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Biofabricated 3D in vitro model of fibrosis-induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver.

ABSTRACT: 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.

SUBMITTER: Brovold M

PROVIDER: S-EPMC8459590 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Biliary atresia (BA), the most common cause of end-stage liver disease and the leading indication for pediatric liver transplantation, is associated with intrahepatic ductular reactions within regions of rapidly expanding periportal biliary fibrosis. Whereas the extent of such biliary fibrosis is a negative predictor of long-term transplant-free survival, the cellular phenotypes involved in the fibrosis are not well established. Using a rhesus rotavirus-induced mouse model of BA, we demonstrate significant expansion of a cell population expressing the putative stem/progenitor cell marker, PROMININ-1 (PROM1), adjacent to ductular reactions within regions of periportal fibrosis. PROM1positive (pos) cells express Collagen-1?1. Subsets of PROM1pos cells coexpress progenitor cell marker CD49f, epithelial marker E-CADHERIN, biliary marker CYTOKERATIN-19, and mesenchymal markers VIMENTIN and alpha-SMOOTH MUSCLE ACTIN (?SMA). Expansion of the PROM1pos cell population is associated with activation of Fibroblast Growth Factor (FGF) and Transforming Growth Factor-beta (TGF?) signaling. In vitro cotreatment of PROM1-expressing Mat1a-/- hepatic progenitor cells with recombinant human FGF10 and TGF?1 promotes morphologic transformation toward a myofibroblastic cell phenotype with increased expression of myofibroblastic genes Collagen-1?1, Fibronectin, and ?-Sma. Infants with BA demonstrate similar expansion of periportal PROM1pos cells with activated Mothers Against Decapentaplegic Homolog 3 (SMAD3) signaling in association with increased hepatic expression of FGF10, FGFR1, and FGFR2 as well as mesenchymal genes SLUG and SNAIL. Infants with perinatal subtype of BA have higher tissue levels of PROM1 expression than those with embryonic subtype.Expansion of collagen-producing PROM1pos cells within regions of periportal fibrosis is associated with activated FGF and TGF? pathways in both experimental and human BA. PROM1pos cells may therefore play an important role in the biliary fibrosis of BA.

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Biofabricated 3D in vitro model of fibrosis-induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver.

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