Synthetic hydrogels to study formation and maintenance of intestinal crypts

摘要 肠道类器官模型作为研究肠道发育和疾病、筛选药物的工具具有广阔的前景 候选者，甚至在体外产生可移植组织。目前肠道生长的培养方法 类器官几乎完全依赖于基质胶，但基质胶的松散定义和可变成分使得 临床转化几乎不可能，并且阻碍了对关键基质因素作用的基础研究 关于类器官的形成。虽然在基质胶中生长的肠道干细胞 (ISC) 具有巨大的自我修复能力。 组织成功能复杂的肠道类器官结构，自组织原理也 负责引入变异性和随机类器官，这些类器官在多个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节的水凝胶基质，用于 ISC 扩张、集落 形成和分化形成隐窝。我们材料的独特之处在于能够调节 ISC 使用光化学反应来研究时空微环境，我们建议使用光适应性 水凝胶测试与 ISC 机械传感及其对类器官生长的影响相关的假设（目标 1）；角色 局部基质刚度对类器官形状、细胞增殖和隐窝形成的影响（目标 2）；和可塑性 隐窝细胞在对压力或损伤做出反应期间（目标 3）。我们假设矩阵的外源控制 特性可用于支持有效的 ISC 类器官生长，并随后模拟细胞介导的隐窝 形成和重塑。所提出的材料系统将使我们不仅能够研究和指导形成 生理相关的隐窝-绒毛结构的研究，而且还测试这些结构的维护 响应与发育过程以及隐窝相对应的基质特性的动态变化 受伤后的再生。具体来说，我们建议： 1. 研究基体力学性能的作用 肠道干细胞（ISC）及其生长成球形类器官的信号传导。 2. 了解如何 水凝胶力学的空间变化允许 ISC 进行祖细胞承诺和后续 分化为功能细胞类型。 3. 研究均匀和空间变异细胞基质的作用 损伤后谱系特异性上皮细胞去分化和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.