A Novel Planar Crypt Microarray for Real-Time Evaluation of Human Intestinal Stem Cell Fate

项目总结 炎症性肠病(IBD)，包括克罗恩病和溃疡性结肠炎，估计影响3.1 百万美国成年人。IBD的病理生理学很复杂，涉及遗传、环境和免疫反应。 以及肠道微生物短链脂肪酸(SCFA)水平的变化。Magness实验室的最新研究 在小鼠回肠器官中，暴露于IL-22比肠道更有利于祖细胞的扩张 干细胞(ISC)的自我更新，这种表型被认为是通过激活信号转导和 转录激活因子(STAT)-3。此外，新的证据表明，肠道微生物的单链脂肪酸，丁酸盐， 增强IL-22受体的表达和IL-22诱导的人结肠癌细胞STAT3的激活。而当 小鼠、类器官和癌细胞系帮助推进了这一领域，在体外更具生理学意义。 需要人体模型来提高实验的准确性和分辨率，目的是预测和 揭示人类肠道间充质干细胞和分化上皮对这些IBD相关因子的反应。 我的目标将使用一种新型的平面加密微阵列(PCM)设备和报告细胞系来现场拍摄初级图像 人结肠干细胞对炎症性肠病相关扰动的反应。相变材料已经进行了优化 用于小鼠间充质干细胞的培养，初步数据表明人类间充质干细胞的兼容性。目标1将调整因素 如PCM膜成分、厚度和改变顶端/基底液的时间以促进融合 干细胞区(Olfm4-EGFP)和分化细胞区(Muc2-BFP)的单层和分隔。vbl.使用 ，我将测试丁酸盐刺激的IL-22信号增强的中心假设 人结肠间充质干细胞的不对称分裂和分化。AIM 2将使用CRISPR/Cas9 INDELS来 诱导IL-22受体α1亚基移码功能丧失突变为四环素诱导型 H_2B：：RFP(Teto-H_2B：：RFP)人结肠干细胞株，以测试IL-22信号是否是必要的 不对称除法。Teto-H2B：：RFP融合蛋白允许对细胞分裂进行可控的、敏感的分析 哺乳动物细胞的动力学，快速分裂的细胞对应于稀释的RFP荧光强度超过 时间到了。将通过免疫荧光法评估RFP标记保留细胞的磷酸化STAT3(PSTAT3) 用FACS-scRNA-seq对未分选的细胞单层和受扰的谱系通路进行成像。LOF模型 将与PCM上的IL22RA1+控制进行比较，以确定是否通过 丁酸刺激的IL-22信号传导。我们的实验室已经有了Olfm4-EGFP和H2B报告线的初步数据。 在北卡罗来纳大学教堂分校Scott Magness博士团队出色的培训环境中 希尔，我将在微制造和肠道干细胞生物学方面获得专业知识，并在小学学习新技术 人类结肠干细胞培养、单细胞生物学/基因组学和生物信息学。我的职业发展培训 Plans专注于研究技能、专业发展和临床技能，以促进我的独立道路 作为一名学术内科医生兼科学家。

PROJECT SUMMARY Inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis, affect an estimated 3.1 million U.S. adults. IBD pathophysiology is complex and involves genetic, environmental, and immune responses as well as alterations in gut microbial short-chain fatty acid (SCFA) levels. Recent studies from the Magness lab in murine ileal organoids demonstrated that exposure to IL-22 favors progenitor cell expansion over intestinal stem cell (ISC) self-renewal, and this phenotype is thought be regulated by activating Signal Transducer and Activator of Transcription (STAT)-3. Further, new evidence has shown that the gut microbial SCFA, butyrate, enhances IL-22 receptor expression and IL-22-induced activation of STAT3 in human colon cancer cells. While mouse, organoid, and cancer cell lines have helped to advance the field, more physiologically relevant in vitro human models are needed to increase accuracy and resolution of experiments aimed at predicting and uncovering the response of human gut ISCs and differentiated epithelium to such IBD-relevant factors. My aims will use a novel planar crypt-microarray (PCM) device and reporter cell lines to live-image primary human colonic stem cells during their response to IBD-related perturbations. PCMs have already been optimized for culture of mouse ISCs, and preliminary data demonstrates human ISC compatibility. Aim 1 will adjust factors such as PCM membrane composition, thickness, and timing of apical/basal media changes to promote confluent monolayers and compartmentalization of stem (Olfm4-EGFP) and differentiated (Muc2-BFP) cell zones. Using human-optimized PCMs, I will test the central hypothesis that butyrate-stimulated IL-22 signaling enhances asymmetric division and differentiation in human colonic ISCs. Aim 2 will use CRISPR/Cas9 indels to induce a frameshift loss-of-function (LOF) mutation in IL-22 receptor subunit alpha 1 into a tetracycline-inducible H2B::RFP (tetO-H2B::RFP) human colon stem cell line to test whether IL-22 signaling is necessary for asymmetric division. The tetO-H2B::RFP fusion protein allows for controlled, sensitive analysis of cell division dynamics in mammalian cells, with rapidly dividing cells corresponding to diluted RFP fluorescence intensity over time. RFP label-retaining cells will be assessed for phosphorylated STAT3 (pSTAT3) via immunofluorescence imaging on unsorted cell monolayers and for perturbed lineage pathways by FACS-scRNA-seq. The LOF model will be compared against IL22RA1+ controls on PCMs to determine if asymmetric division is enhanced by butyrate-stimulated IL-22 signaling. Our lab already has preliminary data for Olfm4-EGFP and H2B reporter lines. In the outstanding training environment in Dr. Scott Magness’ group at the University of North Carolina at Chapel Hill, I will gain expertise in microfabrication and intestinal stem cell biology and learn new techniques in primary human colon stem cell culture, single-cell biology/genomics, and bioinformatics. My career development training plan focuses on research skills, professional development, and clinical skills to promote my path to independence as an academic physician-scientist.