Fig. 7: EpCAM⁺Gli1⁺ organoids differentiate into functional hepatocytes and efficiently repopulate FRG liver after transplantation.

a Schematic illustration of the experimental design. b RT-qPCR analysis of Krt19, EpCAM, Alb, and HNF4α in expansion medium (EM) or differentiation medium (DM). Data are represented as means ± SEM (n = 3). c Immunofluorescence analysis of the hepatocyte marker genes Alb (green) or HNF4α (red) in DM. Scale bar, 50 μm. d Glycogen accumulation was determined by periodic acid-Schiff (PAS) staining in organoids grown in EM or DM for 12 days. Scale bar, 50 μm. e LDL uptake was analyzed using an LDL fluorescent substrate (red) in organoids that were maintained in EM or DM for 12 days. Scale bar, 50 μm. f Albumin secretion in EM or DM for 12 days. Data are shown as means ± SEM (n = 3). g Measurement of the cytochrome activity of CYP3A4 in EM or DM for 12 days. Relative light units (RLUs) per mL per million cells are shown. Data are presented as means ± SEM (n = 3). h Immunofluorescence staining for FAH (green) and tdTomato (red) showing the engraftment of FAH⁺ hepatocytes in FRG recipient livers at 3 months post transplantation. Scale bar, 10 μm. i, j Serum levels of ALT and AST were determined at 3 months post transplantation. Data are shown as means ± SEM (n = 3). k Immunofluorescence staining for tdTomato (red) and HNF4α (green), KRT19 (green), or EpCAM (green) at 3 months after transplantation. Scale bar, 50 μm.