Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocytelike morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.H epatitis B, one of the major infectious diseases worldwide, is caused by a small enveloped DNA virus, the hepatitis B virus (HBV). HBV exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells. It has therefore been assumed that susceptibility to HBV infection is restricted to differentiated cells. Accordingly, it was found that only human hepatocyte primary cultures were susceptible to HBV infection (1-4). However, the use of this model is hampered by the limited availability and the inherent variability of human liver material. Even though several human hepatoma-derived cell lines support HBV replication after HBV DNA transfection (5-9), none of them are susceptible to HBV infection.We describe here a hepatoma-derived cell line that expresses a representative panel of liver-specific genes and is susceptible to HBV infection. This goal was achieved by combining an original selection procedure applied early after the cell line establishment in culture and the use of appropriate culture conditions, allowing the commitment of these cells to an optimal differentiation status. MethodsIsolation of the Cell Line and Culture Conditions. Cells were isolated from a liver tumor of a female patient suffering from hepatocarcinoma and hepatitis C infection. All experimental procedures were conducted in conformity with French laws and regulations and were approved by the National Ethics Committee. The samples were minced into small pieces, washed with Hepes buffer (pH 7.7; 140 mM NaCl͞2.68 mM KCl͞0.2 mM Na 2 HPO 4 ͞10 mM Hepes), and digested with 0.025% collagenase D (Boehringer Mannheim) diluted in the same buffer supplemented with 0.075% CaCl 2 under gentle stirring at 37°C. The cell suspension was washed twice in Hepes buffer and resuspended in a William's E medium supplemented with 10% FCS, 100 units͞ml penicillin, 100 g͞ml streptomycin, 5 g͞ml insulin, and 5 ϫ 10 Ϫ7 M hydrocortisone hemisuccinate. Cell suspension was distributed in several dishes without any coating feeder layer. After several weeks, cell growth was sufficient to fulfill the culture dishes. Cells appeared well differentiated, with a hepatocyte-like ...

ABSTRACT:Most human hepatocyte cell lines lack a substantial set of liverspecific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor ␣), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

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Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) H epatic tumors with combined hepatocellular cholangiocarcinoma have been frequently described for instance, hepatoblastoma with cholangioblastic features in young patients 1 and HCCs with dual expression of hepatocyte and bile duct markers in adult patients suffering from diseases related to HCV and/or HBV infection. 2 Such tumors usually contain mature hepatocytes and so-called transitional areas that consist of undifferentiated cells that have morphological and immunological features of both hepatocytes and cholangiocytes. 3 Co-expression of hepatocytic and biliary markers suggests involvement of hepatic progenitor cells in development of these human tumors and supports the concept of genetic events to explain their abnormal growth during tumor formation. 4 However, mechanisms of occurrence of these progenitors and abnormal control of their expansion and differentiation are still unclear.Hepatic progenitor cells, also referred to as oval cells in rodents, have been defined as immature epithelial cells able to differentiate toward both biliary and hepatocytic lineages. The smallest ramification of the biliary tree in adult liver, the canal of Hering, may constitute the niche for these hepatic progenitor cells. 5 They are few in number, and because bile ductular and hepatocytic cells have a tremendous capacity to proliferate and differentiate, heAbbreviations: BrdU, bromodeoxyuridine; HNF, hepatocyte nuclear factor; RT, reverse transcription.

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G 1 phase of hepatocytes from regenerating liver but not from shamoperated control animals. One occurred in early G 1 (30 min to 4 h), and the other occurred in mid-late G 1 , peaking at around 10.5 h. Interestingly, the mid-late G 1 activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G 1 /S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G 1 MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12-and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G 1 MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G 1 phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

Several hepatocyte mitogens have been identified, but the signals triggering the G0/G1 transition and cell cycle progression of hepatocytes remain unknown. Using hepatocyte primary cultures, we investigated the role of epidermal growth factor/pyruvate during the entry into and progression through the G1 phase and analyzed the expression of cell cycle markers. We show that the G0/G1 transition occurs during hepatocyte isolation as evidenced by the expression of early genes such as c-fos, c-jun, and c-myc. In culture, hepatocytes progress through G1 regardless of growth factor stimulation until a restriction point (R point) in mid-late G1 beyond which they cannot complete the cell cycle without mitogenic stimulation. Changes in cell cycle gene expression were associated with progression in G1; the cyclin E mRNA level is low early in G1 but increases at the G1/S boundary, while the protein is constantly detected during cell cycle but undergoes a change of electrophoretic mobility in mid-late G1 after the R point. In addition, a drastic induction of cyclin D1 mRNA and protein, and to a lesser extent of cyclin D2 mRNA, takes place in mitogen-stimulated cells after the R point. In contrast, cyclin D3 mRNA appears early in G1, remains constant in stimulated cells, but accumulates in unstimulated arrested cells, paralleling the cyclin-dependent kinase 4 mRNA expression. These results characterize the different steps of G1 phase in hepatocytes.

Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features which are found in more than 80% hepatocellular carcinomas (HCC). Activated hepatic stellate cells (HSC) play a critical role in fibrogenesis associated with HCC onset and progression, yet their functional impact on hepatocyte fate remains largely unexplored. Here, we used a coculture model to investigate the crosstalk between hepatocytes (human hepatoma cells) and activated human HSC. Unsupervised genome-wide expression profiling demonstrated that hepatocyte-HSC crosstalk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of pro-inflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC crosstalk also generated a permissive pro-angiogenic microenvironment, particularly by inducing VEGFA and MMP9 expression in HSC. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC crosstalk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCC. Interestingly, the effects of crosstalk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings therefore indicate that the crosstalk between hepatoma cells and activated HSC is an important feature of HCC progression, which may be targeted by epigenetic modulation.

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