Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Abstract

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.

Critical selection of reliable reference genes for gene expression study in the HepaRG cell line

“…As such, the S18 rRNA gene has been used in most studies in which the HepaRG cell line is involved [7][8][9][10][11][12][13]. Next to human primary hepatocytes, this hepatocyte model seems to be a very suitable tool for in vitro toxicity testing of xenobiotics and even has some additional advantages [5,6].…”

“…Expression of a plethora of markers/functions has been 4 evaluated both at the transcriptional and the translational level. With respect to the former, reverse transcription quantitative PCR (RT-qPCR) assays were generally adopted often using the S18 rRNA gene as single reference gene [7][8][9][10][11][12][13].…”

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…As PML expression is frequently deregulated in human tumour cell lines and/ or during oncogenesis Gurrieri et al, 2004;Scaglioni et al, 2006), we analysed several human tumour and pseudoprimary cell lines for endogenous PML expression (Supplementary Figure 1). Consequently, we decided to use human hepatocytic cells (HepaRG) (Gripon et al, 2002;Cerec et al, 2007), as they express PML in the well-defined physiological amounts (Condemine et al, 2006) and represent a suitable system to analyse endogenously PML components during viral infection Lukashchuk and Everett, 2010). Cells were infected with wild-type virus (H5pg4100), E1B-55K null mutant H5pm4149 (Kindsmu¨ller et al, 2009) or previously described H5pm4102/H5pm4101 virus harbouring mutations within the SCM (K104R)/NES (L83/87/91A) of E1B-55K (Kindsmu¨ller et al, 2007).…”

“…HepaRG (Gripon et al, 2002;Cerec et al, 2007), MIO-M1 (Limb et al, 2002;Lawrence et al, 2007), A549 (DSMZ ACC 107; Braunschweig, Germany), H1299 (Mitsudomi et al, 1992), HEK293 (Graham et al, 1977), U2OS (Ponten and Saksela, 1967) and primary BRK-derived cell lines (AB120, AB115) (Endter et al, 2005) were grown in DMEM supplemented with 10% FCS, 100 U of penicillin, 100 mg of streptomycin per ml in a 5% CO 2 atmosphere at 37 1C. For HepaRG cells, the medium was additionally supplemented with 5 mg/ml of bovine insulin and 0.5 mM of hydrocortisone.…”

Pancreatic carcinoma development: new etiological and pathogenetic evidence

“…41,42 A wide series of induction programs have been proposed, on the basis of different experimental conditions and objectives. In particular, upon treatment with dexamethasone and oncostatin M, cellular lines from a pancreatic origin were able to trans-differentiate into permissive hepatocyte-like cells that were able to sustain HBV replication with production of viral antigens, replicative forms and entire genomes, when they were permanently transfected with HBV-DNA.…”