Fig. 3: Promotion of hepatocyte differentiation in hiHepPC aggregates.

a Representative morphologies of hiHepPC aggregates at days 1, 7, 14, 21, and 28 after initiation of 3D culture. b Representative images of hematoxylin and eosin (HE)-stained hiHepPC aggregates at day 7 after initiation of 3D culture. c Co-immunofluorescence staining of ALB with E-CAD and of CYP3A4 with MRP2 were conducted for hiHepPC aggregates at day 7 after initiation of 3D culture. DNA was stained with DAPI. d Representative bright-field and fluorescence images of a living hiHepPC aggregate at day 7 after initiation of 3D culture stained with 5- and 6-carboxy-2′,7′-dichlorofluorescein diacetate (carboxy-DCFDA). Green fluorescence shows functional MRP2 transporter activity in hiHepPC aggregates. e PCA was performed using CEL-seq2 data for HUVECs, hiHepPCs in monolayer and cell aggregation cultures, and human hepatocytes. f GSEA of CEL-seq2 data for hiHepPC aggregates and HUVECs and of those for hiHepPC aggregates and hiHepPC monolayer cultures were performed using the set of top 100 genes specifically upregulated in human hepatocytes. g GOEA was performed for genes with expression levels higher in hiHepPC aggregates than in hiHepPC monolayer cultures (left graph). The liver-enriched genes shown in the left graph were related to hepatic functions associated with xenobiotic metabolism, drug metabolism, and lipid metabolism (right graph). h, i CYP3A4 and CYP2C9 activities (h) and the amounts of ALB and urea in the culture media (i) were measured after culture of HUVECs, two different hiHepPCs, cell aggregates derived from these hiHepPCs, and human hepatocytes. All data shown in h were normalized with the values for hepatocytes in culture without rifampicin, and the fold differences are shown. j The viability of hiHepPCs in monolayer and cell aggregation cultures and primary human hepatocytes was measured after culture of cells with the indicated hepatotoxins. Statistical difference was determined by one-way analysis of variance followed by Tukey–Kramer test. Data represent the mean ± SD (n = 3 (h, i) or n = 4 (j) independent assays). Scale bars, 50 µm. Source data are provided as a Source Data file.