Fig. 5: Single-cell transcriptome analysis demonstrates the PV-BD interactions in BVLO.

A Single cell RNA-seq analysis of dissociated BVLO identified four distinct cell clusters; Hepatocyte cluster contains ALB⁺ cells; Endothelial one contains PECAM-1+ cells; Cholangiocyte cluster contains SOX9⁺, EpCAM⁺, KRT19⁺, HES1⁺, NOTCH1⁺, NOTCH2⁺ cells; Mesenchymal cell cluster contains ACTA2⁺, TAGLN⁺, PDGFRB⁺, JAG1⁺ cells. In addition to these four clusters, part of BVLO component cells were annotated to immature hepatocytes or epithelial cells, which show hybrid phenotypes of cholangiocytes and hepatocytes. B Comparative clustering analysis of BVLO and human adult liver scRNA-seq data from “Human liver Cell Atlas” (GSE124395). This figure highlights the clustering proximity of cholangiocyte and hepatocyte within BVLO and human adult liver. C Heat map visualization of cholangiocyte and hepatocyte markers expressed in BVLO-derived cells compared to those in primary human liver cells highlights the similarities in the corresponding marker expression. D NicheNet analysis suggesting intercellular communication in BVLO. This ligand–target matrixes denote the regulatory potential between 25 ligands in Mesenchymal Cell (MC: upper) or Endothelial cell (EC: lower) and target genes in Org-Cho (the highly expressed genes in Org-Cho against planar cultured HE cells). As highlighted with red boxes, TGFβ1 in ECs and TGFβ1 as well as JAG1 in MCs potentially activate the corresponding downstream targets in cholangiocytes. E Schematic illustration of possible EC and MC contribution to the BVLO signaling pathways that are suggested to be associated with the differentiation of cholangiocyte clusters from HE cells in BVLO and formation of BD.