Fig 2: Differentiation of hESCs into HLCs.a The relative hepatocyte (ALB, AAT, CK18, and ASGPR1), cholangiocyte (SOX9), and hepatoblast (AFP) gene expression levels of day 19 differentiated cells with different treatments (groups A and B) were determined by qPCR. HGF (hepatocyte growth factor, 20 ng/mL); OSM (oncostatin M, 20 ng/mL); Dex (dexamethasone, 10 µM); SB431542 (TGFß inhibitor, 2 µM); and RO4929097 (Notch inhibitor, 1 µM). b Immunofluorescence analysis of ALB, AAT, ASGPR1, and CK18 expression in group B-induced differentiated cells on day 19. c The expression levels of ALB and ASGPR1 in group B-induced differentiated cells were determined by flow cytometry on day 19. Isotype control antibodies were used as controls. d The relative hepatocyte (ALB, AAT, CK18, ASGPR1, and AFP) gene expression levels of differentiated HLCs (group B) compared with those of hESCs and primary human hepatocytes (PHHs) were determined by qPCR. e Albumin secretion of HLCs (black line) on days 12, 14, 16, 18, and 20 and PHHs (dotted line) on day 2 were determined by ELISA. *p < 0.05, **p < 0.01; data are represented as the mean ± SD. Scale bar, 50 µm.

Fig 3: Differentiation of hESCs into hepatoblasts in defined xeno-free conditions.a The relative hepatoblast gene (HNF4a and AFP) expression levels of cells differentiated with a previously reported protocol were determined by qPCR at days 6, 7, 8, 9, and 10. b Four protocols were used to induce PE to hepatoblasts. Trf (transferrin, 5 mg/mL); Vc-Mg (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 50 µg/L); Ins (insulin, 10 µg/mL); SS (sodium selenite, 0.1 ng/mL); and BMP4 (10 ng/mL); bFGF (10 ng/mL). c The relative hepatoblast gene (HNF4a and AFP) expression of day 9 differentiated cells in basal medium (DMEM/F12, 1 × L-GlutaMAX, and 1 × NEAA) with different treatments (groups A, B, C, and D) were determined by qPCR. d The relative hepatocyte gene (ALB, AAT, and CK18) expression of day 19 differentiated cells in basal medium (DMEM/F12, 1 × L-GlutaMAX, and 1 × NEAA) with different treatments (groups A, B, C, and D) were determined by qPCR. e Immunofluorescence analysis of HNF4a and AFP expression in group A-induced differentiated cells on day 9. f The expression levels of HNF4a and AFP in group A-induced differentiated cells were determined by flow cytometry on day 9. Isotype control antibodies were used as controls. *p < 0.05, **p < 0.01; data are represented as the mean ± SD. Scale bar, 100 µm.

Fig 4: Hepatic differentiation of hMSG-EpiPCs in vitro. (a) Picture of induced hMSG-EpiPCs taken on day 4, 8, 12 and 16 from left to right. (b) Indocyanine green (ICG) uptake assay on day 16 of hepatic induction and the control group. (c) Periodic acid-Schiff (PAS) staining on day 16 of hepatic induction and the control group. (d) Immunostaining on day 16 of hepatic induced and uninduced cells for ALB, CK18 and CYP3A4 from left to right. (e) CYP450 activity analysis on day 16 of hepatic induction for CYP3A4 and CYP2C19. (f) Quantitative real-time PCR analysis of relative mRNA expression of hMSG-EpiPCs and induced hepatic cells on day 16. (***P < 0.01; n ? 3).

Fig 5: Hepatic differentiation of hMSG-EpiPCs in vivo. (a) H&E staining of mice liver section on day 1 and 7 post PBS and hMSG-EpiPCs injection (White arrows: erythrocyte diapedesis, yellow arrows: diseased hepatocytes). (b) Immunostaining 1, 2, 3 and 4 weeks after hMSG-EpiPCs transplantation from left to right, green fluorescence signal for AFP, ALB, CK18, CK19 and CYP3A4 from top to bottom, and red for CM-Dil labeling.