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Abstract：
In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.
摘要：
在炎症性肠病（IBD）中，在活动性疾病和缓解状态下，肠上皮的特征是通透性增加。这种肠道通透性增加的遗传基础在很大程度上没有被研究，部分原因是缺乏适当的建模系统。我们的目标是使用诱导多能干细胞（iPSC）衍生的人类肠道类器官（HIOs）和人类结肠类器官（HCOs）来开发肠道通透性的体外模型，以研究屏障功能障碍。iPSCs是从健康对照中产生的，成人发病IBD,和非常早发性IBD（VEO-IBD）患者，并分化为HIOs和HCOs。将EpCAM+选择的细胞接种到Transwell插入物上,并在存在或不存在促炎细胞因子TNFα和IFNγ的情况下进行屏障完整性研究。实时定量PCR(qRT-PCR),透射电子显微镜（TEM），和免疫荧光用于确定改变的紧密和粘附的连接蛋白表达或定位。与HIO相比，向HCO的分化表明CDX2，CD147和CA2的基因表达增加，并且基础跨上皮电阻增加。在HIO和HCO来源的上皮中进行了渗透性研究，当暴露于TNFα和IFNγ时，FD4的渗透性显着增加。TEM和免疫荧光成像表明，在TNFα和IFNγ攻击的类器官中，E-钙黏着蛋白和ZO-1的错误定位，mRNA表达相应降低。HIO-和HCO-上皮之间的比较显示基因表达的差异，电生理学,和形态学：两者都对TNFα和IFNγ刺激有反应，导致渗透性增强，以及紧密和依附连接结构的变化。该数据表明iPSC衍生的HIO和HCO构成了研究屏障功能障碍和上皮在IBD和VEO-IBD中的作用的合适的生理响应模型。
