Understanding the intestinal regenerative response using patterned organoids in photo-tunable PEG hydrogels

使用光可调 PEG 水凝胶中的图案化类器官了解肠道再生反应

• 批准号: 10153343
• 负责人: Max Yavitt
• 金额: $ 4.06万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153343
• 关键词: 3-Dimensional; Affect; Aftercare; Behavior; Biological Assay; Cell Communication; Cell Compartmentation; Cell Count; Cell Proliferation; Cell Shape; Cells; Cessation of life; Chemicals; Chromosome Mapping; Chronic; Clinical; Confocal Microscopy; Constipation; Controlled Environment; Coupled; Custom; Data; Development; Diarrhea; Dimensions; Dose; Dose-Limiting; Doxorubicin; Encapsulated; Epithelial; Evaluation; Event; Exposure to; G-Protein-Coupled Receptors; Gene Expression; Goals; Growth; Heterogeneity; Homeostasis; Hydrogels; Image; In Vitro; Injury; Intestines; Knowledge; LGR5 gene; Lead; Leucine; Light; Malignant neoplasm of gastrointestinal tract; Microscope; Microscopy; Modality; Modeling; Molecular; Morphology; Mus; Natural regeneration; Organoids; Paneth Cells; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Population; Population Distributions; Process; Proliferating; Proteins; Quality of life; Recovery; Recurrence; Regenerative response; Reporting; Reproducibility; Research; Severities; Shapes; Signal Pathway; Specificity; Structure; System; Techniques; Technology; Time; Treatment Effectiveness; Treatment Efficacy; Ultraviolet Rays; Video Microscopy; cancer type; cell dedifferentiation; cell motility; cell regeneration; cell type; chemotherapeutic agent; chemotherapy; confocal imaging; cost; ethylene glycol; gastrointestinal; imaging capabilities; improved; in vivo; in vivo Model; insight; interest; intestinal crypt; intestinal epithelium; intestinal homeostasis; intestinal injury; live cell imaging; migration; mouse model; notch protein; novel; novel therapeutics; regeneration following injury; response to injury; side effect; spatiotemporal; stem cell population; stem cells; tool; transcriptome

Project Summary Nonspecific targeting of highly proliferative, non-cancerous cell types during chemotherapy highlights the limitations of these treatment modalities. In the intestinal tract, chemotherapeutics target highly proliferative intestinal stem cells (ISCs). ISCs are responsible for maintaining homeostasis in the intestinal epithelium, and their loss results in detrimental side effects that limit the efficacy of treatment and affect patient quality of life, often many years after treatment. Following injury, regeneration of the ISC compartment is driven by dedifferentiation of various committed lineages, including secretory Paneth cells, leading to recovery from detrimental side effects. As such, there is interest in understanding the molecular mechanisms that influence regeneration. Such knowledge would motivate the development of novel therapeutics to enhance the rate of regeneration, reducing the time and cost associated with chemotherapeutic induced side effects. While in vivo mouse models have been used to study intestinal regeneration following injury, they afford no evaluation of dynamic and transient processes, due to the inability to conduct live cell imaging. In vitro cultures of intestinal organoids, which recapitulate the structure and function of the intestinal epithelium, allow for real time tracking of cell populations in order to study the dynamic interactions between cell populations. However, the heterogeneity and stochastic growth of intestinal organoid cultures often limits their advantage when imaging. Photodegradable poly(ethylene glycol) (PEG) hydrogels can be used to pattern regions of localized softening to direct the formation of intestinal crypt in vitro, resulting in the reproducible formation of uniform crypts. We propose that this material platform can be used to probe the rapidly changing cell interactions and mechanisms that drive regeneration following injury. In Aim 1, the formation of mature intestinal crypts in vitro is validated under homeostatic conditions. Directed light exposure is used to degrade regions adjacent to 3D encapsulated intestinal organoids, resulting in crypt formation into the degraded regions. Organoids with live cell markers for ISC and Paneth cells will be tracked by live confocal microscopy and custom MATLAB scripts will be used to quantify the migration and interactions of these cell types in real time. Immunostaining for markers of other committed lineages will define the distribution of cell types during homeostasis. In Aim 2, injury is induced by applying doxorubicin, a chemotherapeutic agent, which eliminates the ISC population. Following injury, the drug will be withdrawn, allowing dedifferentiation of remaining cells and the regeneration of the ISC population. During injury and regeneration, live confocal imaging will be used to track and quantify the ISC and Paneth cell populations, affording insight into their real time dynamic behavior. Single cell transcriptome analysis during injury and regeneration will be used as an unbiased assessment to identify novel pathways that influence Paneth cell dedifferentiation and regeneration. Localization of gene expression will be coupled to real time cell tracking data to further understand the spatiotemporal contributions of essential pathways to intestinal regeneration.

项目概要 化疗过程中针对高度增殖的非癌细胞类型的非特异性靶向凸显了 这些治疗方式的局限性。在肠道中，化疗针对高度增殖的细胞 肠干细胞（ISC）。 ISC 负责维持肠上皮的稳态，并且 它们的损失会导致有害的副作用，限制治疗效果并影响患者的生活质量， 通常在治疗后很多年。损伤后，ISC 室的再生由以下因素驱动 各种定型谱系的去分化，包括分泌性潘氏细胞，导致从 有害的副作用。因此，人们有兴趣了解影响的分子机制 再生。这些知识将推动新疗法的开发，以提高治愈率 再生，减少与化疗引起的副作用相关的时间和成本。当在体内时 小鼠模型已被用来研究损伤后的肠道再生，但它们无法评估 由于无法进行活细胞成像，因此存在动态和瞬态过程。肠道体外培养 类器官概括了肠上皮的结构和功能，可以进行实时跟踪 细胞群体，以研究细胞群体之间的动态相互作用。然而， 肠道类器官培养物的异质性和随机生长往往限制了它们在成像时的优势。 可光降解的聚乙二醇 (PEG) 水凝胶可用于对局部软化区域进行图案化 直接在体外形成肠隐窝，导致均匀隐窝的可重复形成。我们 提出该材料平台可用于探测快速变化的细胞相互作用和机制 促进受伤后的再生。在目标 1 中，验证了体外成熟肠隐窝的形成 稳态条件下。使用定向曝光来降低 3D 封装附近的区域 肠类器官，导致退化区域形成隐窝。具有活细胞标记的类器官 ISC 和潘氏细胞将通过实时共聚焦显微镜进行跟踪，并使用自定义 MATLAB 脚本 实时量化这些细胞类型的迁移和相互作用。其他标记物的免疫染色 定型谱系将定义稳态期间细胞类型的分布。在目标 2 中，损伤是由 使用阿霉素（一种化疗药物）可以消除 ISC 群体。受伤后，药物 将被撤回，从而允许剩余细胞去分化和 ISC 群体再生。期间 损伤和再生，实时共聚焦成像将用于跟踪和量化 ISC 和潘氏细胞 人群，深入了解他们的实时动态行为。单细胞转录组分析 损伤和再生将被用作公正的评估，以确定影响帕内斯的新途径 细胞去分化和再生。基因表达的定位将与实时细胞追踪相结合 数据以进一步了解肠道再生重要途径的时空贡献。