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.

项目概要 ADAM17 功能丧失 (LOF) 是一种称为新生儿炎症性皮肤病的小儿肠病的病因 肠道疾病 1 (NISBD1)，与肠道炎症和腹泻有关。虽然鲜为人知 关于 NISBD1，两名患者活检显示肠隐窝/绒毛形态异常。隐窝/绒毛 结构是基本的重复肠道单位，内衬上皮，包含隐窝限制性肠道 干细胞（ISC）。 ISC 产生分化细胞，对于维持上皮功能至关重要。 ISC 由提供 ISC 生态位信号的专门上皮细胞和底层间充质细胞支持， 包括EGF样配体。 ADAM17 是一种普遍表达的蛋白酶，可从 细胞膜。其底物包括 EGF 样配体，表明 ADAM17 在 ISC 生态位中发挥作用。 最近的研究表明，ErbB3 配体神经调节蛋白 1 (NRG1) 可促进人类 ISC 成熟和 差异化。人胎儿肠道组织切片显示间充质限制性表达 NRG1 和 上皮细胞限制其受体 ErbB3 的表达。这表明 ADAM17 介导的模式 间质到上皮信号传导串扰。我将检验 ADAM17 介导细胞的假设 串扰，调节人体肠上皮发育。首先，我正在建立匹配的同基因 一组 NISBD1 患者 iPSC 系，其中一个 CRISPR 已校正为 ADAM17 熟练。我将区分两者 细胞系进入含有有组织的上皮和间质的人类肠道类器官。然后我会审问 使用细胞、转录组和功能分析确定 ADAM17 LOF 表型。接下来我会调查 ADAM17 通过研究 NRG1-ErbB3 信号传导以靶向方法介导细胞串扰。识别 其他候选 ADAM17 底物，我将采用无偏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 用于未来对人类 ISC 生态位中 ADAM17 底物的针对性研究。总而言之，我将执行 使用 iPSC 衍生的类器官来表征 NISBD1 和 研究 ADAM17 在人类肠道发育中的作用。