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

项目摘要 炎症性肠病（IBD），包括克罗恩病和溃疡性结肠炎，影响估计3.1 百万美国成年人IBD的病理生理学是复杂的，涉及遗传、环境和免疫反应 以及肠道微生物短链脂肪酸（SCFA）水平的改变。Magness实验室的最新研究 在小鼠回肠类器官中，证实了暴露于IL-22比肠类器官更有利于祖细胞扩增， 干细胞（ISC）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SCFA，丁酸， 增强人结肠癌细胞中IL-22受体表达和IL-22诱导的STAT 3活化。而 小鼠、类器官和癌细胞系有助于推动该领域的发展， 需要人类模型来提高实验的准确性和分辨率， 揭示了人肠道ISCs和分化上皮细胞对IBD相关因子的反应。 我的目标是使用一种新的平面隐窝微阵列（PCM）设备和报告细胞系来实时成像原发性肝癌。 人结肠干细胞在其对IBD相关扰动的反应期间。PCM已经过优化 用于培养小鼠ISCs，初步数据证明人ISCs相容性。目标1将调整因素 例如PCM膜组成、厚度和顶端/基底培养基改变的时间，以促进融合 干细胞（Olfm 4-EGFP）和分化的（Muc 2-BFP）细胞区的单层和区室化。使用 为了验证丁酸盐刺激的IL-22信号增强的中心假设， 人结肠ISCs的不对称分裂和分化。Aim 2将使用CRISPR/Cas9 indel来 将IL-22受体亚基α 1移码功能丧失（LOF）突变诱导为四环素诱导型 H2 B：：RFP（tetO-H2 B：：RFP）人结肠干细胞系，以测试IL-22信号传导是否是 不对称分裂tetO-H2 B：：RFP融合蛋白允许对细胞分裂进行受控的灵敏分析 在哺乳动物细胞中，快速分裂的细胞对应于稀释的RFP荧光强度， 时间将通过免疫荧光评估RFP标记保留细胞的磷酸化STAT 3（pSTAT 3） 在未分选的细胞单层上成像，并通过FACS-scRNA-seq进行干扰的谱系途径。LOF模型 将与PCM上的IL 22 RA 1+对照进行比较，以确定是否通过 丁酸盐刺激的IL-22信号传导。我们的实验室已经有了Olfm 4-EGFP和H2 B报告细胞系的初步数据。 在北卡罗来纳州查珀尔大学的Scott Magness博士小组出色的培训环境中 希尔，我将获得在微加工和肠道干细胞生物学的专业知识，并在小学学习新技术 人结肠干细胞培养、单细胞生物学/基因组学和生物信息学。我的职业发展培训 计划侧重于研究技能，专业发展和临床技能，以促进我的独立之路 作为一个学术物理学家和科学家。

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.