Fig. 5: Differentiation of expandable G-heporgs into more mature hepatocyte organoid.

A Schematic illustration of the differentiation of G-heporgs treated with GA017 into more mature hepatocyte organoids. Scale bar = 100 μm. B Dynamic gene expression of mature hepatocyte genes was analyzed by RT-qPCR at different stages and PHHs. n = 4 biologically independent experiments. C Representative Factin staining image of G-heporgs in expansion and maturation medium (12 days post switch). Red arrows indicated binucleated hepatocytes. Scale bar = 50 μm. D, E The quantification of cellular features of G-heporgs in expansion and maturation medium based on Factin (phalloidin) staining, including cell area (n = 30 cells) (D) and nucleus-to-cytoplasm ratio (n = 30 cells) (E). F Immunostaining of ALB, Factin, ZO1, Ki67, and E-cad. Nuclei were stained with DAPI. Scale bar = 50 μm. G Representative images of ICG uptake and excretion. Scale bar = 100 μm. H The expression of CYPs after treating with inducers was analyzed by RT-qPCR. n = 4 biologically independent experiments. Analysis for the secretion of ALB (n = 4 biologically independent experiments) (I) and the production of urea (n = 3 biologically independent experiments) (J). K Heatmap of G-heporgs in expansion and maturation medium for genes related to proliferation, mature, drug metabolism, and polarization. L GSEA analysis. M KEGG analysis. Results were presented as mean ± SD. Statistical significance was determined using one‑way ANOVA followed by Tukey post‑test and unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.