Brovold Matthew, Keller Dale, Devarasetty Mahesh, Dominijanni Anthony, Shirwaiker Rohan, Soker Shay
Wake Forest Institute for Regenerative Medicine Wake Forest Baptist Medical Center, Medical Center Boulevard Winston-Salem North Carolina USA.
Department of Industrial and Systems Engineering North Carolina State University Raleigh North Carolina USA.
Bioeng Transl Med. 2021 Jun 5;6(3):e10207. doi: 10.1002/btm2.10207. eCollection 2021 Sep.
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 , , , and 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.
先天性胆道疾病是小儿肝衰竭的主要原因，也是小儿肝移植需求的最终原因。并非所有这些疾病的病因都已完全清楚，但已知许多患者会发生肝纤维化。本研究的目的是建立一个简单而稳健的模型，该模型能够概括由肝星状细胞（HSCs）介导的纤维化的物理力学和细胞方面，以及它们对胆管祖细胞的影响。通过将具有产生轻度至重度纤维化环境独特能力的各种肝星状细胞与未分化的肝祖细胞系HepaRG一起包埋在I型胶原水凝胶中，构建肝脏类器官。通过定量图像分析、流变学和qPCR检测细胞外基质（ECM）重塑，对每个类器官的纤维化状态进行表征。同时，通过组织学评估肝祖细胞的表型和簇形成。与永生化肝星状细胞（LX-2）相比，活化的肝星状细胞（aHSCs）产生了更严重的纤维化状态，表现为细胞外基质收缩更强烈、更坚硬，以及、、和的相对表达更高。在由aHSCs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、细胞密度更高的胆管样簇的形成有关。CK19细胞的扩增，再加上严重的纤维化环境，是患有各种先天性胆道肝脏疾病的患者体内发现的现象。因此，这里提出的模型可以用作一种新型的体外测试平台，用于测试药物和识别可能使患有与多种先天性肝脏疾病相关的胆管发育异常小儿患者受益的新靶点。
