Objective To establ ish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-l ike cells using an adherent culture system with single-step induction. Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days, hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days. Then the induction medium was replaced by hepatocyte induction medium (HIM) supplemented with fibroblast growth factor 1 (FGF-1) and bone morphogenetic protein 4 (BMP-4) for another 6 days. Finally, the cells were treated with HIM adding hepatocyte growth factor (HGF) and Oncostatin M (OSM) for 5-7 days. The characteristics of differentiated cells were determined by morphology, immunofluorescence staining, RT-PCR, and Periodicacid- Schiff (PAS) test. Results Differentiated cells treated with Activin A, FGF-1, BMP-4, HGF, and OSM sequentially were morphologically larger and became spherical, oval or polygon. Some cells had 2 or 3 nuclei, suggesting that the cells have a hepatocyte-l ike morphology. Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead (FOX)A2 after induction by Activin A. Then they turned to be α fetoprotein (AFP) and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4. Finally, the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection. The differentiated cells at various stages also expressed at early (SOX17, FOXA2, and GATA-4),middle (AFP, albumin, and cytokeratin 18), and mature (alcohol dehydrogenase 1C and Cytochrome P4501B1) stage hepatic genes, respectively. Conclusion Using a simple-step induction method and by suppl ied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-l ike cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.
