Regulation of Adherent Cell Proliferation by Matrix Viscoelasticity

细胞增殖是细胞在体内3D环境中经常发生的基本生物学过程，其中 细胞被细胞外基质(ECM)和其他细胞包围，各种应用依赖于 生物材料内细胞的增殖。很早以前就知道，基质硬度的变化会影响细胞 在2D培养中，通过机械转导的行为和僵硬感知的机制现在是 已经成立了。然而，调节基质硬度变化对细胞增殖影响的机制 3D版本仍不清楚。此外，活组织和细胞外基质是粘弹性的，表现出一些弹性特征 固体和一些粘性液体。基质粘弹性是通过机械传递来感知的，我们有 发现基质粘弹性的变化影响细胞的扩散、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的影响，特别是对增殖的影响尚不清楚。的目标是 建议的工作是确定基质刚性和粘弹性对细胞的影响的中介机制 3D矩阵中的扩散。我们的总体假设是机械敏感离子通道介导的通路 而整合素介导的通路相互作用，感受基质的粘弹性和刚性，并随后控制 通过染色质可及性、YAP非依赖性转录和一组分子的变化而实现的增殖 监管机构没有受到2D文化研究的牵连。我们将在三个目标中解决这一假设，使用一种方法 这涉及到使用具有独立可调节的粘弹性、刚性和RGD配体的藻酸盐水凝胶 贴壁细胞的3D培养密度，包括成纤维细胞、上皮细胞和间充质干细胞。在……里面 目标1，我们将确定水凝胶粘弹性、硬度、 3D培养中贴壁细胞增殖的粘附性。在目标2中，我们将阐明转录和 联合使用RNA-seq和atac-seq对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筛选的3D机械转导。这种方法的创新方面包括 3D基质中机械转导和增殖机制的研究，重点是 粘弹性(超越刚性)，发现YAP独立机制的可能性 机械转导，识别表观基因组如何调控机械转导和增殖 在3D中，以及CRISPR屏幕的应用来识别机械转导的新的分子调节因子。 这项工作的意义在于它将确定细胞外基质的生物物理和分子机制 或者，生物材料的硬度和粘弹性在3D中调节细胞的增殖。鉴于细胞的重要性 增殖，基质粘弹性在ECM中的普遍存在，以及已发现的潜在相关性 对于机械转导到其他过程的机制，其意义预计将很高。

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.