Synthetic hydrogels to study formation and maintenance of intestinal crypts

用于研究肠隐窝的形成和维持的合成水凝胶

基本信息

批准号：
9981736
负责人：
KRISTI S. ANSETH
金额：
$ 40.17万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9981736
关键词：
3-Dimensional Ablation Actomyosin Address Adult Architecture Basement membrane Biological Biological Assay Cell Differentiation process Cell Lineage Cell Proliferation Cell Shape Cell physiology Cells Complex Coupled Cues Development Developmental Process Disease Dose Doxorubicin Drug Delivery Systems Drug Screening Encapsulated Enterocytes Epithelial Epithelial Cells Epithelium Event Excision Genetic Growth Hydrogels In Vitro Injury Intestinal Neoplasms Intestines Investigation Kinetics LGR5 gene Laser Scanning Confocal Microscopy Lead Light Link Maintenance Mechanics Mediating Methods Modeling Modification Morphogenesis Mus Natural regeneration Nuclear Organ Organoids Outcome Paneth Cells Peptide Hydrolases Pharmacology Phenotype Physiological Play Process Proliferating Property Reaction Relaxation Research Role Shapes Signal Transduction Stress Structure System Testing Time Tissue Transplantation Variant Verteporfin Villus Withdrawal blebbistatin cell type clinical translation crypt cell drug candidate drug efficacy inhibitor/antagonist innovation intestinal crypt intestinal epithelium matrigel mechanical properties mechanotransduction notch protein progenitor reconstitution response response to injury sarcoma scaffold self organization spatiotemporal stem stem cell population stem cells stemness tool viscoelasticity

项目摘要

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.

抽象的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，用于肠道生长的当前培养方法。类器官几乎完全依赖于 Matrigel，但 Matrigel 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而在基质胶中生长的肠道干细胞 (ISC) 具有巨大的自我生成能力。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在拟议的研究中，我们的目标是开发用于 ISC 扩增、集落的可调节水凝胶基质。我们的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）；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）以及可塑性；隐窝细胞在对压力或损伤做出反应期间（目标 3）。特性可用于支持有效的 ISC 类器官生长，以及随后的模拟细胞介导的隐窝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具体来说，我们建议： 1. 研究基质机械性能的作用。肠道干细胞 (ISC) 及其生长成球形类器官的信号传导 2. 了解如何进行。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性细胞类型；3. 研究均匀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和隐窝再生的相互作用。