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ABSTRACT: The correct functioning of hepatocytes requires the establishment and maintenance of hepatocyte polarity. However, the mechanisms regulating the generation of hepatocyte polarity are not completely understood. The differentiation of human foetal hepatic progenitor cells (hFHPCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing hepatocyte development. In this study, we used a two-stage differentiation protocol to generate functional polarised hepatocyte-like cells (HLCs) from hFHPCs. Global gene expression profiling was performed on triplicate samples of hFHPCs, immature-HLCs and mature-HLCs. When the differential gene expression was compared based on the differentiation stage, a number of genes were identified that might be essential to establishing and maintaining hepatocyte polarity. These genes include those that encode actin filament-binding protein, protein tyrosine kinase activity molecules, and components of signalling pathways, such as PTK7, PARD3, PRKCI, and CDC42. Based on known and predicted protein-protein interactions, the candidate genes were assigned to networks and clustered into functional categories. The expression of several of these genes was confirmed using real-time RT-PCR. By inactivating genes using small interfering RNA, we demonstrated that PTK7 and PARD3 promote hepatic polarity formation and affect F-actin organization. These results provide unique insight into the complex process of polarisation during hepatocyte differentiation, indicating key genes and signalling molecules governing hepatocyte differentiation.
Journal of Cell Science 09/2012; · 6.11 Impact Factor
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ABSTRACT: Differentiation of human hepatic progenitor cells to functional hepatocytes holds great potential to develop new therapeutic strategies for liver disease and to provide a platform for drug toxicity screens and identification of novel pharmaceuticals. We report here that human fetal hepatic progenitor cells (hFHPCs) efficiently differentiate to hepatocyte-like cells by continuous exposure to a combination of soluble factors for 7 days in vitro. We compared the effect of hepatocyte growth factor (HGF), oncostatin M (OSM), dexamethasone (DEX), or a combination on the expression of a liver-specific marker, albumin (ALB). Real-time RT-PCR analysis showed that, upon exposure to a combination of OSM, DEX, and HGF, the expression of ALB gradually increased in a time-dependent manner. In contrast, the level of the hepatic progenitor cell marker alpha-fetoprotein (AFP) decreased as differentiation progressed. Moreover, cells exposed to the combination of OSM, DEX, and HGF gradually featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and cytochrome P450 (CYP) activity. The effect of these factors on the differentiation of hFHPCs may be blocked by U0126, an inhibitor of the ERK1/2 signaling pathway. In conclusion, we demonstrate that a combination of soluble factors facilitates the efficient generation of highly differentiated hepatocyte-like cells from hFHPCs and ERK1/2 signaling pathway involved in this process. Results suggest that this system will be useful for generating functional hepatocytes and, hence, may serve as a cell source suitable for preclinical pharmacological research and testing.
Journal of Cellular Physiology 07/2011; 227(5):2051-8. · 3.87 Impact Factor
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ABSTRACT: Hepatoblasts, which are considered one type of hepatic progenitor cell, reside in the fetal liver. To selectively identify these cells, we transfected primary cultured human fetal liver cells (FLCs) with a pGL3 vector bearing the gene for the enhanced green fluorescence protein (EGFP) under the control of the alpha-fetoprotein (AFP) promoter expressed in hepatoblasts. The FLCs were then sorted by fluorescence-activated cell sorting (FACS) on the basis of AFP promoter-driven EGFP expression. The EGFP-positive cells expressed AFP, albumin, and cytokeratin 19, and could be expanded in vitro. Thus, the AFP promoter-EGFP reporter system is highly useful for identification and isolation of hepatic progenitor cells.
Histochemie 06/2008; 130(2):375-85. · 2.59 Impact Factor
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ABSTRACT: The aim of this study was to investigate the effect of arsenic trioxide (ATO) on the growth of cultured uveal melanoma cells (OCM-1 cell line).
Cultured OCM-1 cells were treated with 0.75-24 microM of ATO for 4-96 h. Cell viability was evaluated with the methylthiazoletetrazolium (MTT) assay as proliferation test. Apoptotic and necrotic cells were quantified using flow cytometry following Annexin-V/PI double stain. The cell morphology alteration was examined by light and electron transmission microscopy. To investigate the underlying mechanism of ATO-induced apoptosis and necrosis, glutathione peroxidase activity was measured, and mitochondrial membrane potentials were quantified using confocal microscopy.
In the MTT assays, OCM-1 cell growth was inhibited at ATO concentrations of 1.5-24 microM. The 50% inhibitory concentration of ATO was 16.8 microM. At ATO concentrations of 12 and 24 microM, apoptosis and necrosis were induced after 24 h of incubation as shown by light and transmission electron microscopy. At ATO concentrations of 12 and 24 microM, the glutathione peroxidase activity was significantly (p < 0.05) reduced after 24 h of incubation, and the mitochondrial membrane potentials were significantly (p < 0.01) decreased after 4, 12 and 24 h of incubation.
ATO inhibited ocular melanoma cell line growth at concentrations of 1.5-24 microM of ATO in a dose- and time-dependent manner by inducing apoptosis and necrosis of the tumor cells. The concurringly decreased glutathione peroxidase activity and the reduced mitochondrial membrane potentials may be possible underlying mechanisms for the apoptosis and necrosis of the tumor cells.
Ophthalmic Research 01/2007; 39(6):302-7. · 1.56 Impact Factor
