Regulation of Adherent Cell Proliferation by Matrix Viscoelasticity

基质粘弹性对贴壁细胞增殖的调节

• 批准号: 10735701
• 负责人: Ovijit Chaudhuri
• 金额: $ 38.6万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735701
• 关键词: 3-Dimensional; ATAC-seq; Actomyosin; Address; Adherent Culture; Adhesions; Adhesives; Alginates; Automobile Driving; Basement membrane; Binding; Biocompatible Materials; Bioinformatics; Biological Assay; Biological Process; Biophysical Process; Biophysics; Breast Epithelial Cells; CRISPR screen; CRISPR/Cas technology; Cell Adhesion; Cell Proliferation; Cell Volumes; Cells; Characteristics; Chromatin; Collagen Type I; Data; Deposition; Disease; Elasticity; Epigenetic Process; Epithelial Cells; Exhibits; Extracellular Matrix; Fibroblasts; Gene Expression; Genes; Genetic Transcription; Goals; Human; Hydrogels; Integrin Binding; Integrins; Ion Channel; Knock-out; Knowledge; Ligands; Liquid substance; MAP Kinase Gene; Mechanics; Mediating; Mesenchymal Stem Cells; Mission; Molecular; Molecular Analysis; Morphogenesis; PIK3CG gene; Pathway interactions; Process; Proliferating; Regulation; Relaxation; Research; Role; Signal Pathway; Solid; Sp1 Transcription Factor; Stress; Tissues; Transcriptional Regulation; United States National Institutes of Health; Viscosity; Work; biophysical techniques; cancer cell; cell behavior; density; disability; epigenetic regulation; epigenome; genome-wide; genome-wide analysis; in vivo; innovation; mechanotransduction; migration; novel; reconstitution; response; stem cell differentiation; three dimensional cell culture; tool; transcriptome sequencing; viscoelasticity; whole genome

Cell proliferation is a fundamental biological process that often occurs for cells in a 3D context in vivo, in which cells are surrounded by extracellular matrix (ECM) and other cells, and various applications rely on the proliferation of cells within a biomaterial. It has long been known that changes in matrix stiffness impact cell behaviors through mechanotransduction, and mechanisms of stiffness-sensing in 2D culture are now established. However, the mechanisms mediating the impact of changes in matrix stiffness on cell proliferation in 3D remain unclear. Further, living tissues and ECMs are viscoelastic, exhibiting some characteristics of elastic solids and some of viscous liquids. Matrix viscoelasticity is sensed through mechanotransduction, and we have found that changes in matrix viscoelasticity impact cell spreading, migration, proliferation, stem cell differentiation, matrix deposition, morphogenesis, and gene expression. However, the mechanisms mediating the impact of matrix viscoelasticity on these processes, particularly proliferation remain unclear. The goal of the proposed work is to determine the mechanism mediating the impact of matrix stiffness and viscoelasticity on cell proliferation in 3D matrices. Our overall hypothesis is that mechanosensitive ion channel-mediated pathways and integrin-mediated pathways interplay to sense matrix viscoelasticity and stiffness, and subsequently control proliferation through changes in chromatin accessibility, YAP-independent transcription, and a set of molecular regulators not implicated from 2D culture studies. We will address this hypothesis in 3 aims, using an approach that involves the use of alginate hydrogels with independently tunable viscoelasticity, stiffness, and RGD ligand density for 3D culture of adherent cells, including fibroblasts, epithelial cells, and mesenchymal stem cells. In aim 1, we will determine the biophysical mechanisms underlying the impact of hydrogel viscoelasticity, stiffness, and adhesivity on the proliferation of adherent cells in 3D culture. In Aim 2, we will elucidate transcriptional and epigenetic regulation of mechanotransduction and proliferation, using RNA-seq and ATAC-seq combined with advanced bioinformatics analyses. In Aim 3, we will identify novel regulators of proliferation and mechanotransduction in 3D using genome-wide CRISPR screening. Innovative aspects of this approach include the study of mechanisms mediating mechanotrasduction and proliferation in 3D matrices, the focus on viscoelasticity (beyond stiffness), the potential for discovering YAP-independent mechanisms of mechanotransduction, the identification of how the epigenome regulates mechanotransduction and proliferation in 3D, and the application of a CRISPR screen to identify novel molecular regulators of mechanotransduction. The significance of this work is that it will determine the biophysical and molecular mechanisms by which ECM or biomaterial stiffness and viscoelasticity regulate cell proliferation in 3D. Given the importance of cell proliferation, the ubiquity of matrix viscoelasticity in ECMs, and the potential relevance of discovered mechanisms of mechanotransduction to other processes, the significance is expected to be high.

细胞增殖是体内 3D 环境中细胞经常发生的基本生物过程，其中 细胞被细胞外基质（ECM）和其他细胞包围，各种应用都依赖于 生物材料内细胞的增殖。人们早就知道基质刚度的变化会影响细胞 通过机械传导的行为，以及二维文化中的刚度感知机制现在 已确立的。然而，介导基质硬度变化对细胞增殖影响的机制 3D 仍不清楚。此外，活组织和 ECM 是粘弹性的，表现出弹性的一些特征 固体和一些粘性液体。基质粘弹性是通过力传导来感知的，我们有 发现基质粘弹性的变化影响细胞扩散、迁移、增殖、干细胞 分化、基质沉积、形态发生和基因表达。然而，调解机制 基质粘弹性对这些过程，特别是增殖的影响仍不清楚。的目标 建议的工作是确定介导基质刚度和粘弹性对细胞影响的机制 3D 矩阵中的增殖。我们的总体假设是机械敏感离子通道介导的途径 和整合素介导的途径相互作用以感知基质粘弹性和刚度，并随后进行控制 通过染色质可及性的变化、YAP 独立转录和一组分子 2D 培养研究未涉及监管机构。我们将使用一种方法通过 3 个目标来解决这个假设 涉及使用具有独立可调的粘弹性、刚度和 RGD 配体的藻酸盐水凝胶 贴壁细胞 3D 培养的密度，包括成纤维细胞、上皮细胞和间充质干细胞。在 目标 1，我们将确定影响水凝胶粘弹性、刚度、 以及粘附性对 3D 培养中贴壁细胞增殖的影响。在目标 2 中，我们将阐明转录和 使用 RNA-seq 和 ATAC-seq 结合机械转导和增殖的表观遗传调控 先进的生物信息学分析。在目标 3 中，我们将确定新的增殖调节因子和 使用全基因组 CRISPR 筛选进行 3D 机械转导。这种方法的创新之处包括 研究 3D 矩阵中介导机械传导和增殖的机制，重点是 粘弹性（超越刚度），发现与 YAP 无关的机制的潜力 机械转导，识别表观基因组如何调节机械转导和增殖 3D 技术，以及应用 CRISPR 筛选来识别机械转导的新型分子调节剂。 这项工作的意义在于它将确定 ECM 的生物物理和分子机制 或生物材料的刚度和粘弹性在 3D 中调节细胞增殖。鉴于细胞的重要性 增殖、ECM 中基质粘弹性的普遍性以及所发现的潜在相关性 机械传导机制对其他过程的意义预计会很高。