[Differentiation of human embryonic stem cells into hepatocyte-like cells in an adherent culture system with single-step induction].
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.


CONCLUSIONS
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.
Hu
Zhixing
Z
Department of Pharmacology, Kunming Medical University, Kunming Yunnan, 650031, P R China.
Luo
Min
M
Liang
Daoming
D
D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology*; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D023822:Tissue Engineering / Q000379:methods