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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.)

Hepatic tumors with combined hepatocellular cholangiocarcinoma have been frequently described for instance, hepatoblastoma with cholangioblastic features in young patients1 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, hepatic progenitors do not massively contribute to liver regeneration. Their number increases during massive or chronic injury in close correlation with damage severity.6 In experimental animal models reproducing liver injuries, inhibition of hepatocyte proliferation contributes to activate this hepatic progenitor cell compartment,7 which is also found to favor hepatocarcinoma development.8 Indeed, pools of small epithelial cells characterized by stem and fetal markers, and immature biliary and hepatocytic cells expressing specific functional markers have been described in tumors.8

The origin of hepatic progenitors during carcinogenesis is still extensively debated. It can occur from aberrant proliferation and differentiation of hepatic progenitors or from their immediate progeny. At least some types of ductular reaction might be attributable to metaplastic differentiation of mature hepatocytes into bile ductules (ductular metaplasia). Indeed, although mature hepatocytes have an extensive proliferation capacity during liver injury and are supposed to have a stable phenotype, the possible reversion program from differentiated hepatocytes to small hepatocytes has been observed.9, 10 In addition, transdifferentiation of mature hepatocytes into biliary cells has been shown to occur in rat liver.11 Thus, possible bipotentiality of mature hepatocytes occurring during transformation is conceivable but remains questioned. Understanding of this high cellular plasticity would help to identify genes involved in growth mechanisms of liver tumors.

Embryonic development and tissue homeostasis depend on a complex interplay among processes involved in cell proliferation, differentiation, migration, adhesion, and cell death, which are coordinated by highly evolutionarily conserved signaling pathways; deregulation of these pathways leads to pathological situations, including cancer. Among them, Notch12 and Wnt/β-catenin13 signaling cascades play decisive roles during developmental processes and cell fate determination. Recently, they have been suggested to be involved in stem cell autorenewal, early biliary lineage commitment of bipotent progenitors, and hepatocyte maturation.14, 15

Among human hepatoma-derived cell lines, HepaRG cells isolated from a hepatic differentiated grade 1 Edmonson tumor consecutively to chronic HCV infection exhibit unique features. They have a pseudodiploid karyotype. They constitutively and synchronously display both hepatocyte-like and biliary-like epithelial phenotypes at confluence, indicating they may have bipotent progenitor features.16, 17 When optimally differentiated, HepaRG cells support HBV infection16 and express a large panel of liver-specific genes, including those expressing drug metabolizing enzymes.18 In this work, we analyze the possible transdifferentiation process of human liver cells using the HepaRG cell line, define the sequential expression of hepatocyte nuclear factor (HNF) family transcription factors, and identify associated signaling mechanisms involved in this process.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

HepaRG Cell Culture.

HepaRG cells were cultured as described.16 Cells (2.7 × 104/cm2) were maintained in William's E medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, and 5 × 10−5 M hydrocortisone hemisuccinate. A 2-step procedure was used to obtain HepaRG differentiation: cells were cultured in the medium for 2 weeks and then in the presence of 2% dimethyl sulfoxide (DMSO) for 2 more weeks.

Purification of Hepatocyte-like Cells.

Hepatocyte-like cells from HepaRG DMSO-treated cultures were either selectively detached using mild trypsinization or isolated by centrifugation on an OptiPrep gradient. Cells were trypsinized, dissociated, and centrifuged at 1500g 5 min on a 30% OptiPrep gradient. Cells localized at the sample/medium interface were collected and centrifuged again on another 15% gradient. Cells from the pellets were washed in medium and plated at either low density (0.1 × 104/cm2) or high density (1.5 × 105/cm2). Purity of hepatocyte-like cell suspension was determined by CYP3A4 labeling. Remaining epithelial cells after mild trypsinization were cultured as described.

HepaRG Cell Transplantation.

uPA/SCID mice (10–20 days old) were injected intrasplenically with 5 × 105 viable HepaRG cells at progenitor or differentiated stages.19 To control nonadaptive defense, mice were treated as described.20 The uPA/SCID mice were killed 6 weeks after transplantation. Blood samples were collected, and livers were removed. Parts of liver were preserved for histologic analysis or frozen for RNA analysis. Alpha-1-antitrypsin was quantified in sera by ELISA.19

Immunochemical Analysis.

Cells fixed with 4% paraformaldehyde or sections of formaldehyde-fixed and paraffin-embedded uPA/SCID mouse liver tissue were used for immunolocalization of human albumin (Kent Laboratories), transferrin (Kent Laboratories), cytochrome P450 3A4 (CYP3A4) (Chemion International), OV-6 (R&D Systems), CK18 (Dako), CK19 (Novocastra), β-catenin, α1-integrin and α6-integrin (Santa Cruz), or E-cadherin (R&D Systems). Endogenous peroxidase activity was blocked and overnight incubation at 4°C with primary antibody was followed by incubation with peroxidase-conjugated or rhodamine-conjugated secondary antibody (Jackson Laboratories). Peroxidase staining was obtained with 3,3′-diaminobenzidin/H2O2 solution. Cells or slices were counterstained with Masson's Hemalun or with Hoechst and observed using a Zeiss microscope. The percentage of CYP3A4-positive cells was quantified from at least 10 fields of each experiment by imaging analysis using SimplePCI software (Compix Inc.).

Bile Canaliculus Labeling and γGT Activity.

Cells were incubated with 1 μg/ml fluorescein diacetate for 15 minutes at 37°C and then fixed with 4% paraformaldehyde for 20 minutes at 4°C. Specific γGT activity was assayed in HepaRG cells as described.21

Flow Cytometry.

Before fixation with formaldehyde 1% in phosphate-buffered saline, cells were stained by antibodies directed against ABCG2/BCRP1, CD34, CD10, CD13, CD29, ICAM-1, CD44, CD49a, CD49b, CD71, GM-CSF-R, Syndecan (Immunotech), CD33, LIF-R, CD49f, CD45, c-Kit, NCAM, Thy-1, Flt3, IL3-Rα, VEGFR2, gp130 (Pharmingen), and G-CSF-R (Serotec). For cytochrome CYP3A4 staining, cells were first fixed with 4% paraformaldehyde, permeabilized using 0.1% saponin, and stained with primary antibody. After washing, cells were incubated by FITC-coupled secondary antibody. Analyses were performed using a FACSCalibur flow cytometer and CellQuest software (Becton-Dickinson).

DNA Synthesis.

Bromodeoxyuridine (BrdU)-positive cells were detected using the Cell Proliferation kit (Amersham).

Western Blotting.

After cell lysis, 50 μg of total proteins or nuclear proteins were resolved on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane and analyzed as described.22 Antibodies directed against HNF-1α, HNF3β, HNF4α, (Santa Cruz Biotechnology), p21CIP1, p27KIP1 (Neomarkers), p53 (Dako), HSC70 (Santa Cruz Biotechnology), and CDK11P110 (obtained from Dr. P. Loyer, INSERMU522) were used.

RNA Isolation, Reverse Transcription PCR, and Real-Time Reverse Transcription PCR Analysis.

Total RNAs were extracted (SV total isolation system, Promega) and cDNA synthesis was performed from 1 μg RNA (Advantage RT-for-PCR kit, BD Bioscience). Human albumin cDNA in uPA/SCID mouse liver was amplified for 40 cycles using the sense primer 5′-AGACAAATTATGCACAGTTG-3′ and the antisense primer 5′-TTCCCTTCATCCCGAAGTTC-3′, human actin cDNA for 25 cycles using the sense primer 5′-GACTACCTCATGAAGATCCT-3′, and the antisense primer 5′-TTGCTGATCCACATCTGCTG-3′ was the endogenous control. Expression of CYP3A4, Notch 1, 2, 3, jagged1, Hes1, p53, CEA, and c-Myc mRNA in HepaRG cells were measured by quantitative reverse transcription (RT)-PCR. Specific primers were designed using the Primer Express 1.0 software (ABI Prism, Applied Biosystems) from Genbank human mRNA sequences: CYP3A4 forward primer 5′-CTTCATCCAATGGACTGCATAAAT-3′, reverse primer 5′-TCCCAAGTATAACACTCTACACAGACAA-3′; p53 forward primer 5′-GCAATAGGTGTGCGTCAGAA-3′, reverse primer 5′-CCAGTGCAGGCCAACTTGTT-3′; CEA forward primer 5′-GGTCTTCAACCCAATCAGTAAGAAC-3′, reverse primer 5′-ATGGCCCCAGGTGAGAGG-3′; c-Myc forward primer 5′-CGGTCCGCA- ACCCTTG-3′, reverse primer 5′-CTCGGGTGTTGTAAGTTCCAGTG-3′; 18S was the endogenous control, forward primer 5′-CGGCTACCACATCCAAGGAA-3′, reverse primer 5′-GCTGGAATTACCGCGGCT-3′. Specific primers for Notch 1, 2, 3, jagged1, and Hes1 were purchased from Applied Biosystems. Real time PCR was performed using PE Biosystems ABI Prism 7700 Sequence Detection System and the qPCR Core Kit for SYBR Green I or for taqman procedure, respectively (Eurogentec). Data were analyzed with the PE Biosystems ABI Prism 7700 Sequence Detector software. Mutation analysis of β-catenin gene was performed as previously described.23

Soft Agar and Tumor Formation Assays.

Soft agar assays were performed using HepaRG cells at progenitor stage and HepG2 and HuH7 cells as positive controls (n = 2). Cells (2.25 × 105) were seeded in complete William's E medium containing 2.5% indubiose in triplicate.

Two independent series of 15 female nude swiss Nu−/Nu− mice (4-week-old, IFFA CREDO, France) were injected with 10 × 106 HepaRG cells. As positive control, F1 epithelial cells were injected. No tumors were detected 1 year after HepaRG cells injection, whereas tumors appeared 4 weeks after F1 epithelial cells injection.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Differentiation of HepaRG Cells Into Hepatocyte-like Cells and Biliary-like Cells.

Confluent differentiated HepaRG cells plated at 2.7 × 104 cells/cm2 in the presence of insulin and corticosteroids gave rise to undifferentiated cells that actively proliferated for 3 to 4 days and committed into hepatocyte and biliary differentiation pathway early after reaching confluence at day 5 (Fig. 1A, B). The cells were organized in either flat clear epithelial cells or cords of granular polygonal cells (Fig. 1C). Maximum cell differentiation was reached after a 2-week DMSO exposure. Hepatocyte-like cells exhibited a phenotype close to that of human hepatocytes (Fig. 1D), with functional bile canaliculus-like structures as evidenced by fluorescein excretion (Fig. 1E). Immunocytochemical analyses showed that hepatocyte-like cells were positive for CYP3A4, whereas surrounding flattened epithelial cells were negative (Fig. 1F). CYP3A4-expressing cells represented 54.5% ± 7.7% of the whole cellular population (n = 4). CK18 was highly expressed in hepatocyte-like cells and to a lesser extent in biliary-like cells (Fig. 1G). In contrast, CK19 was only expressed in biliary-like cells (Fig. 1H). Last, CD49a (α1 integrin) expression was limited to hepatocyte-like cells whereas CD49f (α6 integrin) was associated with biliary-like cells (Fig. 1I–J). Thus, HepaRG cells exhibited cellular plasticity and supported in vitro transition from undifferentiated cells toward hepatocyte and biliary lineages. The question rose as to whether differentiated cells or bipotent progenitors took part in cell expansion.

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Figure 1. Morphology of HepaRG cells and expression of differentiation markers during differentiation process. Phase-contrast photographs of HepaRG cells at (A) 2, (B) 5, (C) 15, and (D) 30 days after seeding. At day 15, the medium was supplemented with 2% DMSO and cells were cultured for 15 more days. Bar, 60 μm. Black arrowhead shows bile canaliculus–like structure. (E) Micrograph of fluorescein diacetate staining. White arrowheads show fluorescein diacetate accumulation in bile canaliculus–like structures. Immunolocalization of (F) CYP3A4, cytokeratins (G) 18, and (H) 19, (I) integrin α1, and (J) α6 (J) in HepaRG-differentiated cells at day 30. Bar: 30 μm. H, hepatocyte, BC, biliary cell. Black arrowheads show positive staining.

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Proliferation of Differentiated Hepatocyte-like Cells Without Loss of Differentiation and Cell Death.

A specific negative control of cell death program has been described in mature hepatocytes allowing them to retain a proliferation potential through the regulation of p53 and 2 main CDK inhibitors, p21CIP1 and p27KIP1.22 Here, we analyzed the accumulation of these proteins during the HepaRG cell differentiation process by Western blotting (Fig. 2A). p53 expression was constantly detected during the differentiation process and dropped in DSMO-treated culture. p21CIP1 was weakly detected on the first day of culture. Its expression transiently increased during the proliferation phase before being strongly diminished in differentiated cells. In contrast, detectable during the sustained first week of culture, p27KIP1 expression increased in cells undergoing commitment to differentiation process. Finally, the dim expression of p21CIP1 associated with the sustained expression of p27KIP1 in differentiated HepaRG cells, as described in quiescent normal hepatocytes, suggested that HepaRG hepatocyte-like cells could divide without losing differentiation.

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Figure 2. Proliferation of differentiated hepatocyte-like cells without loss of differentiation and cell death. (A) Analysis of p53, p27KIP1, p21CIP1, and HSC70 expression by immunoblotting during the differentiation process at the indicated times of culture. Differentiated hepatocyte-like cells (day 30) were plated at high density (1.5 × 105/cm2) and examined by phase-contrast microscopy at (B) 24 hours, (C) 36 hours, (D) 48 hours, and (E) 96 hours after seeding. DNA synthesis was measured by BrdU incorporation during the last 24 hours at days (F) 1 and (G) 4. In parallel, immunolocalization of CYP3A4 was performed at days (H) 1 and (I) 4. Bar: 30 μm. Results represent 3 independent experiments.

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Thus, hepatocytes from differentiated DMSO-treated cultures were isolated and seeded at high density (1.5 × 105/cm2). Evidence was provided that hepatocyte-like cells preserved both their morphology (Fig. 2B–E) and differentiation status as illustrated by the maintenance of CYP3A4 expression (Fig. 2H–I), although they kept their capacity to proliferate as demonstrated by BrdU incorporation (Fig. 2F,G). At day 1, 55% of hepatocytes had entered into S-phase whereas 18% retained their ability to divide at day 4. Therefore, HepaRG cells seeded at high density support unipotent hepatocyte-like cell behavior as normal hepatocytes do during liver regeneration.

Reversion of Hepatocyte-like Cells and Biliary-like Cells Through Bipotent Progenitors.

Meanwhile, purified hepatocyte-like cells (98% positive for CYP3A4, Fig. 3A) seeded at low density (0.1 × 104/cm2) displayed a great plasticity. A gradual decrease of liver specific functions, illustrated by CYP3A4 extinction, was detected through culture time in parallel to an increase of CK19 expression (Fig. 3B,I–M). No cell death was observed, and active cell proliferation was evidenced by BrdU incorporation and cell counts (Fig. 3C). Concomitantly, extended morphological changes occurred within 3 days in the whole cell population that switched into elongated shaped cells (Fig. 3D–H). After onset of divisions, cells reached confluence and cords of granular hepatocytic cells emerged surrounded by biliary-like cells. As expected, CYP3A4 was only detected in hepatocytic cords and CK19 in biliary-like cells (Fig. 3M). CYP3A4-positive cells represented 53% ± 5.8 of the whole cellular population (n = 2).

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Figure 3. Transdifferentiation potential of hepatocyte-like and biliary-like HepaRG cells through bipotent progenitor. (A) Flow cytometry analysis of CYP3A4 expression in isolated differentiated hepatocyte-like cells (day 30). (B) Percentage of cells expressing CYP3A4 protein quantified by imaging analyses (gray bar) and CYP3A4 mRNA level measured by real-time PCR (black line) at the indicated times. (C) At the indicated times, DNA synthesis was measured by BrdU incorporation during the last 24 hours and cellular proliferation was measured by cell counts; results are expressed as a percentage of BrdU-positive cells (gray bar) and cells/cm2 (black line), respectively. Phase-contrast photographs of differentiated hepatocyte-like cells seeded at low density at days (D) 1, (E) 2, (F) 3, (G) 5, and (H) 30 after plating (15 days without DMSO followed by 15 more days of culture with DMSO). Biliary-like cells were observed at days (N) 1, (O) 2, (P) 3, (Q) 15, and (R) 30 (15 days without DMSO followed by 15 more days of culture with DMSO). In the same time, co-immunolocalization of CYP3A4 (green) and CK19 (red) was performed on (I–M) isolated hepatocyte-like cells and (S–W) the remaining biliary-like cells. Results represent at least 2 independent experiments. Black bars: 60 μm. white bars: 50 μm. H, hepatocyte, BC, biliary cell.

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Drastic morphological changes were also observed with biliary-like cells (Fig. 3N–R). Within the first 24 hours, cells became lengthened, actively proliferated, and then differentiated into biliary-like cells or hepatocyte-like-cells expressing CK19 and CYP3A4, respectively (Fig. 3S–W). CYP3A4-positive cells represented 52.2% ± 8 of the whole cell population (n = 2).

Thus, both hepatocyte-like and biliary-like cells can transdifferentiate through a bipotent hepatic progenitor.

Expression and Evolution of Progenitor Cell Markers Pattern During HepaRG Cells Differentiation Process.

When HepaRG cells displayed bipotent progenitor phenotype, they shared with oval cells expression of NCAM, ABCG2, CK18, and CK19. They also expressed hematopoietic stem cell markers such as CD34, Thy1, Flt-3, c-Kit, IL-3Rα, and LIF-R but failed to express CD45. They exhibited receptors such as CD71, gp130, G-CSF-R, and VEGF-R2; adhesion molecules including ICAM-1, CD29, CD44, CD49a, CD49b, CD49f, and syndecan (CD138); and lymphoid and myeloid markers such as CD13, CD33, and CD10 (Fig. 4A).

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Figure 4. Expression and evolution of progenitor cell markers pattern during differentiation of HepaRG cells. (A) Flow cytometry phenotype of HepaRG cells at progenitor state, 2 days after plating (n = 3). (B) Evolution of progenitor cell markers expression at (A) 2, (B) 5, (C) 15, and (D) 30 days after plating. Cells were stained with specific (thick black line histograms) or isotypic control antibodies (thin gray line histograms) and analyzed by flow cytometry. Results are compared with HepG2 cells (E) during proliferation and (F) when differentiated (n = 2).

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Expression of some of these markers was then examined throughout the differentiation process. Cells rapidly lost expression of CD34, Thy1, CD71, LIF-R, and ABCG2 (Fig. 4B) but also CD33 and gp130 (data not shown). The unexpected re-expression of CD34 and Thy1 in differentiated cells was directly associated with DMSO exposure. NCAM staining gradually decreased until disappearing. In contrast, CD29 slightly decreased during the differentiation process. Of note, HepG2 cells deeply differed from HepaRG cells by the lack of LIF-R and ABCG2 expression at the undifferentiated state and the absence of modulation of CD34, Thy1, CD71, N-CAM, and CD29 expression during their differentiation (Fig. 4B).

Differential Expression of HNFs, Notch Signaling Components, and β-Catenin Localization Changes During Hepatobiliary Lineage Commitment of HepaRG Cells.

To further study commitment of bipotent progenitors into hepatic lineage, the expression of HNF transcription factors and Wnt/β-catenin and Notch signaling components was analyzed. During the first 2 to 3 days of active proliferation, HepaRG cells at the progenitor stage expressed HNF-3β protein. Its expression drastically decreased on day 4 before becoming completely silent in differentiated cells (Fig. 5A). In contrast, HNF4α protein weakly expressed in progenitors progressively accumulated to reach a maximum in differentiated cells. Meanwhile, HNF1α exhibited a stable expression profile during differentiation.

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Figure 5. Expression of HNF transcription factors and Notch signaling pathway components and β-catenin localization changes during the differentiation process in HepaRG cells. (A) Immunoblot analyses of HNF4α, HNF3β, and HNF1α expression at the indicated times of culture. CDK11p110 protein kinase was used as loading control. (B) mRNA levels of Notch1 (white bar), Notch2 (gray bar), Notch 3 (black bar), and jagged 1 (hatched bar) were measured by RT-qPCR at the indicated times of culture. Results are expressed as fold induction compared with the first day of culture arbitrarily set at 1. (C) Co-immunolocalization of β-catenin and E-cadherin at 8 hours and at days 1, 10, and 30 after plating. Bar: 20 μm. Results represent three independent experiments.

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In parallel, modulations of Notch signaling component were observed. Notch 1, 2, and 3 mRNA were expressed in proliferating progenitors whereas Notch 4 was absent. Then, expression of Notch 1 and 2 strongly decreased all along the differentiation process to reach a minimum in differentiated cells. Jagged 1, one of the Notch 1 ligands, exhibited the same expression profile. In contrast, expression of Notch 3 transiently and strongly increased during the first days after plating. It reached a maximum between days 3 and 5 before drastically dropping in differentiated cells. Meanwhile, Hes1, one of the target genes of the Notch signaling pathway, had a stable expression pattern (not shown).

Along with the differential expressions of HNFs and Notch signaling components, changes in β-catenin localization were evidenced. Beta-catenin exhibited nuclear localization just after cell plating (Fig. 5C), and 2 days later, its localization became nuclear, cytoplasmic, and membranous in progenitors (Fig. 5D). At confluence and in differentiated cells, β-catenin localization was mainly restricted to the plasma membrane (Fig. 5E, F) and could be related to the detectable membranous localization of E-cadherin (Fig. 5G, H). Of note, no mutation of β-catenin was detected in HepaRG cells, contrasting with other carcinomas, which frequently exhibit such mutation (Dr. Catherine Cavard, Institut Cochin, Paris, personal communication).

Transplantation of HepaRG Cells into uPA/SCID Mouse Liver Favors Hepatocyte Differentiation Lineage.

Because HepaRG is derived from a differentiated hepatic tumor, we studied markers related to liver carcinoma and its in vitro and in vivo tumorigenicity. Whatever the levels of differentiation, γGT activity was constantly observed in HepaRG cells (Fig. 6A). Weak amounts of c-myc and CEA mRNA were detected compared with HepG2 cells; whereas c-myc expression dropped slowly during the culture period, CEA expression increased gradually. In contrast, p53 mRNA and protein amounts were stable all along the differentiation process and only dropped in DMSO-treated cultures (Figs. 2A, 6B).

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Figure 6. Expression of markers related to hepatocellular carcinoma in HepaRG cells. Cytochemical staining of γGT activity was performed in (A) proliferating and (B) differentiated HepaRG cells; bar: 30 μm. c-myc, p53, and CEA RNAs levels were measured by RT-qPCR at the indicated times of culture (C). Results are expressed as fold induction compared with the first day of culture arbitrarily set at 1. Results represent three independent experiments.

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HepaRG cells grew moderately on soft agar (data not shown) and failed to give rise to tumor when injected in nude mice. However, HepaRG cells, either at progenitor stage (n = 7/7) or differentiated into hepatocyte- and biliary-like cells (n = 6/6), exhibited the ability to repopulate uPA/SCID mice damaged liver. Whatever the initial differentiation stage of HepaRG cells, implanted cells had preferentially differentiated toward the hepatocytic pathway 6 weeks after transplantation. Hepatocytic functionality was supported by human albumin expression evidenced in mice livers by immunohistochemistry and RT-PCR (Fig. 7A,B,I) and by human alpha-1-antitrypsin detected by ELISA in mouse sera (10.2 μg/ml ± 6.1). Mouse liver repopulation by HepaRG cells arose by cells creeping into parenchyma without clustering. Moreover, Sirius red staining did not show more type I collagen accumulation or extracellular matrix (ECM) modification in transplanted mice than in their control counterparts (Fig. 7C,D). Finally, only a few CK19-positive cells, localized next to bile ducts and surrounded by inflammatory infiltrates, were detected (Fig. 7G,H).

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Figure 7. Repopulation of injured uPA/SCID mouse liver by HepaRG cells. Immunolocalizations of (A,B) human albumin and (G,H) CK19 and stainings with (C,D) red syrius and (E,F) hematoxylin-eosin were performed on liver slices from mice (A, C, E, G) transplanted with HepaRG progenitor cells or (B, D, F, H) untransplanted. Of note, no cross-reaction with murine albumin and CK19 was detected. (I) Representative expression of human albumin and actin mRNAs in livers from different mice transplanted with HepaRG cells either at progenitor or differentiated stage were analyzed by RT-PCR. HepaRG cells were used as positive control for human albumin primers; untransplanted mouse was used as negative control. Bar: 100 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Involvement of progenitor cells in carcinogenesis is still debated and raises important remaining unanswered questions.24 In particular, how is the pool of hepatic progenitor cells constantly renewed and what are the molecular signals that induce and control the progenitor differentiation? Recent studies have suggested that hepatocytes can function as unipotent stem cells or be a source of hepatic progenitor cells in rodent liver. For instance, in dipin-induced liver damage in mice or retrorsin-treated rats, hepatocytes dedifferentiated, suggesting they could be a possible source of small hepatocyte-like progenitor cells.9, 10 Rat hepatocytes were also described to transdifferentiate into biliary cells via intermediate precursor cells.11 Until now, studies on human hepatic cells have been greatly hampered by the lack of appropriate cell systems suitable to mimic oval cell properties, including hepatobiliary differentiation abilities. Here, using the HepaRG cells derived from a human differentiated hepatoma, we have evidenced in vitro the transdifferentiation of both biliary-like and hepatocyte-like cell populations into hepatocytic and biliary lineages through a hepatic progenitor that actively divides. However, this does not exclude that hepatocyte-like cells remained able to proliferate as normal hepatocytes do during liver regeneration.

Using purified differentiated HepaRG cells, survival and proliferation of hepatocyte-like cells were accurately demonstrated. This led us to consider these cells as unipotent progenitors as suggested for normal hepatocytes.25 Interestingly, this property appeared to depend on apoptotic regulators such as the tumor suppressor gene product P53, and CDK-inhibitors such as p21CIP1 and p27KIP1, as observed in normal hepatocytes in vivo.22 As expected, the decrease in p53 expression in HepaRG cells correlated with growth arrest and differentiation, and the absence of p21CIP1 accumulation supported low apoptotic activity and high proliferation potential of differentiated cells. Indeed, in contrast to other tissues such as skin, p21CIP1 expression was described as remarkably low in liver, primary adult hepatocytes,22 and HBG human hepatoma cells.26 This high potential of proliferation and survival was confirmed in vivo because HepaRG progenitors as well as differentiated cells had the capacity to repopulate uPA/SCID mouse damaged livers. They predominantly gave rise to functional hepatocyte-like cords as observed after massive hepatic cell death in vivo.27

The high proliferation potential and low apoptotic activity of purified differentiated cells could also be associated with a transdifferentiation program when cells were seeded at low density, leading to disruption of cell–cell communications. Cells transdifferentiated through a bipotent progenitor characterized by major oval-like cell markers expression such as CK19, CK18, γGT, ABCG2/BRCP1,28 NCAM,29 Thy1, CD34, Flt-3, and at a weaker level c-Kit.30–32 In addition, they expressed various receptors, adhesion molecules, lymphoid and myeloid markers, and exhibited nuclear accumulation of β-catenin. Of note, contrasting with most hepatocellular carcinoma, β-catenin is not mutated or deleted in HepaRG cells.33 This expression pattern has never been reported because of high heterogeneity of cell populations analyzed in human tumors in vivo or uncontrolled genetic changes induced by experimental cell immortalization in vitro. Like stem cells, active self-renewal property was preserved and differentiation was abrogated when HepaRG cells at progenitor stage were subcultured before confluence. Thus, HepaRG cells exhibited a set of characteristic features that contributes to define the human hepatic bipotent progenitor signature.

This bipotent property, which allows them to undergo 2 distinct differentiation programs leading to either biliary or hepatocytic lineages, resulted in differential expression patterns of many genes. Expression of 2 transmembranous receptors responsible for important transduction signals, α-1 and α-6 integrins, was restricted to hepatocyte-like and biliary-like cells, respectively, as in vivo.34 CYP3A4 was only detected in hepatocyte-like cells. These modulations could be related to a recent report describing the appearance of liver-specific functions in HepaRG cells after growth arrest.35 Moreover, these gradual appearances of liver-specific functions were associated with rapid disappearance of stem cell markers. These last observations contrasted with the results of Parent et al.17 indicating that differentiated HepaRG cells preserved an immature phenotype characterized by maintenance of oval-cell markers. This discrepancy could be partly explained because DMSO exposure enhanced several liver-specific functions18 and unexpectedly re-induced stem cell marker expression, such as CD34 in HepaRG cells as in normal adult human hepatocytes (unpublished results). Only a group of functions, including γGT, CEA, and c-Myc, remained relatively stable regardless of the cell differentiation status, suggesting that they could be related to cancerous markers. Frequently detected in HCC, permanent γGT expression was associated with abnormal hypomethylation of the gene.36 In agreement with Ordonez et al.,37 the lower CEA levels in HepaRG cells compared with those found in HepG2 cells could be correlated to the higher degree of differentiation reached by HepaRG cells and their inability to induce tumor when injected in nude mice.

During the HepaRG progenitor's commitment into hepatobiliary lineages, changes in gene expression were correlated with differential expression of HNF transcription factors. Whereas HNF1α and HNF3β were expressed in HepaRG cells at the progenitor stage as in oval cells, HNF4α, known to be necessary during the final step of hepatocyte differentiation,38, 39 was up-regulated during the differentiation process.40 However, the molecular mechanisms that at an early stage determine if bipotent progenitor cells progress through either hepatocyte or biliary differentiation programs remain to be elucidated. Like the Wnt/β-catenin signaling pathway, researchers have described the Notch signaling pathway as playing a key role in stem cell autorenewal and development of cell fate decisions in multiple organs.12 For instance, those pathways have been involved in hepatobiliary lineage commitment,15 and particularly, the Notch signaling has been shown to promote hepatoblast differentiation by altering expression of liver-enriched transcription factors.41 Here, evidence was provided that changes in β-catenin localization from nucleus to cytoplasm and plasma membrane, described as characteristic of hepatocyte differentiation,42, 43 took place at the onset of HepaRG differentiation commitment. This change correlated with a transient specific upregulation of both Notch3 and HNF3β expression and a progressive down-regulation of Notch1, Notch2, and Jagged1 expression. Thus, our results suggest that Notch3 was a target gene able to exert a critical role in the cell fate of hepatic bipotent progenitors and emphasized the hypothesis of a possible involvement of Notch3 in biliary lineage commitment.44 The effect of activating or inhibiting Notch3 in hepatobiliary commitment should be addressed through HepaRG cell behavior and its relationships with the Wnt/β-catenin pathway, and HNF transcription factors should be further defined.

In conclusion, the human HepaRG cells display unique features because they share with the human fetal liver multipotent progenitor cells, recently isolated and characterized by Dan et al.45: (1) a high proliferative ability; (2) differentiation potential toward biliary and hepatocytic lineage; and (3) the capacity to differentiate into functional hepatocytes in vivo. Moreover, probably related to their tumor origin, HepaRG cells exhibit a transdifferentiation potential through a hepatic bipotent progenitor. This mechanism should be taken into account to further understand development of differentiated tumors and auto-renewal of hepatic progenitor pool within such tumors. Compared with other hepatoma cell lines, HepaRG cells provide a unique tool for analyzing in vitro and in vivo various hepatic physiopathological situations and, overall, identifying signals that control the bipotent progenitor expansion and thus, designing new targets for therapeutic strategies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. C. Perret (Institut Cochin, Département GDPM, Paris) and Pr. A. Guillouzo (INSERM U-620, Faculté de Pharmacie, Rennes) for their helpful collaborations, Dr. Remy Le Guevel for imaging analysis (plateforme puce à cellules, Genopole Ouest), and Dr. B. Birebent from cytometry facilities (plateforme de cytométrie, IFR 140).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References