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; 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.

项目摘要 化学疗法期间非特异性靶向高度增殖的非癌细胞类型突出了 这些治疗方式的局限性。在肠道中，化学治疗剂靶向高度增殖 肠干细胞（ISC）。 ISC负责维持肠上皮中的体内平衡，并且 它们的损失导致有害的副作用限制了治疗的功效并影响患者生活质量， 经常在治疗后很多年。受伤后，ISC室的再生由 在包括分泌的paneth细胞在内的各种承诺的谱系的去分化，导致从 有害的副作用。因此，有兴趣理解影响分子机制 再生。这种知识将激发新型治疗学的发展，以提高 再生，减少与化学治疗诱导的副作用相关的时间和成本。在体内 鼠标模型已用于研究受伤后的肠再生，它们没有评估 由于无法进行活细胞成像，动态和瞬态过程。肠道体外培养 概括肠上皮的结构和功能的器官，可以实时跟踪 细胞群体以研究细胞种群之间的动态相互作用。但是， 肠癌培养物的异质性和随机生长通常会在成像时限制其优势。 可光降解的聚乙二醇）（PEG）水凝胶可用于模拟局部软化区域 在体外指导肠道隐窝的形成，从而导致均匀的隐窝的可再现形成。我们 建议该材料平台可用于探测快速变化的细胞相互作用和机制 受伤后驱动再生。在AIM 1中，体外成熟的肠道隐窝的形成得到了验证 在稳态条件下。定向光暴露用于降解与3D封装相邻的区域 肠癌，导致隐窝形成降解区域。带有活细胞标记的类器官 ISC和Paneth Cell 实时量化这些细胞类型的迁移和相互作用。对其他标记的免疫染色 固定的谱系将定义稳态期间细胞类型的分布。在AIM 2中，受伤是由 应用阿霉素，一种化学治疗剂，消除了ISC群体。受伤后，药物 将撤回，允许剩余细胞的去分化和ISC种群的再生。期间 伤害和再生，实时共聚焦成像将用于跟踪和量化ISC和Paneth Cell 人群，可以深入了解他们的实时动态行为。单细胞转录组分析 伤害和再生将被用作公正的评估，以识别影响Paneth的新型途径 细胞去分化和再生。基因表达的定位将耦合到实时细胞跟踪 数据以进一步了解肠再生基本途径的时空贡献。