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

用于实时评估人类肠道干细胞命运的新型平面隐窝微阵列

基本信息

批准号：
10436268
负责人：
Meryem Tyrrasch Ok
金额：
$ 5.27万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10436268
关键词：
3-Dimensional Adult Affect Apical Bioinformatics Butyrates CRISPR/Cas technology Cancer cell line Cell Culture Techniques Cell Differentiation process Cell Line Cell Lineage Cell Maintenance Cell Nucleus Cell division Cells Cellular biology Chimeric Proteins Clinical Skills Clustered Regularly Interspaced Short Palindromic Repeats Colon Colon Carcinoma Complex Crohn&apos s disease Data Development Devices Environment Epithelial Cells Epithelium Evaluation Exposure to Fluorescence Foundations Functional disorder Gene Expression Genes Genetic Genomics Goals Goblet Cells Growth Factor Human Hybrids Image Immune response Immunofluorescence Immunologic Impairment In Vitro Inflammatory Bowel Diseases Intestines Knock-out Label Learning Light Mammalian Cell Membrane Methods Microfabrication Microscopic Modeling Molecular Mouse Cell Line Mus North Carolina OLFM4 gene Organoids Pathogenesis Pathway interactions Patients Phenotype Phosphorylation Physicians Physiological Porosity Process Public Health RNA analysis Receptor Signaling Reporter Research Resolution Role STAT3 gene Scientist Signal Transduction Specific qualifier value Stat3 protein Surface System Techniques Technology Testing Tetracyclines Thick Time Training Ulcerative Colitis Universities Visualization Volatile Fatty Acids Western Blotting cancer cell career development dysbiosis epithelium regeneration experimental study fabrication human model improved insertion/deletion mutation interleukin-22 intestinal epithelium live cell imaging loss of function loss of function mutation microbial microdevice monolayer novel progenitor receptor receptor expression response single-cell RNA sequencing skills stem stem cell biology stem cell differentiation stem cell division stem cell expansion stem cell fate stem cell proliferation stem cell self renewal stem cells theories tissue regeneration tissue repair tool transcriptomics wound healing

项目摘要

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.

项目摘要炎症性肠病（IBD），包括克罗恩病和溃疡性结肠炎，估计有3.1 美国成年人。以及肠道短链脂肪酸（SCFA）水平的改变。在鼠回肠或ganoids中，暴露辛苦22促进祖细胞细胞的扩张比肠道扩张干细胞（ISC）自我更新，该表型是通过激活信号传感器和转录激活剂（Stat）-3。增强IL-22受体表达和IL-22诱导的人类结肠癌细胞的激活小鼠，器官和癌细胞lins有助于发展领域，更多的生理学在体外相关需要人类模型来提高旨在预测和的实验的准确性和分辨率揭示了人类肠道ISC的反应，并分化了上皮对这种与IBD相关的因素。我的目标将平面颅骨cranar Crypt-Microarray（PCM）设备和记者细胞系进行活图像主体在对IBD相关扰动的反应过程中，人类结肠干细胞。对于小鼠的培养物，预启示数据证明了人类ISC的兼容性。例如PCM膜组成，厚度和顶端/基础介质的变化以促进汇合使用茎（OLFM4-EGFP）和分化（MUC2-BFP）细胞区的单层和分室化人类优化的PCM，我将测试中心假设，即丁酸酯刺激的IL-22信号增强了人类ISC中的不对称分裂和差异在IL-22受体亚基1中诱导功能丧失（LOF）突变，进入四环素诱导 H2B :: RFP（TETO-H2B :: RFP）人类结肠干细胞系以测试IL-22信号是否需要不对称分裂。 Mamalian细胞的动力学，迅速分裂的细胞Corresspond逐渐稀释的RFP荧光强度时间。通过FACS-SCRNA-SEQ对未分类的细胞单层和Fortuster的谱系途径进行成像将与PCMS上的IL22RA1+对照进行比较，以确定是否通过丁酸酯刺激的IL-22信号传导已经具有OLFM4-EGFP和H2B记者线的预启示数据。在北卡罗琳宇宙的Scott Magness博士小组的出色培训环境中希尔，我将获得微加工和肠干细胞生物学的专业知识，并在初级学习新技术人类的干细胞培养，单细胞生物学/基因组学和生物信息学。计划重点介绍研究技能，专业发展和临床技能，以促进我的独立道路作为学术医师科学家。