Fig. 1

Derivation of cultured liver progenitor cells from the hepatic parenchymal and non-parenchymal cells. a Representative immunofluorescence staining of α-SMA, CK19, SOX9, and CD24 in human liver fibrotic tissues. Scale bar: 200 μm. b Liver non-parenchymal cells (NPCs) were isolated from mouse livers after 4 CCl₄ injections and subjected to scRNA-Seq. U-map visualization of CD24⁺ cell clusters is based on 1038 single-cell transcriptomes and bubble plots show the expression levels of Foxa2, Sox9, Ck19, Hnf1b, ASS1, and Alb. The color bar indicates the expression level of scaled genes. c Representative immunofluorescence staining of CD24⁺tdTom⁻ liver progenitor cells in mice treated with CCl₄. ROSA26^tdTomato mice were infected with AAV8-Tbg-Cre and treated with 2 mL/kg CCl₄ twice a week for a continuous period of 6 weeks. Scale bar: 100 μm. d Schema model and representative images for isolation and culture of LPCs. ALB/GFP⁻EpCAM⁻ cells were isolated from Alb^CreERT/R26^GFP mice by FACS and cultured in TEM (resident LPCs-derived LPCs). Mechanically abraded epithelial cells from wild-type intrahepatic bile ducts were FACS-sorted and EpCAM⁺ cells were cultured in TEM (BECs-derived LPCs). Control tdTom⁺ hepatocytes from ROSA26^tdTomato mice with AAV8-Tbg-Cre were cultured in TEM (Hepatocytes-derived LPCs). Scale bar: 200 μm. Representative immunofluorescence staining of Ki67 (Green) in three cultured LPCs at passage 5. Scale bar: 100 μm. e Proliferation measurements of progenitor-like cells in TEM calculated from CCK-8 assay at passage 5. f Progenitor-like cell doubling time at passage 5, measured by cell counting. g Representative immunofluorescence staining of SOX9 (Green) in three cultured LPCs at passage 5. Scale bar: 100 μm. h Quantification of CD44 and CD24 positive cells among three cultured LPCs at passage 5, assessed by flow cytometry. Red peaks represent staining samples and blue peaks represent the isotype control. i Heat map of three cultured LPCs of gene expression levels compared with HCs, BECs, HSCs, LSECs, and KCs. n = 2 independent experiments. Resident LPCs-derived LPCs, reLPCs; Hepatocytes-derived LPCs, HepLPCs; BECs-derived LPCs, BecLPCs; HC, hepatocyte; BEC, biliary epithelial cell; LSEC, liver sinusoidal endothelial cell; HSC, hepatic stellate cell; KCs, Kupffer cells. For panels e and f, data summarize 3 independent experiments. Data are expressed as means ± SD