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很大的前景 候选人，甚至在体外产生可移植的组织。肠道菌生长的现有培养方法 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组成使得 临床转化几乎是不可能的，并且阻碍了对关键基质因子作用的基础研究 关于类器官的形成虽然在基质胶中生长的肠干细胞（ISCs）具有巨大的自我调节能力， 组织成功能复杂的肠类器官结构，自组织原理也是 负责引入变异性和随机类器官，这些类器官在多个方面也不同于天然器官， 方面在所提出的研究中，我们的目标是开发可调的水凝胶基质，用于ISC扩增、集落形成和细胞增殖。 形成和分化以形成隐窝。我们材料的独特之处在于能够调节ISC 微环境时空使用光化学反应，我们建议使用光适应 水凝胶来测试与ISC机械传感及其对类器官生长的影响有关的假设（目的1）; 局部基质硬度对类器官形状、细胞增殖和隐窝形成的影响（目的2）; 隐窝细胞在其对应激或损伤的反应期间（Aim 3）。我们假设外源性基质控制 这些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。提出的材料系统将使我们不仅能够研究和指导形成 的隐窝绒毛结构，是生理相关的，但也测试这些结构的维护， 对与发育过程相对应的基质性质的动态变化的反应，以及隐窝 损伤后的再生具体而言，我们建议：1.研究基体力学性能的作用 以及肠道干细胞（ISCs）的信号传导及其生长为球形类器官。2.了解如何 水凝胶力学的空间变化允许ISC经历祖细胞定型和随后的 分化为功能性细胞类型。和3.研究均匀和空间变化的细胞基质的作用 在损伤后谱系特异性上皮细胞的去分化和隐窝再生上的相互作用。

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.