Har-Zahav, A. | Search bioRxiv

bioRxiv2025

Human biliary atresia extrahepatic cholangiocyte organoids express increased ER and oxidative stress, altered drug metabolism and cell polarity changes

Background & AimsBiliary atresia (BA), the leading cause of liver transplantation in children, presents in neonates with jaundice and progressive extrahepatic bile duct obstruction, yet its etiology and pathogenesis remain unknown. Here, we aimed to investigate the molecular mechanisms underlying BA and the susceptibility of cholangiocytes in the extrahepatic biliary tree using patient-derived extrahepatic cholangiocyte organoids (EHCOs). MethodsEHCOs were derived from common bile ducts remnants of BA patients undergoing Kasai portoenterostomy and from non-BA controls at the time of liver transplantation. Transcriptomic profiling was performed via bulk RNA sequencing, and analyzed in two ways: differentially expressed pathways and perturbation analysis to predict aberrant functions. Key findings were validated through mechanistic assays, immunofluorescence staining, qPCR and transmission electron microscopy (TEM). ResultsTranscriptomic analysis predicted significant alteration in endoplasmic reticulum (ER) stress, dysregulations of drug metabolism, alongside pronounced alterations in cellular adhesion and polarity-related genes in BA-derived EHCOs. Cell-to-cell alterations were observed with various proteins including E-cadherin, RhoU, Sox17 and CFTR. BA EHCOs had an increased endoplasmic reticulum (ER) stress response, exemplified by elevated PERK, BiP, and ATF4 along with abnormal ER on TEM. Furthermore CHOP, ERO1A, WFS1, and SOD3 were decreased suggestive of abnormal ER stress response. BA EHCOs displayed increased toxicity to biliatresone-induced injury and inhibition of cytochrome P450 resulted in attenuation of the ER stress markers PERK, BiP and ATF4. Finally, liver hilum biopsies from BA patients undergoing Kasai portoenterostomy confirmed elevated PERK and PGR78(BiP) consistent with the EHCOs analysis. ConclusionsBA EHCOs exhibit disrupted polarity, ER stress, and increased susceptibility to drug toxicity. These findings highlight key pathogenic mechanisms in BA and suggest that targeting these pathways may help mitigate cholangiocyte injury in BA. Impact and implicationsThis study provides the first transcriptomic and functional analysis of human extrahepatic cholangiocyte organoids (EHCOs) derived from biliary atresia (BA) patients. By focusing on the extra-hepatic biliary tree, we identified key mechanisms of cholangiocyte injury, including persistent ER stress, impaired stress response pathways, altered drug metabolism and disrupted epithelial polarity. These findings highlight ER stress and metabolic vulnerability as potential therapeutic targets and establish EHCOs as a tractable model for investigating BA pathogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/649927v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@150ab7org.highwire.dtl.DTLVardef@172953forg.highwire.dtl.DTLVardef@1a46e4eorg.highwire.dtl.DTLVardef@45c5c6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst transcriptomic profiling of extrahepatic cholangiocyte organoids (EHCOs) from BA patients, C_LIO_LIrevealing distinct molecular alterations compared to controls. C_LIO_LIBA EHCOs exhibit disrupted epithelial polarity, with downregulation of E-cadherin and Sox17 and upregulation of CFTR. C_LIO_LIER stress is a hallmark of BA cholangiocytes, with elevated PERK, BiP, and ATF4, and C_LIO_LIdysregulation of downstream effectors including CHOP, ERO1A, and SOD3. C_LIO_LIBA EHCOs are more susceptible to biliatresone-induced injury, with enhanced ER stress and structural damage. C_LIO_LIInhibition of cytochrome P450 activity (CYP4A) reduces ER stress markers. C_LI

molecular biology↗

bioRxiv2023

Human Vascularized Bile Duct-on-a Chip: A multi-cellular micro-physiological system for studying Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease in which the bile ducts of the liver become inflamed and scarred. Scarred bile ducts eventually narrow and obstruct and can cause additional liver pathology including liver failure, repeated infections, and tumors. The pathogenesis of PSC remains largely unknown, partly due to difficulty in obtaining cholangiocytes and partly due to a paucity of in vitro models that capture the various factors contributing to disease progression. Here we report the development of a human vascularized bile duct-on-a-chip that models blood vessels and bile ducts structurally and functionally in three dimensions and includes cholangiocytes derived from control and PSC patient tissue and bile. The flow of blood and bile was modeled by perfusion of cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to perfusion. Normal and PSC cholangiocytes polarized normally, formed mature tight junctions and displayed similar permeability, comparable to ex vivo measurements. The model with PSC cholangiocytes, however, became more inflammatory than the normal under the stimulation of IL-17A, which induced PBMC and differentiated Th17 cells in the vascular channel to transmigrate more through the endothelial layer of the vascular compartment. In sum, this human vascularized bile duct-on-a-chip recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholangiopathies such as PSC.

bioengineering↗

bioRxiv2022

Periductal bile acid exposure causes cholangiocyte injury and fibrosis

IntroductionBile duct integrity is essential for the maintenance of the structure and function of the biliary tree. We previously showed that cholangiocyte injury in a toxic model of biliary atresia leads to increased monolayer permeability. Increased epithelial permeability was also shown in other cholangiopathies. We hypothesized that after initial cholangiocyte injury, leakage of bile acids into the duct submucosa propagates cholangiocyte damage and fibrosis. We thus aimed to determine the impact of bile acid exposure on cholangiocytes and the potential therapeutic effect of a non-toxic bile acid. Materials and methodsExtrahepatic bile duct explants were isolated from adult and neonatal BALB/c mice. Explants were cultured with or without glycochenodeoxycholic acid and ursodeoxycholic acid. They were then fixed and stained. ResultsExplants treated with glycochenodeoxycholic acid demonstrated cholangiocyte injury with monolayer disruption and partial lumen obstruction compared to control ducts. Massons trichrome stains revealed increased collagen fibers. Myofibroblast marker -SMA stains were significantly elevated in the periductal region. The addition of ursodeoxycholic acid resulted in decreased cholangiocyte injury and reduced fibrosis. ConclusionsBile acid leakage into the submucosa after initial cholangiocyte injury may serve as a possible mechanism of disease propagation and progressive fibrosis in cholangiopathies.

physiology↗