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

Abstract Background & Aims Biliary 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). Methods EHCOs 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). Results Transcriptomic 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. Conclusions BA 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 implications This 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 Highlights First transcriptomic profiling of extrahepatic cholangiocyte organoids (EHCOs) from BA patients, revealing distinct molecular alterations compared to controls. BA EHCOs exhibit disrupted epithelial polarity, with downregulation of E-cadherin and Sox17 and upregulation of CFTR. ER stress is a hallmark of BA cholangiocytes, with elevated PERK, BiP, and ATF4, and dysregulation of downstream effectors including CHOP, ERO1A, and SOD3. BA EHCOs are more susceptible to biliatresone-induced injury, with enhanced ER stress and structural damage. Inhibition of cytochrome P450 activity (CYP4A) reduces ER stress markers.