[Differentiation of human embryonic stem cells into hepatocyte-like cells in an adherent culture system with single-step induction].
摘要
OBJECTIVE: To establish a new induction method from human embryonic stem cells (hESCs) differentiating into hepatocyte-like 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 Periodic acid-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-like 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 a fetoprotein (AFP) and al antitrypsin 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 positive 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 supplied with cytokines consequently, hESCs can be induced to differentiate into hepatocyte-like cells. The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.