细胞的差异，无论是材料与可变模量或材料，空间上限制了细胞的形状，通过粘附位点分布。我们提出，一个共同的机制，控制hES细胞自我更新和细胞命运的决定是收缩状态的细胞核形态，整合素的参与和集群。因此，我们希望探索细胞粘附结构域的空间排列（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，改变一个多能hES细胞的物理状态，通过其粘附与表面的空间聚类，将影响自我更新和分化为一个特定的表型。提出了以下具体目标。具体目标1：开发和表征纳米图案化细胞培养基质，其中整合素接合结构域的大小、肽配体密度、数量/细胞体和空间排列将变化以控制细胞和集落形态。具体目标2：评价纳米图案化基质支持人ES细胞在化学成分确定的培养基中的长期生长（5-10代）。公共卫生相关性：该申请将专门关注工程设计一个可调和明确定义的环境，为hES细胞提供完全合成的细胞培养表面和化学成分确定的培养基，以促进自我更新。其结果将是一个合成的微环境，既可以作为大规模hES细胞扩增的再生医学技术平台，也可以提供一个新的高度模块化的系统，用于解剖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.