Prom1+ Biliary Progenitor Cells Display Abnormal Cellular Polarity and Ciliopathy in Murine Biliary Atresia

INTRODUCTION: Although the pathogenesis of the extrahepatic bile duct (EHBD) occlusion in biliary atresia (BA), the leading cause of liver failure, is unclear, BA has been associated with genetic polymorphisms linked to ciliopathies. Prominin-1 (Prom1) promotes intrahepatic fibrosis in BA. PROM1+ biliary progenitor cells (PCs) regenerate the EHBD after injury, whereas Prom1 knockout (KO) mice have impaired EHBD regeneration. Prom1 has been shown to maintain PC ciliary structure. Thus, we hypothesize that Prom1 regulates PC polarity and ciliary structure integral to EHBD regeneration. METHODS: Wild-type (WT) and KO EHBDs 18 days after murine BA rhesus rotavirus (RRV) or saline injection at day of life 3 were collected and sectioned. WT and KO EHBDs were dissociated and grown in Matrigel. All were stained via immunofluorescence and imaged with confocal microscopy. Images were processed with Arivis Vision4D. Paired t-tests were performed for statistical analyses (p < 0.05). RESULTS: EHBDs of WT mice after RRV injury demonstrate normal cell polarity with cytoskeletal protein F-actin localized to the apical membrane, but KO EHBD do not. Further analysis of WT organoids demonstrates apical co-localization of PROM1 with F-actin, phosphorylated EZRIN, and cystic fibrosis transmembrane conductance regulator (CFTR). In contrast, KO organoids exhibit less apical expression of F-actin, pEZRIN, and CFTR compared with WT (F-actin: 12% vs 84%, p = 0.0367). KO also manifest shorter and fewer cilia by α-acetyl-tubulin staining (Figure).CONCLUSION:Prom1 maintains cellular polarity and ciliary structure of biliary PCs that may support EHBD regeneration. Prom1 dysfunction may be associated EHBD obliteration in BA.