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

项目摘要 化疗期间对高增殖、非癌细胞类型的非特异性靶向突出了 这些治疗方式的局限性。在肠道中，化疗药物的靶点是高度增殖的 肠道干细胞(ISC)。ISCs负责维持肠上皮的动态平衡，并且 它们的丢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， 通常是在治疗多年后。损伤后，ISC小室的再生是由 包括分泌潘氏细胞在内的各种承诺谱系的去分化导致从 有害的副作用。因此，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发展，以提高 再生，减少与化疗副作用相关的时间和成本。在活体内 小鼠模型已经被用来研究损伤后的肠道再生，它们没有提供对 动态和瞬变过程，由于无法进行活细胞成像。肠道的体外培养 有机类化合物概括了肠道上皮的结构和功能，允许实时跟踪 以研究细胞群体之间的动态相互作用。然而， 肠道器官培养的异质性和随机性往往限制了它们在成像时的优势。 可光降解的聚乙二醇水凝胶可用于将局部软化区域图案化以 在体外指导肠隐窝的形成，导致可重复形成均匀的隐窝。我们 提出该材料平台可用于探索快速变化的细胞相互作用和机制 这推动了受伤后的再生。在目标1中，验证了成熟肠腺在体外的形成。 在动态平衡条件下。定向光曝光用于降级3D封装附近的区域 肠道器官类物质，导致隐窝形成进入退化区域。带有活细胞标记的有机类化合物 ISC和Paneth细胞将由实时共焦显微镜跟踪，并将使用定制的MatLab脚本来 实时量化这些细胞类型的迁移和相互作用。其他肿瘤标志物的免疫组化染色 承诺的谱系将定义细胞类型在动态平衡期间的分布。在目标2中，伤害是由 应用阿霉素，一种化疗药物，它消除了ISC群体。受伤后，这种药物 将被撤回，从而允许剩余细胞的去分化和ISC群体的再生。在.期间 损伤和再生，将使用实时共聚焦成像来跟踪和量化ISC和Paneth细胞 种群，提供对其实时动态行为的洞察。单细胞转录组分析 损伤和再生将被用作一种公正的评估，以确定影响Paneth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跟踪相结合 进一步了解关键途径对肠道再生的时空贡献的数据。

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.