Extended Data Fig. 6: Human portal fibroblasts from patient donors expand in vitro while retaining the expression profile of in vivo portal fibroblasts.
From: Human assembloids recapitulate periportal liver tissue in vitro
a, Mesenchymal cell populations in human healthy and cirrhotic livers from the publicly available scRNAseq data from Ramachandran et al.8. The dataset was explored for the expression of specific portal fibroblast markers in human healthy liver using the interactive site https://shiny.igc.ed.ac.uk/livercellatlas/ provided by the authors. tSNE map (left) and corresponding violin plots (right) depict THY1 (CD90) transcript distribution and expression levels across mesenchymal subpopulations. b, FACS gating strategy for isolating PFs from human liver tissue. Immune and endothelial cells are excluded through negative gating for CD31, CD45, and CD11b markers. hPFs are isolated based on EpCAMneg/THY1pos sorting. c, Immunofluorescence staining of human liver tissue reveals the distribution of different liver cell types (CD90, PF marker, magenta; pan cytokeratin, PanCK, bile duct marker, green). Membranes are marked with phalloidin (white) nuclei with DAPI (blue). Dotted boxes, magnifications showing midzonal tissue parenchyma (1) and liver portal tract (2) region. Note that CD90 is exclusively expressed in the portal region. n = 3 independent experiments. Scale bar, 50 µm. d, Representative images of PFs monoculture, cultured in Basal medium, Basal medium with 3% FBS and Wnts, Basal medium with 3% FBS and DMEM with 20% FBS. n = 2 independent experiments. Scale bar, 100 µm. e, RT-qPCR gene expression analysis for portal fibroblast (THY1, PDGFRA, MFAP4, COL1A1, COL3A1), HSC (RGS5, LRAT), cholangiocytes (EPCAM) and VSMC (ACTA2, MYH11) markers in cultured PDGFRA+CD90+ cells at passage 1 and 3 (P1-P3) and human liver tissue (h-liver). Graph presents mean ± SD (n = 3). Values are presented relative to the house-keeping gene HPRT. f, Heatmap showing the expression of hepatocyte, cholangiocyte and mesenchymal markers from the indicated subpopulations (PFs, HSC, mesothelia, VSMC) in cultured portal fibroblasts (PF, magenta) and fresh isolated hepatocytes (primary, brown) and cholangiocytes (h-Chol, green) from different donors. PF, portal fibroblast; HSC, hepatic stellate cell; VSMC, vascular smooth muscle cell. Column, donor. g, Gene expression levels for the indicated secreted molecules in cultured PDGFRA+CD90+ cells and h-liver analyzed by RT-qPCR. Data are expressed as mean ± SD (n = 3). h, Heatmap of all differentially expressed genes in cultured hPFs and freshly isolated hepatocytes (PHH). Note that genes showing expression of mesenchymal markers are highly expressed in the PF population. i, Immunofluorescence staining for the portal fibroblast marker, CD90 (yellow) and the pan-mesenchymal marker, Vimentin (magenta) in cultured portal fibroblasts, n = 3 independent experiments in n = 1 donor. Note that all the cells are positive for the PF marker, CD90. Scale bar, 50 µm. j, To identify the PFs and cholangiocyte cells after assembly in periportal assembloids, PFs and human cholangiocytes were isolated from human donors (matching whenever possible) and cultured as described in Methods. After several passages, the cells were infected with lentiviral particles containing nuclear-GFP or nuclear-RFP to generate reporter fibroblast lines. Left, schematic diagram of viral infection for h-CholOrg (top) and hPFs (bottom). Representative images of h-CholOrg (top) and PFs (bottom) following transduction with specific nuclear-GFP or nuclear-RFP viral vectors. n ≥ 3 independent experiments. Scale bars, 100 µm. Panel j created in BioRender. Yuan, L. (2025) https://BioRender.com/g8uci15.
