Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis

Portal mesenchymal cells represent a very small amount of cells. They are tightly bound to the bile duct and portal vascular structures, relying on basement membranes. In the present study, we set up a specific procedure to isolate portal mesenchymal cells, for scRNAseq analysis. We identified 16 distinct cell clusters, including fibroblasts (5 clusters), vascular smooth muscle cells (VSCMs, 5 clusters), endothelial cells (4 clusters), HSCs (1 cluster) and mesothelial cells (1 cluster). First, we isolated fragments of the bilio-vascular tree from mouse liver. Next, bilio-vascular fragments were submitted to enzymatic digestion, resulting in a single-cell suspension. The cell suspension was depleted in cells expressing the lineage (Lin) markers of cholangiocytes (EpCAM), endothelial cells (CD31), and hematopoietic cells (CD45 and CD11b) by means of cell sorting. Lin-negative single-cell suspension was processed to generate a scRNAseq cDNA library using the 10x Genomics technology.