Multi-gene biomarkers reveal spatial organization and subpopulation-specific damage response in biliary epithelial cells.

Biliary epithelial cell (BEC) heterogeneity remains challenging to define. Here, we performed scRNA-seq on Sox9EGFP+ liver epithelium from mice subjected to bile duct ligation (BDL) and sham controls. We identified 4 BEC subpopulations in sham controls, each associated with specific enrichment of a minimal, 1-2 gene biomarker ("metagene"). Metagenes were validated by hybridization chain reaction (HCR) FISH, which also facilitated spatial analysis of intrahepatic bile ducts. BECs belonging to a given subpopulation were most likely to have neighbors of the same identity, forming homogenous cellular compartments within otherwise heterogeneous ducts. Subpopulation specific differential gene expression (DGE) analysis revealed that BDL decreases transcriptomic heterogeneity among BECs by suppressing subpopulation specific genes. BDL samples also included a proliferative subpopulation not found in controls, which contained populations enriched for three of the four metagenes. All BEC subpopulations were observed in vitro in monolayer cultures, where they clustered spatially with BECs of the same subtype. Together, our data demonstrate that metagene biomarkers facilitate spatial localization of BEC subpopulations in situ, identify subpopulation specific injury responses, and confirm that BEC heterogeneity is preserved in vitro.