Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells’ fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

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用于肠道炎症和纤维化体外研究的人类肠道类器官模型的开发

炎症性肠病 (IBD) 的特征是慢性肠道炎症和纤维化，后者是长期并发症的主要分母。上皮和间充质 2D 培养物在体外模型中被高度利用，用于抗炎和抗纤维化治疗的临床前评估。最近，人类肠道类器官 (HIO)，一种新的体外3D源自多能干细胞的模型具有与肠粘膜结构非常相似的优势。然而，在 HIO 发展过程中研究炎症和纤维化反应的适当时机尚未得到充分研究。我们从人类胚胎干细胞系 H1 中开发了 HIO，并检查了它们成熟过程中间充质标志物的表达。我们还研究了炎症刺激对纤维化和免疫介质表达的影响。对间充质和细胞外基质 (ECM) 标记表达的连续评估表明，HIO 具有充分发育的间充质成分，通过培养通道逐渐下降。具体来说，CD90、I型胶原蛋白、III型胶原蛋白、和纤连蛋白在早期传代中高度表达，但在晚期传代中逐渐减少。促炎细胞因子 IL-1α和 TNF- α诱导纤连蛋白、I 型和 III 型胶原蛋白、组织因子 (TF) 和 α-平滑肌肌动蛋白 ( α-SMA) 主要出现在早期段落中。类似地，HIO 在早期而非晚期传代中引发强烈的 mRNA 和蛋白质间充质 (CXCL10) 和上皮 (CXCL1、CCL2、CXCL8 和 CCL20) 趋化因子反应。相比之下，上皮紧密连接成分 CLDN1 和 JAMA 对炎症刺激做出反应，而与培养通道无关。我们的研究结果表明，该 HIO 模型在早期传代期间包含功能性间充质成分，并强调了间充质细胞在炎症和纤维化反应中的适应性的重要性。因此，我们建议该模型适用于间充质成分活跃时早期传代中上皮间充质相互作用的研究。