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

Biliary atresia (BA), the leading cause of pediatric liver transplantation, is characterized by neonatal jaundice and progressive extrahepatic bile duct obstruction, yet its pathogenesis remains elusive. To uncover extrahepatic cholangiocyte injury, we generated patient-derived extrahepatic cholangiocyte organoids (EHCOs) from bile duct remnants of BA and controls. We performed discovery bulk RNA-seq followed by targeted molecular and functional validation. Transcriptomic profiling and pathway/perturbation analyses revealed dysregulation of ER stress pathways, xenobiotics/drug metabolism, and genes regulating cell adhesion and polarity. BA EHCOs exhibited altered expression of E-cadherin, RhoU, SOX17, and CFTR. ER stress markers PERK, BiP, and ATF4 were elevated, and TEM demonstrated abnormal ER morphology. Conversely, CHOP, ERO1A, WFS1, and SOD3 were downregulated, suggesting impaired ER stress resolution. Functionally, BA EHCOs were more susceptible to toxic injury, and CYP450 inhibition attenuated ER stress genes expression. Immunostaining of BA liver hilum biopsies confirmed elevated PERK and BiP. Together, these data identify unknown arms of ER stress and epithelial dysfunction as disease-relevant injury process of the extrahepatic biliary tree and potentially implicate xenobiotic metabolism as a modifiable driver.