Figure 2.

(a) Stage-specific protein expressions of hiPSC-EBs during the differentiation process. SOX17 and FOXA2 for the definitive endoderm stage; HHEX and GATA4 for the foregut endoderm; AFP and HNF-4α for the hepatic progenitor cells; ALBUMIN and CK-18 for the mature HLCs. DAPI stains for cell nuclei. Scale bar 100 μm. (b) Stage-specific gene expression analysis by Real-Time PCR. The relative quantities of stage-specific genes were measured at the mRNA level to follow the progression of the differentiation process. Sox17 as the definitive endoderm marker; Gata4 as the foregut endoderm marker; HNF-4α as the hepatic progenitor cells marker; Albumin was used to determine the final maturation for the hepatocyte-like cells (HLCs). Undifferentiated cells were used as negative control. (c) quantitative RT-PCR displayed the presence of mRNA for AFP, five P450 isoforms (Cyp3A4, Cyp2C9, Cyp3A7, Cyp1B1, and Cyp2B6), Albumin, and CK18 in the terminally differentiated hiPSC-EB-HLCs with and without inhibitors. Gene expression for the condition with inhibitors was greater compared with the one without inhibitors for any gene tested; (d,e) Following the differentiation program, terminally differentiated hiPSC-EB-HLCs expressed mature hepatocyte-specific markers, as evidenced by co-staining of ALBUMIN and HNF-1α, and ALBUMIN and C-MET. Scale bar 100 μm. (f) FACS analysis for albumin positive cells showed a higher percentage of albumin producing cells in the condition with inhibitors compared with the one without inhibitors (80% vs 68%).