Anne Corlu

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

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) the reversible differentiation fate of HepaRG cells is related to the absence of p21CIP1 and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of β‐catenin localization and HNF3β expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. Conclusion: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors. (HEPATOLOGY 2007;45:957–967.)

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.

Hepatocyte–Stellate Cell Cross-Talk in the Liver Engenders a Permissive Inflammatory Microenvironment That Drives Progression in Hepatocellular Carcinoma

Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features that 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 cross-talk between hepatocytes (human hepatoma cells) and activated human HSCs. Unsupervised genome-wide expression profiling showed that hepatocyte-HSC cross-talk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of proinflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC cross-talk also generated a permissive proangiogenic microenvironment, particularly by inducing VEGFA and matrix metalloproteinase (MMP)9 expression in HSCs. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC cross-talk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCCs. Interestingly, the effects of cross-talk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings, therefore, indicate that the cross-talk between hepatoma cells and activated HSCs is an important feature of HCC progression, which may be targeted by epigenetic modulation.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

P-glycoprotein induction in rat liver epithelial cells in response to acute 3-methylcholanthrene treatment

Expression of P-glycoprotein (P-gp), a plasma membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in nonparenchymal rat liver epithelial (RLE) cells in response to acute exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). High levels of mdr mRNAs were evidenced by Northern blotting in two independent RLE cell lines after treatment by either 3-methylcholanthrene (MC) or benzo-(a)pyrene. MC-mediated mdr mRNA induction was demonstrated to be dose-dependent; it occurred through enhanced expression of the mdr 1 gene, as indicated by reverse transcriptase-polymerase chain reaction analysis using rat mdr gene-specific primers and paralleled an induction of a 140 kDa P-gp as demonstrated by Western blotting. In addition, MC-induced P-gp appeared to be fully functional because RLE cells exposed to MC displayed enhanced cellular efflux of rhodamine 123, a known P-gp substrate, compared to their untreated counterparts. Analysis of time-course induction revealed that mdr mRNA levels were maximally increased when RLE cells were treated for 48 to 96 hr and returned to low levels after the PAH was removed. In contrast to P-gp, both cytochrome P-450 1A1 and cytochrome P-450 1A2 were not detected after exposure to MC, thus indicating that these liver detoxification pathways are not coordinately regulated with P-gp in RLE cells. In addition, MC-mediated P-gp regulation was not associated with major cellular disturbances such as alteration of protein synthesis and, thereby, differed from the known mdr mRNA induction occurring in response to cycloheximide. Moreover, cotreatment with MC and cycloheximide led to a superinduction of mdr mRNAs, thus suggesting that the effects of the two xenobiotics were, at least partly, additive. In contrast to MC and benzo(a)pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(e)pyrene were unable to increase P-gp expression. These results indicate that some PAHs can act as potent inducers of P-gp in RLE cells and may be interpreted as an adaptive reaction of these cells in lowering cellular accumulation of toxic drugs, including carcinogens transported by P-gp and, therefore, conferring protection on these compounds.

Generation of functional cholangiocyte‐like cells from human pluripotent stem cells and HepaRG cells

Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder‐free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin‐6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG‐derived hepatoblasts. hESC‐ and HepaRG‐derived cholangiocyte‐like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca2+. We demonstrated by integrative genomics that the expression of genes, which signed hESC‐ or HepaRG‐cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte‐like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. Conclusion: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte‐like cells, which display structural and functional similarities to bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches. (Hepatology 2014;60:700–714)

Characterization of Cyclin L1 and L2 Interactions with CDK11 and Splicing Factors INFLUENCE OF CYCLIN L ISOFORMS ON SPLICE SITE SELECTION

Although it has been reported that cyclin L1α and L2α proteins interact with CDK11p110, the nature of the cyclin L transcripts, the formation of complexes between the five cyclin L and the three CDK11 protein isoforms, and the influence of these complexes on splicing have not been thoroughly investigated. Here we report that cyclin L1 and L2 genes generate 14 mRNA variants encoding six cyclin L proteins, one of which has not been described previously. Using cyclin L gene-specific antibodies, we demonstrate expression of multiple endogenous cyclin L proteins in human cell lines and mouse tissues. Moreover, we characterize interactions between CDK11p110, mitosis-specific CDK11p58, and apoptosis-specific CDK11p46 with both cyclin Lα and -β proteins and the co-elution of these proteins following size exclusion chromatography. We further establish that CDK11p110 and associated cyclin Lα/β proteins localize to splicing factor compartments and nucleoplasm and interact with serine/arginine-rich proteins. Importantly, we also determine the effect of CDK11-cyclin L complexes on pre-mRNA splicing. Preincubation of nuclear extracts with purified cyclin Lα and -β isoforms depletes the extract of in vitro splicing activity. Ectopic expression of cyclin L1α, L1β, L2α, or L2β or active CDK11p110 individually enhances intracellular intron splicing activity, whereas expression of CDK11p58/p46 or kinase-dead CDK11p110represses splicing activity. Finally, we demonstrate that expression of cyclins Lα and -β and CDK11p110 strongly and differentially affects alternative splicing in vivo. Together, these data establish that CDK11p110 interacts physically and functionally with cyclin Lα and -β isoforms and SR proteins to regulate splicing.

Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells

Acquisition of drug resistance by cancer cells is attributed to various factors including alterations in apoptotic pathways, enhanced expression of multidrug resistance-associated proteins, altered drug metabolism or uptake and/or overexpression of cytoprotective genes. Thus, potential induction of defence pathways by anticancer drugs might have a marked incidence on cancer cell resistance. 5-Fluorouracil (5-FU) remains the most commonly used anticancer drug for the treatment of colorectal cancer, although objective response rates are as low as 20%. The aim of our study was to investigate the effects of 5-FU on cytoprotective systems in human colon HT-29 cells. Our results demonstrate that 5-FU induced the expression of mRNAs encoding glutathione transferases and antioxidant enzymes. To further determine the mechanisms involved in 5-FU effects, we investigated whether it activates the Nrf2/antioxidant response element pathway which is implicated in the regulation of several genes involved in cytoprotection. Translocation of Nrf2 into the nucleus after 5-FU exposure was demonstrated by immunocytochemistry and western blotting. Using an ARE-driven reporter gene assay, activation of the luciferase activity by 5-FU was also evidenced. Moreover, transfection of HT-29 cells with siRNA directed against Nrf2 inhibited induction of Nrf2 target genes and increased 5-FU cytotoxicity. In conclusion, we demonstrate for the first time that 5-FU activates the Nrf2/ARE pathway which in turn induces cytoprotective genes and modulates chemosensitivity of HT-29 colon cancer cells. Therefore, we postulate that Nrf2 might represent a potential therapeutic target in 5-FU treatment of colon cancer.

Stem cell-derived hepatocytes and their use in toxicology.

Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.

Improvement of HepG2/C3a cell functions in a microfluidic biochip

Current developments in tissue engineering and microtechnology fields allow the use of microfluidic biochip as microtools for in vitro investigations. In the present study, we describe the behavior of HepG2/C3a cells cultivated in a poly(dimethylsiloxane) (PDMS) microfluidic biochip coupled to a perfusion system. Cell culture in the microfluidic biochip for 96 h including 72 h of perfusion provoked a 24 h delay in cell growth compared to plate cultures. Inside the microfluidic biochip, few apoptosis, and necrosis were detected along the culture and 3D cell organization was observed. Regarding the hepatic metabolism, glucose and glutamine consumptions as well as albumin synthesis were maintained. A transcriptomic analysis performed at 96 h of culture using Affymetrix GeneChip demonstrated that 1,025 genes with a fold change above 1.8 were statistically differentially expressed in the microfluidic biochip cultures compared to plate cultures. Among those genes, phase I enzymes involved in the xenobiotics metabolism such as the cytochromes P450 (CYP) 1A1/2, 2B6, 3A4, 3A5, and 3A7 were up‐regulated. The CYP1A1/2 up‐regulation was associated with the appearance of CYP1A1/2s activity evidenced by using EROD biotransformation assay. Several phase II enzymes such as sulfotransferases (SULT1A1 and SULT1A2), UDP‐glucuronyltransferase (UGT1A1, UGT2B7) and phase III transporters (such as MDR1, MRP2) were also up‐regulated. In conclusion, microfluidic biochip could and provide an important insight to exploring the xenobiotics metabolism. Altogether, these results suggest that this kind of biochip could be considered as a new pertinent tool for predicting cell toxicity and clearance of xenobiotics in vitro. Biotechnol. Bioeng. 2011; 108:1704–1715.