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

项目摘要 在化疗期间高度增殖的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，化疗药物靶向高度增殖的 肠干细胞（ISCs）。ISCs负责维持肠上皮的稳态， 它们的损失导致限制治疗功效和影响患者生活质量的有害副作用， 通常是在治疗多年后。损伤后，ISC区室的再生由以下驱动： 各种定型谱系的去分化，包括分泌型潘氏细胞，导致从 有害的副作用因此，有兴趣了解影响的分子机制， 再生这些知识将促进新疗法的开发，以提高癌症的发生率。 再生，减少与化疗诱导的副作用相关的时间和成本。在体内 小鼠模型已被用于研究损伤后的肠再生，但它们没有提供对 由于不能进行活细胞成像，动态和瞬时过程。肠上皮细胞的体外培养 类器官再现了肠上皮的结构和功能，允许真实的时间跟踪 为了研究细胞群体之间的动态相互作用，但 肠类器官培养物的异质性和随机生长通常限制了它们在成像时的优势。 可光降解的聚（乙二醇）（PEG）水凝胶可用于图案化局部软化的区域， 在体外指导肠隐窝的形成，导致均匀隐窝的可重复形成。我们 我认为这种材料平台可以用来探测快速变化的细胞相互作用和机制 在受伤后驱动再生。在目的1中，验证了体外成熟肠隐窝的形成 在稳态条件下。定向光曝光用于降解与3D封装材料相邻的区域。 肠类器官，导致在降解区域形成隐窝。具有活细胞标志物的类器官， ISC和Paneth细胞将通过实时共聚焦显微镜进行跟踪，自定义MATLAB脚本将用于 真实的量化这些细胞类型的迁移和相互作用。免疫染色用于其他 定型谱系将确定稳态期间细胞类型的分布。在目标2中，损伤是由 应用阿霉素，一种化疗剂，其消除了ISC群体。受伤后，药物 将被撤回，允许剩余细胞的去分化和ISC群体的再生。期间 损伤和再生，活体共聚焦成像将用于跟踪和定量ISC和潘氏细胞 人口，提供洞察他们的真实的时间动态行为。单细胞转录组分析 损伤和再生将作为一个公正的评估，以确定新的途径，影响帕内特 细胞去分化和再生。基因表达的定位将与真实的时间细胞跟踪相结合 数据，以进一步了解时空的重要途径肠道再生的贡献。

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.