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

项目摘要 ADAM17功能丧失(LOF)是一种称为新生儿炎症性皮肤和肠病的儿科肠道疾病的原因。 肠道疾病1(NISBD1)，与肠道炎症和腹泻有关。虽然我们知之甚少 关于NISBD1，两名患者的活组织检查显示肠隐窝/绒毛形态异常。墓穴/绒毛 结构是基本的重复的肠道单位，由含有隐窝限制的肠道的上皮组织构成。 干细胞(ISC)。ISCs产生分化的细胞，对维持上皮功能是不可或缺的。ISCS 由提供ISC生态位信号的特化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水解酶，它能从 细胞膜。它的底物包括EGF样配体，这表明ADAM17在ISC利基中发挥了作用。 最近的研究表明，ErbB3配体NeuRegin 1(NRG1)促进人ISC成熟和 差异化。人胎肠组织切片显示间充质细胞限制NRG1和NRG1的表达 上皮细胞限制其受体ErbB3的表达。这表明了ADAM17介导的一种模式 间充质向上皮间充质传递串扰信号。我将测试ADAM17介导细胞 串扰，调节人类肠道上皮发育。首先，我要建立一个匹配的同源基因 一组NISBD1患者IPSC系，其中一个CRISPR更正为ADAM17熟练。我会将两者区分开来 含有有组织的上皮和间充质的人体肠道器官。然后，我会审问 使用细胞、转录和功能分析的ADAM17 LOF表型。接下来，我将调查 ADAM17通过研究NRG1-ErbB3信号，有针对性地介导细胞串扰。要确定 其他候选ADAM17底物，我将采用无偏质谱和蛋白质组学方法 以确定所有人类肠道ADAM17底物，即Sheddome。此分析将建立数据集 用于未来对人类ISC利基中ADAM17底物的定向研究。总而言之，我将执行一个 创新的患者特定疾病建模研究，使用IPSC衍生的有机化合物来表征NISBD1和 研究ADAM17在人体肠道发育中的作用。

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.