The staining of these cells by CK suggested a similar phenotype as that of the neighboring epithelial cells of the duct mucosa, but the unique anatomical organization raised the possibility that they may display other distinct cellular phenotypes. Based on the CK-19+ staining detected in individual

PBG cells, we first explored whether they express the primary cilium of mature Acalabrutinib cholangiocytes. Confocal images showed CK-19+ PBG cells also expressing α-tubulin similarly to CK-19+ cells in the epithelium (Fig. 4A,B). α-tubulin (staining the cholangiocyte cilium) is expressed in most, but not all, PBG cells, as demonstrated by a detailed survey of several EHBDs by serial confocal sections (data not shown). Based on these findings and on previous work reporting the existence of cells producing mucin or expressing other cell markers in PBGs,[9,

12] we stained bile duct sections using PAS and Alcian blue. Both stains produced similar signals in some, but not all, PBG cells, but no signal was noted in the duct epithelium (Fig. 4C,D). To examine a different type of secretory function, we performed dual staining with CK-19 and CgA, which marks neuroendocrine cells, and found that a small population of PBG cells expresses both markers (Fig. 4E). A similar staining pattern for all three assays was present in cells of the peribiliary network (data not shown). Given that PBGs have been proposed to be a niche for multipotent stem cells within the bile duct and the well-described role of the transcription factors, Sox17 and Pdx1, RG7204 in vitro in differentiation of the extrahepatic biliary tree from the endoderm,[8, 18] we determined the expression of both of these transcription factors in PBGs along the entire anatomy of gallbladder and EHBDs. Sox17 was expressed predominantly in the gallbladder (61%-82% of the epithelial cells of

the gallbladder, depending on age) and less this website frequently in the cystic duct (3%-15% of epithelial cells and 12%-30% of PBG cells) and in the CBD (<10% of epithelial and PBG cells; Figs. 5A and Fig. 6A-C). In contrast, epithelial cells of the gallbladder rarely expressed Pdx1 (<5%), but Pdx1 was expressed in ∼50% of epithelial cells and ∼75% of PBG cells of both the cystic duct and CBD (Figs. 5A and 6A-C). To identify cells with a biliopancreatic progenitor phenotype, we also quantified cells that are double stained for Sox17 and Pdx1 (Sox17+/Pdx1+) in all three segments of EHBDs. We found that Sox17+/Pdx1+ cells were rare in the gallbladder and represented <20% of epithelial and PBG cells of the cystic duct and CBD (Fig. 6A-C and Supporting Fig. 2). Sox17 and Pdx1 signals were also detected in CK-19+ cells of the peribiliary network (data not shown).