Congenital disorders of the biliary tract are the primary reason for pediatric liver failure and ultimately for pediatric liver transplant needs. Not all causes of these disorders are well understood, but it is known that liver fibrosis occurs in many of those afflicted. The goal of this study is to develop a simple yet robust model that recapitulates physico-mechanical and cellular aspects of fibrosis mediated via hepatic stellate cells (HSCs) and their effects on biliary progenitor cells. Liver organoids were fabricated by embedding various HSCs, with distinctive abilities to generate mild to severe fibrotic environments, together with undifferentiated liver progenitor cell line, HepaRG, within a collagen I hydrogel. The fibrotic state of each organoid was characterized by examination of extracellular matrix (ECM) remodeling through quantitative image analysis, rheometry, and qPCR. In tandem, the phenotype of the liver progenitor cell and cluster formation was assessed through histology. Activated HSCs (aHSCs) created a more severe fibrotic state, exemplified by a more highly contracted and rigid ECM, as well higher relative expression of TGF-β, TIMP-1, LOXL2, and COL1A2 as compared to immortalized HSCs (LX-2). Within the more severe fibrotic environment, generated by the aHSCs, higher Notch signaling was associated with an expansion of CK19⁺ cells as well as the formation of larger, more densely populated cell biliary like-clusters as compared to mild and non-fibrotic controls. The expansion of CK19⁺ cells, coupled with a severely fibrotic environment, are phenomena found within patients suffering from a variety of congenital liver disorders of the biliary tract. Thus, the model presented here can be utilized as a novel in vitro testing platform to test drugs and identify new targets that could benefit pediatric patients that suffer from the biliary dysgenesis associated with a multitude of congenital liver diseases.

中文翻译：

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纤维化诱导的异常肝母细胞/胆道祖细胞发育中肝脏扩张的生物制造 3D 体外模型

先天性胆道疾病是小儿肝功能衰竭的主要原因，最终需要小儿肝移植。并非所有这些疾病的原因都得到了很好的理解，但众所周知，许多患者都会发生肝纤维化。本研究的目标是开发一个简单而强大的模型，该模型概括了肝星状细胞 (HSC) 介导的纤维化的物理机械和细胞方面及其对胆道祖细胞的影响。肝脏类器官是通过嵌入各种 HSC 制造的，这些 HSC 具有产生轻度至重度纤维化环境的独特能力，以及未分化的肝祖细胞系 HepaRG，嵌入胶原蛋白 I 水凝胶中。每个类器官的纤维化状态的特征是通过定量图像分析、流变测量和 qPCR 检查细胞外基质 (ECM) 重塑。同时，通过组织学评估肝祖细胞的表型和簇形成。活化的 HSC (aHSC) 产生了更严重的纤维化状态，例如高度收缩和刚性的 ECM，以及更高的相对表达与永生化 HSC (LX-2) 相比，TGF-β、TIMP-1、LOXL2和COL1A2。在由 aHSC 产生的更严重的纤维化环境中，与轻度和非纤维化对照相比，更高的 Notch 信号与 CK19 ⁺细胞的扩增以及更大、更密集的细胞胆管样簇的形成有关。CK19 ⁺的扩展细胞，加上严重的纤维化环境，是在患有胆道的各种先天性肝病的患者中发现的现象。因此，这里介绍的模型可以用作一种新的体外测试平台来测试药物并确定新的目标，这些目标可以使患有与多种先天性肝病相关的胆道发育不全的儿科患者受益。