O1117: Disruption of prom1-mediated Cell Polarity and Ciliary Structure in Biliary Progenitor Cells Decreases Biliary Regeneration in Murine Biliary Atresia

Background: Biliary Atresia (BA) is the leading cause of pediatric liver failure. While the pathogenesis of the extrahepatic bile duct (EHBD) occlusion in BA is unclear, BA has been associated with distorted biliary epithelial cilia and genetic polymorphisms linked to ciliopathies. Ciliary function is also a known driver of biliary proliferation. Prominin-1 (Prom1) promotes fibrosis in BA. PROM1+ biliary progenitor cells (PC’s) regenerate the EHBD after injury. Prom1 is involved in maintaining ciliary structure in PC’s in other epithelial systems. Thus, we hypothesize that Prom1 regulates PC polarity and ciliary structure integral to biliary epithelial proliferation and EHBD regeneration.

Methods: Prom1 knockout (KO) mice (Prom1cre-ert2/cre-ert2) were bred with Rosa26Gfp Balbc reporter mice. Both Wild-type (WT) and KO EHBD’s were dissociated and grown in Matrigel. Both WT and KO EHBD tissue were collected and sectioned 18 days after murine BA rhesus rotavirus (RRV) or saline injection at day of life 3. Organoids and tissue sections were stained via immunofluorescence and imaged with confocal microscopy. Both WT and KO organoids derived from murine EHBD were then grown in Matrigel at a starting concentration of 1000cells/ 40ul of Matrigel under a brightfield live imaging microscope for 2 weeks. Images were processed with Arivis Vision4D®. Paired t-tests were performed for statistical analyses (p < 0.05).

Results: PROM1 co-localizes with cytoskeletal proteins F-ACTIN, Phosphorylated EZRIN, and CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) at the apical cellular surface in WT organoids. F-ACTIN, pEZRIN, and CFTR are diffusely expressed in KO organoids (% diffuse F-ACTIN staining- 12.21% vs. 84.42%, p=0.0367). alpha-ACETYL-TUBULIN demonstrates shorter cilia in KO than WT, indicating distorted cilia structure in KO organoids (Fig. 1A). F-ACTIN expression in vivo after RRV injury localizes to the apical membrane in WT but lacks localization in KO (Fig. 1B). WT organoids grew to larger maximal surface area than KO organoids over 2 weeks (68,865 +/- 176,427 um2 vs. 12,805 +/- 39,376 um2, p< 0.0001) (Fig. 2A). WT organoids also resulted in a larger cell population than KO organoids (597,500 +/- 116,6773 vs. 81,667 +/- 119.826 cells, p=0.0176) (Fig 2B) after 2 weeks of culture.

Conclusion: Prom1 functions by maintaining cellular polarity and ciliary structure of biliary PC’s. Loss of Prom1 in biliary progenitor cells is associated with altered cellular polarity and ciliopathy with resulting decreased cellular and organoid proliferation. Prom1 dysfunction may lead to abnormal cellular polarity in cholangiocytes and ciliopathy associated with EHBD obliteration in BA.