Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients
Characterization of Human Colon Organoids From Inflammatory Bowel Disease Patients
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DOI：
10.3389/fcell.2020.00363
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发表时间：
2020-06-04
影响因子：
5.5
通讯作者：
Vergnolle, Nathalie
中科院分区：
文献类型：
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作者：
d'Aldebert, Emilie;Quaranta, Muriel;Vergnolle, Nathalie
炎症性肠病（IBD）是慢性炎症性疾病，其中上皮缺陷至少部分地驱动一些病理。我们利用人结肠类器官的三维培养，构建了人肠上皮器官。我们的目的是表征对照（非IBD）类器官的形态和功能表型，与IBD患者的发炎类器官相比。所产生的结果描述了与IBD相关的上皮缺陷在原代类器官培养，并评估使用该模型的抗炎方法的药理学测试。人结肠组织从手术切除或活组织检查中获得，均在非炎症区收获。从对照（非IBD）和IBD患者分离隐窝，并培养长达12天。通过免疫组织化学、RT-qPCR或Western印迹测量形态学（大小、出芽形成、极化、管腔内容物）、细胞组成（增殖、分化、未成熟标记物表达）和功能（趋化因子和紧密连接蛋白表达）参数。分别在对照和IBD类器官培养物中研究了炎性鸡尾酒或抗炎治疗的效果。来自对照或IBD患者的类器官培养物在培养10至12天后具有相同的细胞组成，但IBD类器官培养物显示出炎性表型，具有减小的尺寸和出芽能力、增加的细胞死亡、管腔碎片和反向极化。在IBD类器官培养物中，紧密连接蛋白也显著减少。炎性细胞因子混合物在非IBD类器官中再现了这种炎性表型。临床使用的治疗（5-阿萨，糖皮质激素，抗TNF）减少了一些，但不是所有的参数。炎症表型与IBD上皮相关，并且可以在类器官培养物中进行研究。该模型构成了可靠的人类临床前模型，以研究靶向上皮修复的新策略。
Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.