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Am J Physiol Gastrointest Liver Physiol 293: G944-G955, 2007. First published August 23, 2007; doi:10.1152/ajpgi.00263.2006
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LIVER AND BILIARY TRACT

Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro

Rahul Kuver, Christopher E. Savard, Sung Koo Lee, W. Geoffrey Haigh, and Sum P. Lee

Division of Gastroenterology, Department of Medicine, University of Washington, and Puget Sound Veterans Affairs Health Care System, Seattle Division, Seattle, Washington

Submitted 14 June 2006 ; accepted in final form 21 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4{alpha}, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, {alpha}-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.

stem cells; cell culture; transgenic mice; transdifferentiation; biliary epithelium

HEPATOCYTES AND EPITHELIAL cells of the intrahepatic biliary tract are capable of remarkable plasticity in response to certain forms of severe liver injury. Hepatocytes can alter their differentiation state so as to undergo a metaplastic conversion to biliary epithelial cells. Conversely, biliary epithelial cells appear capable of changing their phenotype and giving rise to hepatocyte-like cells, small intestinal mucosal cells, or pancreatic acinar cells (8, 46, 50, 51). Two mechanisms can be envisioned to explain such changes in differentiation. On the one hand, terminally differentiated cells could change their gene expression profiles and "transdifferentiate" into terminally differentiated cells of another type (6). Such transdifferentiation has been reported to occur with rat hepatocytes, which change into bile duct-like cells in vitro (30) and in vivo (28). Similar mechanisms have been proposed to explain the conversion of rat pancreatic exocrine cells to hepatocyte-like cells (24, 44).

On the other hand, the presence of cells with stem cell properties in the liver or pancreas could explain this phenomenon. A substantial body of evidence supports the existence of a facultative pluripotent stem-like cell compartment in the liver that becomes activated after certain types of hepatic injury in which the normal regenerative capacity of hepatocytes is impaired (5, 39, 42). Oval cells, found in the canals of Hering and terminal bile ductules, are capable of differentiating into hepatocytes and biliary epithelial cells (12). Oval cells express the hematopoietic cell marker CD34, whereas hepatocytes and biliary epithelial cells do not (36). In transplanting cell/tissue preparations, "contamination" with a small number of such pluripotential stem cells capable of differentiating into liver cells may have occurred (52). This concept is further complicated by the phenomenon of hepatocyte regeneration following liver injury, in which hepatocytes themselves repopulate the damaged liver without a significant contribution from bone marrow-derived pluripotential stem cells or oval cells (11).

A more direct approach to demonstrate differentiation from one terminally differentiated cell type to another would be to establish an in vitro model with cells that can be well characterized so as to exclude pluripotential stem cells and hepatocytes. Extrahepatic biliary epithelial cells, such as those derived from the gallbladder, would fulfill such a requirement, inasmuch as there would be no possibility that such cell preparations would be "contaminated" with any cells possessing a hepatocytic lineage or stem cell properties (including hepatocytes, oval cells, or bone marrow-derived progenitor cells). This "clean" cell model could then be used to investigate whether such changes in differentiation occur. We and others (3, 14, 17, 22, 31, 32, 35) have reported the establishment of cell lines from normal gallbladder epithelia of adult dogs, humans, and mice. We therefore used genetically labeled well-differentiated murine gallbladder epithelial cells (GBEC) to examine whether differentiation into hepatocyte-like cells can be induced in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Analytic-grade chemicals and tissue culture supplies were obtained from Sigma (St. Louis, MO), except where noted. Vitrogen (bovine dermal collagen) was purchased from Cohesion (Palo Alto, CA), Transwell inserts (24-mm diameter, 3-µm pore size) from Corning (Acton, MA), and Matrigel, a murine extracellular matrix mixture, and dispase from BD Biosciences (Bedford, MA).

Cell isolation and culture. Epithelial cells were isolated from the gallbladders of MT-lacZ mice (38). These mice contain a modified beta-galactosidase (beta-Gal) gene attached to the murine metallothionein (MT)-1 gene promoter, which is only activated in the liver of the transgenic mouse, conferring hepatocyte-specific beta-Gal expression. GBEC were also isolated from green fluorescent protein (GFP) transgenic mice [strain C57BL/6-Tg(ACTbEGFP)10sb/J, Jackson Laboratory, Bar Harbor, ME]. GBEC were isolated and serially cultured as described elsewhere (22). Briefly, the cells were cultured on Vitrogen-coated Transwell inserts suspended above human gallbladder myofibroblast feeder cells. The cells were fed Eagle's minimum essential medium containing 10% fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin plus insulin-transferrin-sodium selenite supplement (ITS), and MEM vitamins and nonessential amino acid solution. Trypsin-EDTA (0.25%-0.1%) was used to passage cells. The Institutional Animal Care Committee approved all animal experiments.

Experimental culture conditions. Murine GBEC were trypsinized and plated onto 35-mm tissue culture plates that had been coated with Vitrogen gel (1 ml of 1:1 Vitrogen-medium). The cells were allowed to attach to the Vitrogen overnight. Media were removed from the plates along with unattached cells. Cold Matrigel (1 ml per 35-mm plate) was then layered on top of the cells and allowed to solidify at 37°C for 15 min–1 h. The cells were then fed medium consisting of Dulbecco's minimum essential medium, 2 mM L-glutamine, 20 mM HEPES, 0.54 mg/l ZnCl2, 0.75 mg/l ZnSO4·7H2O, 0.2 mg/l CuSO4·5 H2O, 0.025 mg/l MnSO4, 3 g/l glucose, 2 g/l galactose, 2 g/l BSA, 0.1 g/l ornithine, 0.03 g/l proline, 0.6 g/l nicotinamide, and 0.1 µM dexamethasone plus ITS medium supplement. This medium was modified with various concentrations of hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transforming growth factor. Cells were fed three times per week with medium freshly supplemented with growth factors. Media from each feeding were saved and frozen for later use. At specified times, cells were analyzed for morphology and transgene expression, and RNA was isolated for PCR analysis.

Measurement of beta-Gal activity. Increased zinc concentration upregulates the activity of the MT promoter by 10-fold (38). Therefore, to maximally induce beta-Gal expression, GBEC from MT-lacZ mice were treated with 100 µM ZnSO4 for 24 h, harvested, and then fixed with 0.5% glutaraldehyde in PBS for 10 min at room temperature. Cells were washed three times with PBS. Stock solutions of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal, 50 mg/ml in DMSO), KFeCN [100 mM K4Fe(CN)6 and 100 mM K3Fe(CN)6 in PBS], and 1 M MgCl2 were mixed to yield a final concentration of 1 mg/ml X-Gal, 5 mM KFeCN, and 2 mM MgCl2. One milliliter of this staining solution was added to each well, and the cells were placed in a 37°C oven for 6–24 h. Fixed and stained cells were then photographed at x20–100 magnification using an Olympus phase contrast microscope and camera. A similar protocol was used for GBEC from control (non-MT-lacZ) mice.

Bile acid analysis. Media from GBEC isolated from MT-lacZ and GFP transgenic mice cultured under standard and experimental conditions were analyzed for the presence of bile acids. Lyophilized samples were redissolved in distilled water and applied to solid-phase extraction cartridges (I300mg C18 Bond Elut, Varian, Walnut Creek, CA). The cartridges were washed with 10 ml of water and 3 ml of hexane, and the samples were eluted with 10 ml of methanol. The methanol was evaporated to dryness, and the residue was taken up in 1 ml of methanol for HPLC analysis. HPLC analysis of free and conjugated bile acids was carried out by a modification of the method of Scalia (40) in which ammonium acetate is substituted for the phosphate buffer. Using a Rainin Dynamax system (Rainin/Varian, Walnut Creek, CA), the sample was applied to a 4.6 x 250 mm Spherisorb ODS-2 5-µm column (Phase Separations, Clwyd, UK) and eluted with a gradient of 65% methanol-35% 0.03 M ammonium acetate, pH 4.5, to 90% methanol-10% ammonium acetate buffer followed by 100% methanol. For detection and quantification, an evaporative light-scattering detector (model Mk.III, Alltech, Deerfield, IL) was used.

LDL uptake assay. Cells were cultured under standard or experimental conditions for 2 wk. Acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA) was added to cells at 10 µg/ml, and the cells were incubated at 37°C for 4 h. Cytospin cell preparations were made (100 µl per slide, centrifuged for 3 min at 500 rpm) after the cells were harvested with dispase or trypsin-EDTA. Aqueous mounting medium containing the nuclear stain Hoechst 33342 was used to apply coverslips to the slides. Intracellular DiI-Ac-LDL was visualized using a fluorescence microscope with a rhodamine excitation-emission filter.

Diazepam metabolism assay. Media collected from cells cultured for 2 wk under standard or experimental conditions and incubated for 4 and 24 h at 37°C with 50 µg/ml diazepam were analyzed for the diazepam metabolite temazepam by isocratic HPLC as described elsewhere (1). A Rainin Dynamax system with a 4.5 x 250 mm, 5-µm Spherisorb ODS-2 column (Phase Separation, Deeside, UK) and a Rainin model UV-1 detector was used for HPLC analysis.

RT-PCR. RNA from GBEC isolated from MT-lacZ and GFP transgenic mice was isolated using RNeasy columns (Qiagen, Valencia, CA). RNeasy Mini spin columns were used for cells cultured under standard conditions, and Midi columns were used for cells cultured under experimental conditions. RT was performed using the SuperScript first-strand synthesis system, and PCR was performed using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) for 30 cycles. The sequences for the primer pairs and PCR conditions used for {alpha}-fetoprotein, aldolase B, albumin, and GAPDH were as described elsewhere (25). The primer sequences for Notch 1–4, Jagged 1, and Delta 1 have been described previously (45).

Relative quantitative real-time PCR. {alpha}-Fetoprotein, aldolase B, albumin, hepatocyte nuclear factor (HNF)-4{alpha}, aldehyde dehydrogenase 1, glutamine synthetase, and Sca-1 mRNA levels were evaluated by real-time PCR. All mRNA samples were reverse transcribed as described above and then diluted 1:10 in diethylpyrocarbonate-treated autoclaved H2O (Sigma) and stored in aliquots at –70°C. Relative quantitative real-time PCR was performed using a sequence detection system (ABI 7000, Applied Biosystems, Foster City, CA). The primers and fluorogenic probes were designed and synthesized by Applied Biosystems. Assays were performed in MicroAmp optical reaction tubes and caps. Reactions were set up in triplicate. Briefly, 1 µl of cDNA in 8 µl of water was mixed with 1 µl of primer and probe mix and 10 µl of 2x TaqMan universal master mix (Applied Biosystems) in a 96-well plate. Normal murine liver cDNA served as a positive control sample, and three cDNA negative controls (no RT, no RNA, and water control) were run in parallel with the sample cDNA. The threshold cycle and the standard curve method were used to calculate the relative amount of the target RNA. The PCR sequence was as follows: hold at 50°C for 2 min, run at 95°C for 10 min, and 50 cycle repeats at 95°C for 15 s and 60°C for 1 min. Some samples were also analyzed by multiplex real-time PCR using a primer-limited beta-actin TaqMan endogenous control labeled with VIC reporter dye (Applied Biosystems). This procedure is outlined in the Predeveloped TaqMan Assay Reagents Gene Quantification Protocol (Applied Biosystems). SDS version 1.0 software (Applied Biosystems) was used to analyze real-time and end-point fluorescence. Equal amounts from each sample were pooled to generate a standard curve for each gene. Data were analyzed using Microsoft Excel and calculated using the relative standard curve method. The expression levels of the target genes were normalized to internal beta-actin control samples. The results from one cDNA sample in each run from the standard culture group were chosen as the reference sample and set to a value of 1, and the other samples in the run were related to that reference sample.

Flow cytometry. Subconfluent GBEC isolated from GFP transgenic mice cultured on Transwell inserts were harvested by trypsin-EDTA treatment. Cells were aliquoted into microfuge tubes at 1–2 x 106 cells/tube and resuspended in 1 ml of PBS-0.1% BSA (PBS-BSA). Rat anti-murine CD34 antibody (RAM34, BD Biosciences) was added at 20 µg/ml for 40 min at room temperature. Cells were washed twice with PBS-BSA and then resuspended in 1 ml of PBS-BSA. Alexa 633-labeled goat anti-rat antibody (Molecular Probes, Eugene, OR) was added at 4 µg/ml for 30 min at room temperature. Pelleted cells were fixed in 0.5 ml of 2% paraformaldehyde for 10 min, washed twice with PBS-BSA, and then resuspended in 1 ml of PBS-BSA. The presence of CD34 in the cells was evaluated by influx flow cytometry (Cypopeia, Seattle, WA) followed by population comparison analysis using FlowJo (Tree Star, San Carlos, CA). The same procedure was used with NIH/3T3 cells (American Type Culture Collection, Manassas, VA). NIH/3T3, a murine fibroblast cell line that expresses CD34 (21), was used as a positive control. Cells to which no primary or secondary antibodies were added were used as negative controls.

Cytokeratin 19 immunofluorescence. GBEC cultured under standard or experimental conditions were harvested by scraping, frozen in optimal cutting temperature compound, and cut into 10-µm sections. Liver and gallbladder tissue sections were used as controls. Slides were fixed in formalin for 5 min. Primary antibody was cytokeratin 19 (CK19) at 1:50 dilution (catalog no. sc-33111, Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibody was Alexa 633 donkey anti-goat at 10 µg/ml (Invitrogen). Mounting medium contained Hoechst 33342 nuclear dye. Slides were viewed and digitally captured using a Nikon Eclipse microscope with appropriate filters.

Connexin-32 immunofluorescence. Cells were cultured for 2–3 wk and then harvested after treatment with 4 ml of dispase for 1.5 h at 37°C. For removal of released extracellular matrix, cells were washed with cold PBS. The sample was then diluted in 1 ml of PBS and used to make cytospin slides. Slides were stored at –70°C. Immunohistochemistry was performed using a 1:200 dilution of a rabbit polyclonal anti-connexin-32 antibody (Zymed/Invitrogen, San Francisco, CA). Murine liver and gallbladder tissue were used as controls. Secondary antibody was a 1:200 dilution of a rhodamine-labeled donkey anti-rabbit IgG (catalog no. sc-2095, Santa Cruz Biotechnology). Mounting medium contained Hoechst 33342 nuclear dye.

Statistical analysis. Values are means ± SD. Student's unpaired t-test was used to assess the significance of differences between two groups using Prism version 4.0 (GraphPad, San Diego, CA). Significance levels were established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GBEC from MT-lacZ mice can be induced to express hepatocyte-specific beta-Gal. MT-lacZ mice contain a modified beta-Gal gene attached to the murine MT-1 gene promoter. This promoter is only activated in the liver of the transgenic mouse, conferring hepatocyte-specific beta-Gal expression (38). A simian virus-40 large T-antigen nuclear localization signal sequence targets the expression of beta-Gal to the nucleus. We used this model to determine whether in vitro conditions could be manipulated so that beta-Gal expression was induced in GBEC. When GBEC from MT-lacZ mice were cultured on Transwell inserts above a feeder layer of human myofibroblasts (i.e., standard culture conditions) (22), they formed confluent monolayers of tall columnar cells with characteristics of normal epithelial cells, and they retained this morphology over many (up to 20) passages. When cultured between a layer of Vitrogen and a layer of Matrigel and fed medium that contained HGF and EGF (i.e., experimental culture conditions), the cells had a modified growth pattern, with many cells invading the Matrigel layer and dividing to form distinct multicellular spherical structures, often with a central lumen (Fig. 1, A–F). Some cells remained in the interface between the two layers and formed sheets of cells (not shown). HGF alone promoted the development of the spherical structures higher into the Matrigel layer, whereas EGF alone enhanced the formation of sheets of cells.


Figure 1
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  		Fig. 1. Photomicrographs of gallbladder epithelial cells (GBEC) cultured under experimental conditions. A: macroscopic view of GBEC from MT-lacZ mice cultured under experimental conditions in 35-mm culture plate stained with X-Gal. B: GBEC from MT-lacZ mice cultured under experimental conditions and stained with X-Gal. Magnification x40. C: GBEC from MT-lacZ mice cultured under experimental conditions and stained with X-Gal for 24 h. Magnification x200. D: GBEC from MT-lacZ mice cultured under experimental conditions and stained with X-Gal for 4 h. Magnification x200. E and F: thin (10-µm) section of GBEC from MT-lacZ mouse cultured under experimental conditions and stained with X-Gal (no counterstain). Magnification x400. G: thin (10-µm) section of GBEC from MT-lacZ mouse cultured under experimental conditions and stained with X-Gal and eosin. Magnification x400. H: GBEC from MT-lacZ mice grown on Transwell inserts with media + 40 ng/ml hepatocyte growth factor + 25 ng/ml epidermal growth factor (no Matrigel). Magnification x100. I: thin section of MT-lacZ mouse liver stained with X-Gal. Magnification x400. J: GBEC from MT-lacZ mice grown on Transwell inserts under standard conditions and stained with X-Gal for 24 h. Magnification x40. For all cultures, cells were also treated with 100 µM ZnSO4 for 24 h.

 
GBEC from MT-lacZ mice were stained for beta-Gal expression using X-Gal after 24 h of treatment with 100 µM ZnSO4. No blue staining was observed when cells cultured on Transwell inserts were fed regular media (Fig. 1J), and blue staining was very rarely detected when cells were cultured with media containing HGF and EGF (Fig. 1H). Blue staining was also sparse in cells cultured with media containing HGF or EGF. In contrast, blue staining was prominent when cells were grown between Vitrogen and Matrigel and cultured with media containing HGF and EGF (Fig. 1, A–D) and was distinctly nuclear in localization (Fig. 1, E–G), suggesting differentiation toward a hepatocyte-like phenotype, inasmuch as the appearance was similar to X-Gal staining of liver from MT-lacZ mice (Fig. 1I). Various concentrations of HGF and EGF and various times in culture resulted in 1–50% blue-stained cells. Treatment with 40 ng/ml HGF and 25 ng/ml EGF consistently showed the highest percentage of blue cells. If transforming growth factor-{alpha} (25 ng/ml) was added along with HGF but without EGF, the number of blue cells increased slightly (data not shown). The majority of the blue cells were located in the spherical structures, whereas there was also notable staining of cells in the sheets. When GBEC from MT-lacZ mice were not covered with Matrigel, far fewer cells stained blue, although this number was still consistently higher than the number of blue-staining cells cultured on Transwell inserts without Matrigel. After digestion of the extracellular matrix by dispase, harvested cells were tested for viability by trypan blue exclusion. The cell viability was 75–95% (n = 6). Many of these cells demonstrated two nuclei per cell, although none showed more than two nuclei per cell; 4.6% ± 1.48 (n = 6) of the cells were binucleated. In contrast, GBEC cultured on Transwell inserts under standard conditions invariably demonstrated one nucleus per cell. The finding of double nuclei in the cells cultured under experimental conditions is consistent with a hepatocyte-like phenotype, inasmuch as hepatocytes are known to exhibit this characteristic. GBEC from control (non-MT-lacZ) mice cultured under standard or experimental conditions did not stain blue.

GBEC cultured under experimental conditions produce bile acids. We looked for additional evidence of hepatocyte differentiation in GBEC from MT-lacZ mice cultured under experimental conditions. Bile acid synthesis is a function of hepatocytes that is not shared with GBEC. We therefore used HPLC to analyze media from these cells for bile acids. Cells cultured under experimental conditions produced bile acids, with the highest total concentration measured at 67.3 µM, whereas bile acids were not detectable in media from GBEC cultured under standard conditions.

To show that the results obtained with GBEC from MT-lacZ mice were not due to an artifact of beta-Gal expression, we repeated these studies in GBEC isolated from GFP mice and conducted more detailed analysis of bile acid production. In these mice, all cells express the reporter gene GFP. Therefore, GBEC and hepatocytes would be indistinguishable on the basis of GFP fluorescence. Nevertheless, GBEC from GFP mice mimicked the growth characteristics and morphology of GBEC from MT-lacZ mice when cultured between collagen and Matrigel and incubated with media containing 40 ng/ml HGF and 25 ng/ml EGF (i.e., experimental culture conditions; data not shown).

Media harvested from GBEC isolated from GFP transgenic mice cultured under experimental conditions were tested for the presence of bile acids. After 8 days in culture, substantial concentrations of various bile acids were detected in the media. Total bile acids were 46.4 ± 11.7 µM (n = 4) between days 8 and 10 in culture. Taurocholic acid was the most common bile acid (15.7 ± 10.0 µM, n = 4). The amount of taurocholic acid released into the media decreased over time but was still measurable after 23 days in culture. Glycocholic acid concentration was 7.5 ± 2.6 µM (n = 5) by day 8 and was not significantly changed after 23 days. Taurochenodeoxycholic acid (8.8 ± 2.6 µM), glycochenodeoxycholic acid (4.3 ± 1.5 µM), cholic acid (1.6 ± 0.8 µM), and deoxycholic acid (1.5 ± 1.8 µM) were also detected (n = 5 for each bile acid). Media from GBEC from GFP mice cultured on Transwell inserts under standard conditions did not show detectable quantities of any bile acid. Interestingly, cells cultured under experimental conditions did not acquire the ability to synthesize urea (data not shown).

GBEC cultured under experimental conditions take up LDL and upregulate diazepam metabolism. We sought additional functional evidence for a hepatocyte-like phenotype in GBEC cultured under experimental conditions. LDL uptake is a function of hepatocytes that is not found in normal murine GBEC (41). We confirmed absence of DiI-Ac-LDL uptake in GBEC cultured under standard conditions (Fig. 2, A–C). DiI-Ac-LDL uptake was evident in cells cultured under experimental conditions (Fig. 2, D–F).


Figure 2
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  		Fig. 2. LDL uptake in GBEC cultured under standard or experimental conditions. LDL uptake was measured using DiI-labeled acetylated LDL via fluorescence microscopy. Representative images from 3 separate experiments are shown. GBEC were cultured under standard (A–C) or experimental (D–F) conditions. A and D: DiI-Ac-LDL fluorescence signal. B and E: nuclear stain alone. C and F: merged DiI-Ac-LDL and nuclear signals. Original magnification x400.

 
Benzodiazepine metabolism is mediated by cytochrome P-450 3A4, which is expressed in hepatocytes (4). Although biliary epithelial cells also express cytochrome P-450 family members, including cytochrome P-450 3A4, levels of expression are 5- to 20-fold lower than in autologous hepatocytes (23). We therefore determined whether the capacity of GBEC cultured under experimental conditions to metabolize benzodiazepines increased compared with that of GBEC cultured under standard conditions. Cells were incubated with diazepam for 4 and 24 h, and HPLC was used to analyze the media for the presence of the diazepam metabolite temazepam. A time-dependent increase in temazepam was observed in GBEC cultured under both conditions. Temazepam levels were significantly higher in GBEC cultured under experimental conditions (area under the curve = 11,348 ± 2,925 at 4 h and 38,123 ± 11,640 at 24 h) than in GBEC cultured under standard conditions (1,502 ± 359 at 4 h and 3,720 ± 1,271 at 24 h). The differences were significant (P = 0.003 for the 4-h comparison and P = 0.0045 for the 24-h comparison), representing a 7- to 10-fold increase in the capacity to metabolize diazepam.

Genes expressed in hepatocytes, but not in GBEC, are induced in murine GBEC cultured under experimental conditions. We next performed RT-PCR to look for expression of genes normally expressed in hepatocytes, but not in gallbladders, in the GBEC cultured under experimental conditions. We began with an examination of aldolase B, which is the isoform of aldolase found predominantly in the liver and expressed at lower levels in the kidneys; it is not expressed in the gallbladder (34). We used total RNA from GBEC isolated from GFP and MT-lacZ mice that were cultured under experimental conditions and compared the results with total RNA isolated from GBEC from GFP and MT-lacZ mice cultured under standard conditions on Transwell inserts. Aldolase B mRNA was expressed in GBEC cultured under experimental conditions obtained from separate isolations (Fig. 3).


Figure 3
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  		Fig. 3. Aldolase B mRNA expression in GBEC cultured under standard or experimental conditions. RT-PCR for murine aldolase B was performed after isolation of total RNA from GBEC cultured under standard conditions (a–c), GBEC cultured under experimental conditions (f–m), and green fluorescent protein transgenic (GFP) mouse liver (d and e). Base pair standards are shown in h and n.

 
Aldolase B mRNA expression levels were also quantitatively assessed using real-time PCR, with expression of beta-actin used as a control. Aldolase B mRNA expression was 0.813 ± 0.24 (n = 3) in GBEC cultured under experimental conditions compared with 0.018 ± 0.008 (n = 3, P < 0.05) in GBEC cultured under standard conditions, representing a 45-fold increase in gene expression. Albumin mRNA expression was also increased in GBEC cultured under experimental conditions (3.442 ± 1.41, n = 3) compared with native GBEC (0.124 ± 0.042, n = 3, P < 0.05), which represents a 28-fold increase in gene expression. High levels of aldolase B and albumin mRNA were observed by real-time PCR in normal murine liver, serving as a positive control for this assay (data not shown). {alpha}-Fetoprotein mRNA expression was low in the murine liver controls, but not in GBEC cultured under standard or experimental conditions.

We also performed quantitative multiplex real-time PCR analysis with probes for additional genes expressed in hepatocytes, but not in GBEC. We again analyzed aldolase B expression to confirm our earlier findings and also included HNF-4{alpha}, aldehyde dehydrogenase 1, and glutamine synthetase (Fig. 4). The preponderance of cells cultured under experimental conditions showed higher levels of expression of these genes than did the cells cultured under standard conditions. Again, we found that cells cultured under experimental conditions expressed hepatocyte-specific genes at high or low levels compared with cells cultured under standard conditions; in the majority of cases, the differences in fold increases in gene expression between the experimental culture groups and the standard culture group was significant.


Figure 4
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  		Fig. 4. Relative mRNA expression of genes normally expressed in hepatocytes, but not in GBEC. Murine GBEC were cultured under standard or experimental conditions for up to 18 days, and RNA was extracted for subsequent RT and multiplex real-time PCR analysis. Data are shown for separate sets of cells analyzed for expression of aldolase B (A), hepatocyte nuclear factor(HNF)-4{alpha} (B), aldehyde dehydrogenase 1 (C), and glutamine synthetase (D). Results from up to 7 separate experiments are shown. When cells were cultured under experimental (Exp) conditions, mRNA levels of each hepatocyte-specific gene was found at high (Exp High) or low (Exp Low) levels of expression compared with signals obtained from cells cultured under standard (Std) conditions. *P = 0.0007; +P = 0.01; **P = 0.03; ++P = 0.051; #P = 0.0016; {dagger}P < 0.0001; {dagger}{dagger}P = 0.016.

 
Corresponding mRNA levels of each marker in murine liver were invariably higher than those measured in GBEC cultured under experimental conditions and quite variable with respect to each other (expressed as fold increase from levels detected in GBEC cultured under standard conditions): 4.0 x 106 ± 1.4 x 106 for aldolase B, 148.8 ± 54.6 for HNF-4{alpha}, 9,650.9 ± 5,101.0 for aldehyde dehydrogenase 1, and 530 ± 52.6 for glutamine synthetase. When expressed as a percentage of levels in GBEC cultured under experimental conditions relative to levels expressed in normal murine liver, the values were 0.007% for aldolase B, 5.4% for HNF-4{alpha}, 12.8% for aldehyde dehydrogenase 1, and 16.3% for glutamine synthetase.

Loss of expression of Notch family genes occurs when GBEC are cultured under experimental conditions. The Notch signaling pathway has been implicated in cell fate determination, and mutations in Jagged 1, the ligand for Notch 2, leads to Alagille syndrome, which is characterized by biliary atresia (26, 33). This led us examine whether changes in expression of Notch family genes occurred when GBEC were cultured under experimental conditions. As shown in Table 1, all the Notch family members tested were expressed in GBEC cultured under standard conditions. Expression of Notch 1, Notch 3, and Jagged 1 was lost when cells were cultured under experimental conditions, whereas expression of Notch 2 was downregulated. Overall, the mRNA levels of Notch gene family members from GBEC cultured under experimental conditions approximated those in murine liver.


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  		Table 1. mRNA expression of Notch family members

 
The hematopoietic stem cell marker CD34 and the oval cell marker Sca-1 are not present in GBEC isolated from GFP mice. CD34-positive bone marrow-derived stem cells resident in the gallbladder at the time of cell isolation could theoretically be a source of cells predestined to differentiate into hepatocyte-like cells (49). To rule out this possibility, we performed flow cytometry using antibodies against murine CD34 on GBEC isolated from GFP mice cultured under standard conditions. The data were analyzed using population comparison and the Overton cumulative histogram subtraction algorithm to determine the percentage of CD34-positive cells in a population compared with a negative control (no primary antibody). We used murine NIH/3T3 cells as a positive control for CD34 expression, inasmuch as these cells are known to express this marker (21). In two separate experiments, 42.3 ± 0.9% of NIH/3T3 cells were CD34 positive, whereas only 0.1 ± 0.1% of GBEC isolated from GFP mice and cultured under standard conditions were CD34 positive. The latter result is in the range of background readings and is a false positive (i.e., the percentage of CD34-positive GBEC was the same as that in samples that had no primary or secondary antibody). Figure 5 shows representative plots of the flow cytometry results. We also performed real-time multiplex PCR experiments from murine GBEC for the hepatic oval cell marker Sca-1. No Sca-1 mRNA was detectable in multiple different sets of murine GBEC. Furthermore, {alpha}-fetoprotein, a marker of fetal hepatoblasts that can also be a marker for oval cells (43), was not found in murine GBEC by real-time PCR. We conclude that GBEC isolated from GFP mice do not express CD34, Sca-1, or {alpha}-fetoprotein and, therefore, do not contain stem cells derived from the hematopoietic system or oval cells.


Figure 5
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  		Fig. 5. Absence of CD34 expression in GBEC cultured under standard conditions. Flow cytometry was performed using an antibody specific for murine CD34. Top: GBEC isolated from GFP mice and cultured under standard conditions. Bottom: NIH/3T3 cells. Results represent values from 2 experiments with similar results. Secondary antibody was Alexa 633. Histograms depicting cell populations with and without primary antibody completely overlap for GBEC isolated from GFP transgenic mice.

 
CK-19 is selectively downregulated in GBEC cultured under experimental conditions. Expression levels of the biliary epithelial cell marker CK-19 are uniformly high in murine GBEC, as shown by immunohistochemical staining in murine GBEC cultured under standard conditions (Fig. 6A). When murine GBEC are cultured under experimental conditions, there is selective loss of expression of CK-19 (Fig. 6C), as shown by comparison with staining by a nuclear marker on the same section (Fig. 6D). These images also show that the loss of expression of CK-19 is not uniform.


Figure 6
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  		Fig. 6. Cytokeratin 19 (CK-19) expression in murine GBEC cultured under standard and experimental conditions. Immunohistochemistry using a CK-19 antibody was performed on murine GBEC cultured under standard (A) and experimental (C) conditions. B: section shown in A stained with nuclear marker Hoechst 33342. D: section shown in C stained with Hoechst 33342. Large arrows point to regions where no CK-19 expression is evident and small arrows to a region where cells express CK-19. Original magnification x200.

 
Connexin-32 is expressed in GBEC cultured under experimental conditions. Connexin-32, a component of gap junctions in hepatocytes, has been used as a marker of hepatocyte-like cells (2, 15, 20). Normal murine liver showed abundant staining with a specific anti-connexin-32 antibody near or on the sinusoidal membranes of hepatocytes (Fig. 7A). Biliary epithelia cells in murine liver do not express connexin-32 (arrow, Fig. 7B). Furthermore, no connexin-32 was observed in murine gallbladder, either in the epithelial cells or in cells in the subepithelial regions (Fig. 7C). GBEC cultured under standard conditions similarly did not show any expression of connexin-32 (Fig. 7D). In contrast, GBEC cultured under experimental conditions showed abundant connexin-32 expression in a distribution reminiscent of that seen in native hepatocytes (Fig. 7E). The heterogeneous expression pattern of connexin-32 in cells cultured under experimental conditions is also shown in Fig. 7E.


Figure 7
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  		Fig. 7. Connexin-32 expression in normal murine liver, normal murine gallbladder, and GBEC cultured under standard or experimental conditions. An anti-connexin-32 antibody was used for immunohistochemical analysis of normal murine liver sections (A and B), normal murine gallbladder (C), and GBEC cultured under standard (D) and experimental (E) conditions. Connexin-32 expression is evident as punctate red signals. Arrow in B points to bile duct epithelial cells within the murine liver that do not express connexin-32. Original magnification x400 (A, D, and E) and x200 (B and C).

 

   DISCUSSION
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 MATERIALS AND METHODS
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In the present study, we have shown that GBEC isolated from genetically labeled mice can be induced to differentiate into hepatocyte-like cells. The use of in vitro conditions and cells derived from the extrahepatic biliary system precludes contamination with putative stem cells derived from the liver or with hepatocytes. Furthermore, the lack of expression of the hematopoietic stem cell and oval cell markers CD34, Sca-1, and {alpha}-fetoprotein in well-differentiated murine GBEC argues against a stem cell niche as a source for the cells that differentiated into a hepatocyte-like phenotype. In addition, when our GBEC are seeded onto a culture plate, at any given time, except when confluency is attained, most cells take part in active proliferation. Flow cytometry analysis showed 40–70% of cells in active DNA synthesis (S phase) during days 1–10 of culture. When the cells are confluent, the percentage of cells in the S phase is reduced to <1% (22, 31, 32). These data argue against a model wherein our cell culture system contains a small subpopulation of progenitor cells. Taken together, the evidence suggests that GBEC can differentiate toward a hepatocyte-like phenotype under suitable in vitro conditions.

The ability of adult well-differentiated mammalian cells to change their characteristics and become a different type of cell has been well documented. For example, hepatocytes are able to develop in the pancreas under certain conditions (37), whereas pancreatic cells can repopulate damaged livers of mice (7, 10, 16, 46, 52). More recently, studies have shown that hepatocytes can be induced to differentiate into cells with the characteristics of biliary epithelial cells in vitro and in vivo (28, 30). In all these studies, the possibility that stem cells resident within the donor cell population could have been the source of the transdifferentiated cells was not definitively ruled out. Therefore, in none of these studies was the concept that terminally differentiated cells from one organ changed into terminally differentiated cells of another organ proven. In contrast, the use of an extrahepatic epithelial cell line as the starting point for our studies and the lack of expression of CD34, Sca-1, and {alpha}-fetoprotein in these cells argue that differentiation toward a hepatocyte-like cell occurred without the participation of a stem cell compartment. We considered performing single-cell experiments, wherein individual murine GBEC are cultured under experimental conditions, as an additional means to prove that biliary epithelial progenitor cells were not involved in the evolution toward hepatocyte-like cells. This approach would not prove the absence of stem cells, however, inasmuch as it would not be possible to characterize the starting cell, because stem cell markers in GBEC are not defined. Furthermore, because GBEC, when cultured under standard or experimental conditions, did not grow when isolated into single cells, such experiments are impossible to perform.

Two different epithelial cell lines isolated from the murine gallbladder were used in these experiments. GBEC isolated from MT-lacZ mice were used to establish the growth conditions that allowed well-differentiated murine GBEC to change into hepatocyte-like cells (38). Since GBEC from MT-lacZ mice contain a hepatocyte-specific promoter controlling expression of the beta-Gal gene, this model allowed an efficient means to screen different growth conditions and incubation times. These studies showed that a combination of HGF and EGF in the culture medium on a collagen-Matrigel "sandwich" led to the most pronounced expression of hepatocyte-specific beta-Gal. We then switched to GBEC isolated from GFP mice and found that identical phenotypic and gene expression changes could be induced when these cells were cultured under the same experimental conditions. A hepatocyte-like phenotype induced in the cells from GFP mice cultured under experimental conditions was supported by the marked increase in bile acid synthesis in these cells, by the ability to take up LDL particles, and by the enhancement of the capability to metabolize diazepam.

Striking cellular morphological changes were observed when cells were cultured under experimental conditions. Instead of the tall columnar cells with prominent microvilli characteristic of well-differentiated GBEC (22), cells assumed a spherical configuration and/or grew in sheets, depending on the types of extracellular matrix and growth factors used. The spherical structures often developed a central lumen. These findings suggested that morphological features of GBEC and hepatocytes were retained in the cells cultured under experimental conditions. This intermediate morphology is consistent with that observed when exocrine pancreas cells differentiated into hepatocyte-like cells (24) and hepatocytes differentiated into bile duct-like cells (30).

Genes normally expressed in hepatocytes, but not in GBEC, were induced in the cells cultured under experimental conditions at variable levels. Conversely, not all cells cultured under experimental conditions lost expression of the biliary cell marker CK-19. These observations suggest that the transformation from a biliary epithelial cell phenotype to a hepatocyte phenotype was not a complete "all-or-none" phenomenon. Rather, this finding argues for a continuum of changes in gene expression, rather than a "master switch," in the differentiation process. One candidate master switch that could turn on hepatocyte-specific genes is CCAAT enhancer-binding protein-beta (C/EBP-beta). Several groups reported that pancreatic exocrine cells differentiate into hepatocyte-like cells after treatment with dexamethasone, which induces C/EBP-beta expression (11, 12, 24, 44). Whereas dexamathasone was present in the experimental culture medium, induction of C/EBP-beta in GBEC was not investigated. Whether a threshold level of C/EBP-beta expression could achieve more complete phenotypic conversion to hepatocytes requires further investigation. Furthermore, the lack of a complete and absolute switch to the hepatocyte phenotype argues against contamination of our cell preparation with murine hepatocytes.

We are careful to avoid the use of the term "transdifferentiation" to describe the process that occurred in our cells, inasmuch as these cells acquired some, but not all, phenotypic characteristics of hepatocytes. The latter finding raises the philosophical question: When is a hepatocyte a hepatocyte? Our study shows that the answer is likely to be complex, inasmuch as certain features of "hepatocyte-ness," such as bile acid synthesis, are acquired when other features, such as urea synthesis, are not. Bile acid synthesis is a specific function of hepatocytes that is not shared with GBEC; that GBEC cultured under experimental conditions acquired the ability to synthesize bile acids is, from a functional standpoint, one of the most compelling findings of the present study. A spectrum of changes in differentiation, therefore, is more likely to occur with in vitro manipulation, rather than the complete switch in phenotype that is implied by the term transdifferentiation. Our study also discounts the involvement of cell fusion as a mechanism, because cells changed phenotype over time in an in vitro system.

Adult human liver expresses all four Notch receptors, with expression noted in hepatocytes, medium-sized bile ducts, and the sinusoidal epithelium (29). Although mRNA for Notch 1, 2, and 4 was detectable in normal mouse liver tissue, we did not detect Notch 3. This finding was not unexpected, inasmuch as in human liver tissue the expression levels of Notch 3 are extremely low compared with the other Notch receptors (29). Additionally, a similar absence of Notch 3 mRNA expression is found in embryonic and neonatal mouse livers (Fig. 1 in Ref. 47).

Notch receptors and Jagged 1 have been implicated in bile duct formation in the liver (48). Notch 1 and Jagged 1 are also highly expressed in the rodent intrahepatic and extrahepatic biliary systems (19, 47). The robust expression of Notch receptors and Jagged 1 in well-differentiated GBEC argue for a role in the maintenance of the biliary epithelial cell phenotype. Downregulation of expression of Jagged 1 and Notch receptors in GBEC cultured under experimental conditions may allow cells to differentiate away from the biliary epithelial cell phenotype, analogous to the biliary atresia phenotype seen in patients with Alagille syndrome (9, 13, 26, 27, 33). An intriguing question is whether downregulation of Notch 1 and Jagged 1 is simply a marker of a change in differentiation from biliary epithelial cells to hepatocytes or whether such downregulation is causally related to this change in differentiation. The latter possibility is supported by several studies. For example, Notch 2/Jagged 1 signaling is important in the formation of intrahepatic bile ducts (18), and Notch 1 and Jagged 1 are upregulated during liver regeneration following partial hepatectomy (19). Hes-1, a downstream target of Notch 1 and Notch 2/Jagged 1 signaling, is expressed in the extrahepatic biliary system, and hes1-null mice display gallbladder agenesis and abnormalities in intrahepatic bile duct development (47). Furthermore, exposure to Jagged 1 induced transactivation of the Hes-1 promoter and increased expression of biliary epithelial cell markers in the bipotential liver precursor cell line WB-F344 (18). Therefore, loss of expression of Notch 1, Notch 2, and Jagged 1 and subsequent loss of Hes-1 activation may have had a causal role in differentiation of murine GBEC away from a biliary epithelial cell phenotype. Interestingly, in the study by Sumazaki et al. (47), loss of Hes-1 expression led to a conversion from a biliary to a pancreatic phenotype. Additional studies are needed to determine whether 1) Hes-1 expression or function was compromised under our experimental conditions and 2) GBEC cultured under experimental conditions upregulated pancreas-specific genes.

The significance of our work does not lie in a quantitative assessment of the rate of differentiation of cells cultured under experimental conditions (whether assessed by the percentage of cells acquiring hepatocyte-like changes or by comparison with the extent of expression in bona fide murine hepatocytes); rather, the significance lies in the demonstration that terminally differentiated murine GBEC have the capability to acquire hepatocyte-like features, including functional attributes, such as bile acid synthesis. Our data suggest that when this occurs, it does so in a continuum, so that a mixed cell population results. Many cells cultured under experimental conditions remained terminally differentiated GBEC, whereas some acquired hepatocyte-like properties to varying degrees. Indeed, it is more difficult to demonstrate loss of expression of biliary epithelial cell markers than gain of expression of hepatocyte markers for the simple reason that a mixed population of cells ranging from native GBEC to cells with variable levels of expression of biliary and hepatocyte markers is observed when cells are cultured under experimental conditions. Preliminary data from ongoing studies in our laboratory addressing the feasibility of transplanting such cells into recipient mouse livers show that such cells successfully engraft and continue to express hepatocyte-like phenotypic characteristics over many months.

Our studies raise the possibility of a therapeutic use for gallbladders in patients with chronic liver disease. More than 500,000 cholecystectomies are performed annually in the United States. Isolation of GBEC from these gallbladders followed by in vitro culture under conditions similar to those described in the present study could provide a supply of cells with therapeutic potential if transplanted into the livers of patients with chronic liver disease. Additionally, inasmuch as laparoscopic cholecystectomy is a relatively safe procedure, harvesting of the gallbladder for the purpose of isolating GBEC that can be induced to differentiate into cells with phenotypic characteristics of hepatocytes for subsequent liver cell replacement therapy could become a viable alternative to orthotopic liver transplantation.


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REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-061157 and a Merit Review Award from the Department of Veterans Affairs.


   ACKNOWLEDGMENTS
 
We thank the Peter Rabinovitch Laboratory (Department of Pathology, University of Washington) for assistance with flow cytometry. We also thank Jonathan Rhim (Department of Pathology, University of Washington) for assistance with the transgenic mice.

Present address of S. K. Lee: Asian Medical Center, University of Ulsan, College of Medicine, Pungnapdong, Songpagu, Seoul, Korea.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Kuver, Div. of Gastroenterology, Box 356424, Univ. of Washington, 1959 NE Pacific St., Seattle, WA 98195 (e-mail: kuver{at}u.washington.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 

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