PROM1+ Biliary Progenitor Cells Reside in Peribiliary Glands and Proliferate into Cholangiocytes in Response to Cholestatic Injury in Murine Biliary Atresia Models

Background: Biliary atresia (BA) is a congenital obliterative extrahepatic bile duct (EHBD) injury. In BA, intrahepatic PROM1+ hepatic progenitor cells (HPCs) give rise cholangiocytes comprising ductular reactions. Hypothesis: PROM1+ HPCs, present in peribiliary glands (PBGs) outside the EHBD lumen, differentiate into cholangiocytes following injury.

Methods: EHBD immunofluorescence was performed in: (1) neonatal wildtype (WT) and Prom1 knockout (KO) Balbc mice with rhesus rotavirus (RRV)-mediated BA and (2) Prom1cre-ert2;Rosa26Gfp(Prom1-Gfp) B57BL/6 adult mice, wherein GFP+ denotes Prom1-expressing cell lineage, after bile duct ligation (BDL). Epithelial organoids from Prom1cre-ert2;Rosa26mTmG WT and KO EHBDs were grown in Matrigel® for 9 days. Confocal imaging was analyzed using ImageJ® and Arivis Vision4D®. Unpaired t-tests were performed for statistical analyses (p < 0.05).

Results: At baseline, Prom1-Gfp mice expression was localized mostly to PBGs. After BDL, GFP+ cell lineage replaced nearly the entire EHBD lumen (Figure). A similar pattern was observed in RRV-mediated BA. PBGs of RRV-treated KO mice were larger than WT PBGs (1484.41156 vs. 126.186.3μm2, p=0.002). After 9 days in vitro, KO EHBD organoids were larger than WT (0.300.87 vs. 0.090.20 mm3, p=0.02), similar to in vivo findings. All organoids were mG+ (green), not mT+ (red), suggesting that only HPCs gave rise to viable organoids, even with Prom1 KO.

Conclusion: PROM1+ HPCs in PBG’s give rise to EHBD cholangiocytes after BDL and RRV-mediated BA. Larger PBGs in RRV KO mice suggests a cholangiocyte differentiation defect, paralleled in vitro with larger organoids. We conclude that PROM1 HPCs promote EHBD epithelial repair; this process may be disrupted in BA.