Investigating a rare pediatric enteropathy caused by ADAM17 loss of function using iPSC-derived human intestinal organoids

使用 iPSC 衍生的人肠道类器官研究由 ADAM17 功能丧失引起的罕见儿童肠病

• 批准号: 10823723
• 负责人: Shane Williams
• 金额: $ 3.94万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-11 至 2026-09-10
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10823723
• 关键词: Architecture; Binding; Biological Assay; Biological Models; Biopsy; CRISPR correction; Cell Differentiation process; Cell Maturation; Cell Membrane Proteins; Cell Separation; Cells; Childhood; Chronic diarrhea; Data Set; Development; Diarrhea; Disease; Disease model; Disintegrins; EGF gene; Environment; Epithelial Cells; Epithelium; Family; Fetal Development; Future; Genetic Transcription; Homeostasis; Human; Immunofluorescence Immunologic; Impairment; In Vitro; Inflammatory; Intestines; Investigation; Knockout Mice; Ligands; Mass Spectrum Analysis; Mediating; Membrane; Mesenchymal; Mesenchyme; Metalloproteases; Modeling; Molecular; Morphogenesis; Morphology; Neonatal; Neuregulin 1; Organoids; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Permeability; Phenotype; Proteins; Proteomics; Receptor Activation; Role; Sampling; Signal Transduction; Signaling Molecule; Specialized Epithelial Cell; Structure; Study models; Supporting Cell; Testing; Tissues; Villus; Work; cell type; directed differentiation; fetal; gut inflammation; human disease; improved; induced pluripotent stem cell; infancy; innovation; insight; intestinal crypt; intestinal epithelium; loss of function; loss of function mutation; mutation correction; receptor; skin disorder; stem cell niche; stem cells; transcriptome sequencing; transcriptomics

Project Summary ADAM17 loss of function (LOF) is the cause of a pediatric enteropathy termed Neonatal Inflammatory Skin and Bowel Disease 1 (NISBD1), which is associated with intestinal inflammation and diarrhea. While little is known about NISBD1, two patient biopsies revealed abnormal intestinal crypt/villus morphology. The crypt/villus structure is the fundamental repeating intestinal unit lined by an epithelium that contains crypt restricted intestinal stem cells (ISCs). ISCs give rise to differentiated cells and are integral to maintaining epithelial function. ISCs are supported by specialized epithelial cells and underlying mesenchymal cells that provide ISC niche signals, including EGF-like ligands. ADAM17 is a ubiquitously expressed protease that cleaves and sheds proteins from the cell membrane. Its substrates include EGF-like ligands, suggesting a role for ADAM17 in the ISC niche. Recent work has revealed that ErbB3 ligand neuregulin 1 (NRG1) promotes human ISC maturation and differentiation. Human fetal intestinal tissue sections reveal mesenchyme restricted expression of NRG1 and epithelium restricted expression of its receptor ErbB3. This suggests a pattern of ADAM17 mediated mesenchyme to epithelium signaling crosstalk. I will test the hypothesis that ADAM17 mediates cellular crosstalk, regulating human intestinal epithelial development. First, I am establishing a matched isogenic set of NISBD1 patient iPSC lines with one CRISPR corrected to be ADAM17 proficient. I will differentiate both lines into human intestinal organoids containing organized epithelium and mesenchyme. Then, I will interrogate the ADAM17 LOF phenotype using cellular, transcriptomic, and functional analyses. Next, I will investigate ADAM17 mediated cellular crosstalk in a targeted approach by studying NRG1-ErbB3 signaling. To identify additional candidate ADAM17 substrates, I will take an unbiased mass spectrometry and proteomics approach to determine all human intestinal ADAM17 substrates, i.e. the sheddome. This analysis will establish datasets for future targeted investigation of ADAM17 substrates in the human ISC niche. In summary, I will perform an innovative patient specific disease modeling study using iPSC-derived organoids to characterize NISBD1 and study the role of ADAM17 in the developing human intestine.

项目摘要 ADAM17功能丧失（LOF）是儿科肠病的原因，称为新生儿炎症性皮肤和 肠病1（NISBD1），与肠炎和腹泻有关。虽然鲜为人知 关于NISBD1，两名患者活检显示肠道隐窝/绒毛形态异常。地下/绒毛 结构是由包含隐窝限制的上皮衬里的基本重复肠单元 干细胞（ISC）。 ISC产生分化的细胞，并且是维持上皮功能不可或缺的一部分。 ISC 由专门的上皮细胞和提供ISC利基信号的基础间充质细胞支持， 包括类似EGF的配体。 ADAM17是一种普遍表达的蛋白酶，从 细胞膜。它的底物包括类似EGF的配体，这表明ADAM17在ISC利基市场中的作用。 最近的工作表明，ERBB3配体神经调节蛋白1（NRG1）促进了人类ISC成熟和 分化。人类胎儿肠道组织切片揭示了间质限制NRG1和 其受体ERBB3的上皮限制表达。这表明ADAM17介导的模式 间充质到上皮信号传导串扰。我将测试ADAM17介导细胞的假设 串扰，调节人类肠上皮发育。首先，我建立了一个匹配的等源性 一组NISBD1患者IPSC系列具有一个CRISPR校正为ADAM17熟练。我会区分 线进入含有有组织上皮和间质的人肠癌。然后，我会审问 使用细胞，转录组和功能分析的ADAM17 LOF表型。接下来，我将调查 通过研究NRG1-ERBB3信号传导，ADAM17在靶向方法中介导的细胞串扰。识别 其他候选ADAM17底物，我将采用公正的质谱和蛋白质组学方法 确定所有人类肠道ADAM17底物，即Sheddome。该分析将建立数据集 对于未来的针对人类ISC利基市场ADAM17底物的针对性研究。总而言之，我将执行 创新的患者特定疾病建模研究使用IPSC衍生的类器官来表征NISBD1和 研究ADAM17在发展中的人类肠道中的作用。