Synthetic hydrogels to study formation and maintenance of intestinal crypts

摘要 肠道器官模型作为研究肠道发育和疾病、筛选药物的工具前景广阔 候选人，甚至在体外产生可移植的组织。目前肠道生长的培养方法 有机化合物几乎完全依赖于Matrigel，但Matrigel松散定义和可变的组成使 临床翻译几乎是不可能的，阻碍了对关键基质因子作用的基础研究 关于有机体的形成。而在Matrigel中生长的肠道干细胞(ISCs)具有巨大的自我修复能力 组织成功能复杂的肠道器官结构，自组织原理也是 负责引入变异性和随机类器官，这些器官在许多方面也不同于天然器官 方面。在拟议的研究中，我们的目标是开发可调的水凝胶基质，用于ISC的扩张、克隆 形成，分化形成隐窝。我们材料的独特之处在于能够规范ISC 利用光化学反应的时空微环境，我们提出了利用光适应性 用于测试与ISC机械传感及其对类有机物生长的影响的假说的水凝胶(目标1)；作用 局部基质硬度对类器官形状、细胞增殖和隐窝形成的影响(目标2)；以及 隐窝细胞对压力或损伤的反应(目标3)。我们假设矩阵的外源控制 特性可用于支持高效的ISC类器官生长，并随后模拟细胞介导的隐窝 形成和重塑。拟议的材料系统将使我们不仅能够研究和指导形成 生理上相关的隐窝绒毛建筑，但也测试这些结构的维护 对与发育过程相对应的基质属性的动态变化的反应以及隐窝 受伤后的再生。具体地说，我们建议：1.研究基质力学性能的作用 以及对肠道干细胞(ISCs)及其生长为球形器官的信号。2.了解如何 水凝胶力学的空间变化允许ISCs经历祖细胞承诺和随后的 分化为功能细胞类型。3.研究均匀的和空间变异的细胞基质的作用 损伤后谱系特异性上皮细胞去分化与隐窝再生的相互作用。

ABSTRACT Intestinal organoid models hold great promise as a tool to study intestinal development and disease, screen drug candidates, or even produce transplantable tissue in vitro. Current culture methods for growth of intestinal organoids rely almost exclusively on Matrigel, but Matrigel’s loosely-defined and variable composition makes clinical translation nearly impossible and obstructs fundamental investigations into the role of key matrix factors on organoid formation. While intestinal stem cells (ISCs) grown in Matrigel have a tremendous capacity for self- organization into functionally sophisticated intestinal organoid structures, the self-organization principles are also responsible for introducing variability and stochastic organoids that also differ from the native organ in multiple aspects. In the proposed research, we aim to develop tunable hydrogel matrices for ISC expansion, colony formation, and differentiation to form crypts. Unique to our materials is the ability to regulate the ISC microenvironment spatiotemporally using photochemical reactions, and we propose to use photoadaptable hydrogels to test hypotheses related to ISC mechanosensing and its effects on organoid growth (Aim 1); the role of local matrix stiffness on organoid shape, cell proliferation, and crypt formation (Aim 2); and the plasticity of crypt cells during their response to a stress or injury (Aim 3). We hypothesize that exogenous control of matrix properties can be used to support efficient ISC organoid growth, and subsequently mimic cell-mediated crypt formation and remodeling. The proposed material systems will allow us to not only study and direct the formation of the crypt-villus architectures that are physiologically relevant, but also test maintenance of these structures in response to dynamic changes in matrix properties corresponding to developmental processes, as well as crypt regeneration after injury. Specifically, we propose to: 1. Investigate the role of matrix mechanical properties and signaling on intestinal stem cells (ISCs) and their growth into spherical organoids. 2. Understand how spatial changes in hydrogel mechanics permit ISCs to undergo progenitor commitment and subsequent differentiation into functional cell types. and 3. Investigate the role of uniform and spatially variant cell-matrix interactions on the de-differentiation of lineage specific epithelial cells and crypt regeneration after injury.