Transdifferentiation of Mature Rat Hepatocytes into Bile Duct-Like Cells in Vitro
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62328-0
ISSN1525-2191
AutoresYuji Nishikawa, Yuko Doi, Hitoshi Watanabe, Takuo Tokairin, Yasufumi Omori, Mu Su, Toshiaki Yoshioka, Katsuhiko Enomoto,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoWe investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways. We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways. Bile ductules are known to increase in the portal area in chronic liver disease associated with portal fibrosis and inflammation. This tissue reaction has been referred to as the atypical ductular reaction, which can be distinguished from the typical ductular reaction because of regular proliferation of pre-existing bile duct cells seen in acute biliary obstruction.1Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Pathol Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (171) Google Scholar, 2Popper H The relation of mesenchymal cell products to hepatic epithelial system.in: Popper H Schaffner F Progress in Liver Diseases. W. B. Saunders, Philadelphia1990: 27-38Google Scholar The progressive permeation of the liver lobules by irregular ductular structures appears to further hamper the function of the liver. The cellular origin of the newly formed bile ductules in the atypical ductular reaction has been a subject of debate. It has been speculated that emergent ductules in the reaction might be derived from putative liver stem cells.3Haque S Haruna Y Saito K Nalesnik MA Atillasoy E Thung SN Gerber MA Identification of bipotential progenitor cells in human liver regeneration.Lab Invest. 1996; 75: 699-705PubMed Google Scholar, 4Theise ND Saxena R Portmann BC Thung SN Yee H Chiriboga L Kumar A Crawford JM The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (639) Google Scholar Liver stem cells include transit-amplifying ductular cells (oval cells) and periductal stem cells, which reside in the terminal bile ductules and periductal areas of the adult liver, respectively.4Theise ND Saxena R Portmann BC Thung SN Yee H Chiriboga L Kumar A Crawford JM The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (639) Google Scholar, 5Alison M Liver stem cells: a two compartment system.Curr Opin Cell Biol. 1998; 10: 710-715Crossref PubMed Scopus (95) Google Scholar, 6Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (386) Google Scholar They are considered to be bipotential (having a capacity to differentiate into both hepatocytes and bile ductular cells) or multipotential, and some of them might be derived from the bone marrow. Although mature hepatocytes have an extensive self-renewal capacity during liver injury and are regarded as functional stem cells,7Overturf K al-Dhalimy M Ou CN Finegold M Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 151: 1273-1280PubMed Google Scholar, 8Rhim JA Sandgren EP Degen JL Palmiter RD Brinster RL Replacement of diseased mouse liver by hepatic cell transplantation.Science. 1994; 263: 1149-1152Crossref PubMed Scopus (525) Google Scholar, 9Sandgren EP Palmiter RD Heckel JL Daugherty CC Brinster RL Degen JL Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (341) Google Scholar it has been generally supposed that their phenotype is fixed, thereby constituting a unipotential stem cell system.5Alison M Liver stem cells: a two compartment system.Curr Opin Cell Biol. 1998; 10: 710-715Crossref PubMed Scopus (95) Google Scholar, 6Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (386) Google Scholar However, as discussed extensively by Desmet and colleagues,1Desmet V Roskams T Van Eyken P Ductular reaction in the liver.Pathol Res Pract. 1995; 191: 513-524Crossref PubMed Scopus (171) Google Scholar it is also possible that at least some types of ductular reaction might be because of metaplastic differentiation of mature hepatocytes into bile ductules (ductular metaplasia). We previously reported that aggregates of adult rat hepatocytes cultured within type I collagen gels underwent branching morphogenesis with expression of bile duct-specific cytokeratin (CK) 19.10Nishikawa Y Tokusashi Y Kadohama T Nishimori H Ogawa K Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix.Exp Cell Res. 1996; 223: 357-371Crossref PubMed Scopus (69) Google Scholar Other investigators also provided evidence of possible phenotypic changes of mature hepatocytes to bile duct-like cells.11Block GD Locker J Bowen WC Petersen BE Katyal S Strom SC Riley T Howard TA Michalopoulos GK Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.J Cell Biol. 1996; 132: 1133-1149Crossref PubMed Scopus (428) Google Scholar, 12Cable EE Isom HC Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures.Hepatology. 1997; 26: 1444-1457Crossref PubMed Scopus (37) Google Scholar, 13Michalopoulos GK Bowen WC Zajac VF Beer-Stolz D Watkins S Kostrubsky V Strom SC Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor.Hepatology. 1999; 29: 90-100Crossref PubMed Scopus (109) Google Scholar, 14Michalopoulos GK Bowen WC Mule K Stolz DB Histological organization in hepatocyte organoid cultures.Am J Pathol. 2001; 159: 1877-1887Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Michalopoulos GK Bowen WC Mule K Lopez-Talavera JC Mars W Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures.Hepatology. 2002; 36: 278-283Crossref PubMed Scopus (75) Google Scholar Although these data strongly suggest the bipotentiality of mature hepatocytes, the detailed process and mechanism of the transformation have not been described, and true bile ductular structures, ie, small round ductules surrounded by basement membranes, have not been demonstrated to appear in hepatocytic cultures. Furthermore, it has not been clear whether these changes are transdifferentiation of hepatocytes to bile duct cells, or are mediated by dedifferentiation of hepatocytes to more primitive (stem-like) cells, such as oval cells or hepatoblasts. In the present study, we have shown that hepatocytes express several bile duct markers, including CK19, soon after being cultured, but they do not express Delta-like, a recently identified marker for oval cells and hepatoblasts.16Jensen CH Jauho EI Santoni-Rugiu E Holmskov U Teisner B Tygstrup N Bisgaard HC Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.Am J Pathol. 2004; 164: 1347-1359Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 17Tanimizu N Nishikawa M Saito H Tsujimura T Miyajima A Isolation of hepatoblasts based on the expression of Dlk/Pref-1.J Cell Sci. 2003; 116: 1775-1786Crossref PubMed Scopus (297) Google Scholar We have also suggested that transdifferentiation of hepatocytes is associated with the activation of the Notch signaling pathway, which is crucial in normal bile duct differentiation,18Crosby HA Nijjar SS de Goyet Jde V Kelly DA Strain AJ Progenitor cells of the biliary epithelial cell lineage.Semin Cell Dev Biol. 2002; 13: 397-403Crossref PubMed Scopus (50) Google Scholar and that bile duct-like differentiation is dependent on specific protein tyrosine phosphorylation pathways, such as those mediated by MEK1 and PI 3 kinase. Finally, we have demonstrated that hepatocytes actually form round ductular structures, which are morphologically and immunocytochemically indistinguishable from bile ductules, within type I collagen gels in long-term cultures. Hepatocytes of male F344 rats (6 to 10 weeks old) were isolated by the two-step collagenase perfusion method, followed by repeated low-speed centrifugation at 70 × g. Isolated hepatocytes were plated onto positively charged plastic dishes (Primaria; Becton-Dickinson Labware, Franklin Lakes, NJ) to form spheroidal aggregates. In some experiments, cells were also plated on collagen-coated plastic dishes. Cells were cultured in serum-free Williams’ E medium supplemented with 10 mmol/L nicotinamide, 10 ng/ml mouse epidermal growth factor (EGF) (Roche Diagnostics, Mannheim, Germany), and 10−7 mol/L insulin (Sigma Chemical Company, St. Louis, MO). After 4 or 5 days, formed spheroidal aggregates were harvested and used for three-dimensional cultures. In some experiments, periportal and perivenular hepatocytes were selectively isolated by the digitonin-collagenase perfusion technique.19Lindros KO Penttila KE Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes.Biochem J. 1985; 228: 757-760Crossref PubMed Scopus (165) Google Scholar Hepatocytes were also isolated from Ws/Ws rat, a mutant for the c-kit receptor tyrosine kinase20Tsujimura T Hirota S Nomura S Niwa Y Yamazaki M Tono T Morii E Kim HM Kondo K Nishimune Y Kitamura Y Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene.Blood. 1991; 78: 1942-1946PubMed Google Scholar that is known to be defective in oval cell proliferation.21Matsusaka S Tsujimura T Toyosaka A Nakasho K Sugihara A Okamoto E Uematsu K Terada N Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model.Hepatology. 1999; 29: 670-676Crossref PubMed Scopus (77) Google Scholar Three-dimensional cultures of hepatocytic spheroids within the collagen gel matrix were performed using a type I collagen solution (Cellmatrix Type I-A; Nitta Gelatin, Osaka, Japan) as described previously.10Nishikawa Y Tokusashi Y Kadohama T Nishimori H Ogawa K Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix.Exp Cell Res. 1996; 223: 357-371Crossref PubMed Scopus (69) Google Scholar After embedding the spheroidal aggregates within the matrix, cells were cultured in Williams’ E medium supplemented with 10 mmol/L nicotinamide and 10% fetal bovine serum (standard medium), with or without insulin (10−7 mol/L) and EGF (10 ng/ml). To examine the effects of increased protein tyrosine phosphorylation on hepatocytic differentiation, a protein tyrosine phosphatase inhibitor, sodium orthovanadate (OV; Wako Pure Chemical Industries, Osaka, Japan) was added to the medium at various concentrations. In some experiments, dexamethasone (Sigma), a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059; Cell Signaling, Beverly, MA), or a phosphatidyl inositol (PI) 3-kinase inhibitor (LY294002; Cell Signaling) was also added to the medium. To examine the presence of ductular structures, long-term cultured cells within the collagen gels were fixed in acetic acid-ethanol (1:99), paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). For observation of the ultrastructures, cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, and embedded in Epon resin. Ultrathin sections were stained with uranylate and lead and observed under an electron microscope (JEOL, Tokyo, Japan). Gels containing cells were washed with phosphate-buffered saline and homogenized in a lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 158 mmol/L sodium chloride in 10 mmol/L Tris-HCl buffer, pH 7.5) containing protease inhibitors. Protein samples were also prepared from the nonparenchymal cells, which were present in the supernatant after the first low-speed centrifugation during hepatocyte isolation, and undigested portal tissues. Samples (40 μg protein per lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes. Primary antibodies used were anti-albumin (rat-specific; Nordic Immunological Laboratories, Tilburg, Netherlands), anti-CK19 (Amersham-Pharmacia Biotech UK, Buckinghamshire, UK), anti-CK8 (American Research Products, Belmont, MA), anti-CK20 (Biomeda, Foster City, CA), anti-proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-hepatocyte nuclear factor (HNF)-1 (Santa Cruz), anti-HNF-4α (Santa Cruz), anti-SE-1 (a specific antibody against rat sinusoidal endothelial cells),22Ohmura T Enomoto K Satoh H Sawada N Mori M Establishment of a novel monoclonal antibody, SE-1, which specifically reacts with rat hepatic sinusoidal endothelial cells.J Histochem Cytochem. 1993; 41: 1253-1257Crossref PubMed Scopus (53) Google Scholar PY-20 (TaKaRa, Ohtsu, Japan), and anti-actin (Sigma) antibodies. Detection was performed with enhanced chemiluminescence reagents (Amersham-Pharmacia Biotech UK). We examined gene expression of several hepatocytic and bile duct markers, as well as deleted in malignant brain tumor 1 (DMBT1), a marker for atypical bile ductular reaction,23Bisgaard HC Holmskov U Santoni-Rugiu E Nagy P Nielsen O Ott P Hage E Dalhoff K Rasmussen LJ Tygstrup N Heterogeneity of ductular reactions in adult rat and human liver revealed by novel expression of deleted in malignant brain tumor 1.Am J Pathol. 2002; 161: 1187-1198Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar and Delta-like, a marker for oval cells and hepatoblasts,16Jensen CH Jauho EI Santoni-Rugiu E Holmskov U Teisner B Tygstrup N Bisgaard HC Transit-amplifying ductular (oval) cells and their hepatocytic progeny are characterized by a novel and distinctive expression of delta-like protein/preadipocyte factor 1/fetal antigen 1.Am J Pathol. 2004; 164: 1347-1359Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 17Tanimizu N Nishikawa M Saito H Tsujimura T Miyajima A Isolation of hepatoblasts based on the expression of Dlk/Pref-1.J Cell Sci. 2003; 116: 1775-1786Crossref PubMed Scopus (297) Google Scholar in cultured hepatocytes by RT-PCR. We also examined gene expression of the ligands, receptors, and primary targets of the Notch signaling system, which is thought to be crucial in the development of intrahepatic bile ducts.24Louis AA Van Eyken P Haber BA Hicks C Weinmaster G Taub R Rand EB Hepatic Jagged1 expression studies.Hepatology. 1999; 30: 1269-1275Crossref PubMed Scopus (74) Google Scholar, 25Li L Krantz ID Deng Y Genin A Banta AB Collins CC Qi M Trask BJ Kuo WL Cochran J Costa T Pierpont ME Rand EB Piccoli DA Hood L Spinner NB Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.Nat Genet. 1997; 16: 243-251Crossref PubMed Scopus (1045) Google Scholar, 26Oda T Elkahloun AG Pike BL Okajima K Krantz ID Genin A Piccoli DA Meltzer PS Spinner NB Collins FS Chandrasekharappa SC Mutations in the human Jagged1 gene are responsible for Alagille syndrome.Nat Genet. 1997; 16: 235-242Crossref PubMed Scopus (968) Google Scholar Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was amplified to evaluate the input RNA for each reaction. Total RNA from hepatocytes and portal tissues was prepared by using TRIzol reagent (Life Technologies, Grand Island, NY). A one-step RT-PCR kit (TaKaRa) was used for the reaction, in which RNA (0.5 μg) was reverse-transcribed using AMV reverse transcriptase for 30 minutes and then amplified for 25 cycles of 95, 60, and 72°C for 30, 30, and 90 seconds. The specific primers used and the expected fragment sizes were as follows. Albumin: forward, 5′-TTGCCAAGTACATGTGTGAG-3′; reverse, 5′-GGTTCTTCTACAAGAGGCTG-3′; 373 bp. Transthyretin: forward, 5′-GGGTAGAACTGGACACCAAATC-3′; reverse, 5′-AGAACGT-TTCACGGCATCTTCC-3′, 294 bp. CYP2B2: forward, 5′-GAGTTCTTCTCTGGGTTCCTG-3′; reverse, 5′-ACTGTG-GGTCATGGAGAGCTG-3′; 550 bp. CYP8B1: forward, 5′AGCACGCAGAAAGTGCTAGAC-3′; reverse, 5′-TGTCC-TTGGTGCAGCCAAACA-3′; 301 bp. CK19: forward, 5′-GACTTCCTATAGCTATCGCC-3′; reverse, 5′-TCTGGT-ACCAGTCGCGAATC-3′; 359 bp. Glutathione S-trans-ferase placental form (GST-P): forward, 5′-TCAAGTC-CACTTGTCTGTATG-3′; reverse, 5′-CTGTTTACCGCCG-TTGAT-3′; 500 bp. DMBT1: forward, 5′-GGCACAAATA-ACGATGTGTCC-3′; reverse, 5′-AGTTGTCCACCAATCG-GCTAT-3′; 510 bp. α-Fetoprotein (AFP): forward, 5′-TGAAA-TTTGCCACGAGACGG -3′; reverse, 5′-TGTCATACTGA-GCGGCTAAG-3′; 272 bp (specific to the 2.1 kb transcript).27Miura K Nagai H Ueno Y Goto T Mikami K Nakane K Yoneyama K Watanabe D Terada K Sugiyama T Imai K Senoo H Watanabe S Epimorphin is involved in differentiation of rat hepatic stem-like cells through cell-cell contact.Biochem Biophys Res Commun. 2003; 311: 415-423Crossref PubMed Scopus (34) Google Scholar Delta-like (preadipocyte factor 1): forward, 5′-ATGTCTGCAGGTGTGAGCCTG-3′; reverse, 5′-GGC-TTGCACAGACACTCGAAG-3′; 599 bp. Jagged1: forward, 5′-TCCAGCCTCCAGCCAGTGAA-3′; reverse, 5′-GGAAGGCTCACAGGCTATGT-3′; 201 bp.24Louis AA Van Eyken P Haber BA Hicks C Weinmaster G Taub R Rand EB Hepatic Jagged1 expression studies.Hepatology. 1999; 30: 1269-1275Crossref PubMed Scopus (74) Google Scholar Jagged2: forward, 5′-ACATGCTATGACAGCGGCGA-3′; reverse, 5′CCAGGAACTCCCATGTGGGA-3′; 480 bp. Notch1: forward, 5′-ATCCATGGCTCCATCGTCTA-3′; reverse, 5′TTCTGATTGTCGTCCATCAG-3′; 422 bp. Notch2: forward, 5′-TTTGCTGTCGGAAGACGACC-3′; reverse, 5′-GCCCATGTTGTCCTGGGCGT-3′; 403 bp. Notch3: forward, 5′-TTCCAGATTCTCATCAGGAA-3′; reverse, 5′-TCCTGGTGGGCAGAGCAATG-3′; 480 bp. Hes (hairy/enhancer of split) 1: forward, 5′-CAACACGACACCGGACAAACC-3′; reverse, 5′-AGTGCGCACCTCGGTGTT-AAC-3′; 349 bp. Hes2: forward, 5′-CTTCTGTTTTCGCT-CTGCCTT-3′; reverse, 5′-TCGAGGTAGCTATCCAA-AGAC-3′; 352 bp. Hes3: forward, 5′-TCAGCATCTTTAT-CAGGCCTC-3′; reverse, 5′-CTAGCCAAAGTCCTTGC-AATG-3′; 302 bp. Hes5: forward, 5′-CAAGGAGAAAAA-TCGACTGCG-3′; reverse, 5′-ATGCAGGGTCAGGAACT-GCAC-3′; 280 bp. HERP (Hes-related repressor protein) 1: forward, 5′-ACCCAATGGACTCCACACATC-3′; reverse, 5′-TGTTCCACTGCTTGTCTGCTG-3′; 379 bp. HERP2: forward, 5′-GAGATCTTGCAGATGACTGTG-3′; reverse, 5′-TGTCGAAGGACTCAGTAGATG-3′; 561 bp. GAPDH: forward, 5′-ACCACAGTCCATGCCATCAC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′; 452 bp. Immunocytochemistry for albumin, CK19, CK7, laminin, type IV collagen, Notch1, and Jagged1 was performed on paraffin sections of cells embedded within the gels. The primary antibodies used were as follows. Anti-albumin (Nordic Immunological Laboratories), anti-CK19 (mouse monoclonal, 1:200 dilution; Amersham-Pharmacia Biotech UK), anti-CK7 (mouse monoclonal, 1:20 dilution; ICN Pharmaceuticals, Aurora, OH), anti-laminin (rabbit polyclonal, 1:1000 dilution, LSL; Cosmo Bio, Tokyo, Japan), anti-collagen type IV (mouse monoclonal, 1:100; Chemicon, Temecula, CA), anti-Notch1 (goat polyclonal, 1:100 dilution, C-20; Santa Cruz), and anti-Jagged1 (rabbit polyclonal, 1:100 dilution, H-114; Santa Cruz). Detection was performed with Envision+ system HRP (DAKO, Carpinteria, CA) for albumin, CK19, CK7, laminin, collagen type IV, and Jagged1, and with ImmunoCruz staining system (Santa Cruz) for Notch1. Cells in paraffin sections were also examined for binding sites of Dolichos biflorus agglutinin, a bile duct-specific lectin, using biotin-conjugated Dolichos biflorus agglutinin (EY Laboratories, San Mateo, CA). After plating on Primaria dishes, isolated hepatocytes formed spheroidal aggregates within 3 days (Figure 1A). After being embedded within a collagen gel matrix, hepatocytes began to extend cellular processes into the matrix and, after 7 days, some of the spheroids showed a branching morphology in the presence of insulin and EGF, but not in the absence of these factors (Figure 1B). We examined the expression of several hepatocytic markers and CK in hepatocytes cultured within the collagen gels by Western blot analysis. Hepatocytic markers (albumin, HNF-1, and HNF-4α), as well as CK8, which is known to be normally expressed in hepatocytes, were expressed in the spheroids either before or after being embedded within the gels (Figure 1C). Although there was no detectable protein expression of CK19 and CK20, another bile duct-specific CK,28Faa G Van Eyken P Roskams T Miyazaki H Serreli S Ambu R Desmet VJ Expression of cytokeratin 20 in developing rat liver and in experimental models of ductular and oval cell proliferation.J Hepatol. 1998; 29: 628-633Abstract Full Text PDF PubMed Scopus (35) Google Scholar in freshly isolated hepatocytes and 3-day- and 5-day-cultured spheroids, the cells in the spheroids began to express both CKs 2 days after being embedded within the collagen gel matrix (Figure 1C). A total period of 7 days in culture was necessary to obtain a detectable level of protein expression of the bile duct-specific CK, irrespective of culture forms (monolayer, spheroid, or collagen gel cultures) (data not shown). Actin was expressed in both freshly isolated and cultured hepatocytes, whereas there was an increase in expression after culture (Figure 1C). As shown in Figure 1D, bile duct-specific CK19 expression was found in the portal tissues (containing bile ducts and blood vessels), but absent in freshly isolated hepatocytes and a nonparenchymal cell fraction (containing Kupffer cells, stellate cells, and sinusoidal endothelial cells). CK8 was detected in both hepatocytes and portal tissues, and expression of the specific endothelial cell marker, SE-1,22Ohmura T Enomoto K Satoh H Sawada N Mori M Establishment of a novel monoclonal antibody, SE-1, which specifically reacts with rat hepatic sinusoidal endothelial cells.J Histochem Cytochem. 1993; 41: 1253-1257Crossref PubMed Scopus (53) Google Scholar was only found in the nonparenchymal cell fraction (Figure 1D). Although there was no detectable CK19 protein (Figure 1, C and D) or CK19-immunopositive cells in the isolated hepatocytes,10Nishikawa Y Tokusashi Y Kadohama T Nishimori H Ogawa K Hepatocytic cells form bile duct-like structures within a three-dimensional collagen gel matrix.Exp Cell Res. 1996; 223: 357-371Crossref PubMed Scopus (69) Google Scholar there was a possibility that the phenotypic changes in cultured hepatocytes described above were because of contaminated bile duct cells or liver stem cells in the initial cell fraction. Because bile duct cells and stem cells are present in the periportal areas of the liver lobules, the perivenular areas should be devoid of these cells. Therefore, to test the possible contribution of contaminated cells to the appearance of bile duct markers, we selectively isolated perivenular and periportal hepatocytes by the digitonin infusion method19Lindros KO Penttila KE Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes.Biochem J. 1985; 228: 757-760Crossref PubMed Scopus (165) Google Scholar and compared the morphogenesis and CK protein expression. As shown in Figure 2A, there was no difference between the perivenular hepatocytes in branching morphogenesis. Furthermore, the hepatocytes from both areas expressed bile duct-specific CK (CK19, CK20) to the same extent after being embedded within the collagen gel matrix (Figure 2B). Although our results showed that mature hepatocytes could demonstrate a bile duct-like phenotype soon after culture, it is possible that hepatocytes might have transformed themselves to stem-like cells, such as oval cells, at least transiently. To address the possibility, we examined hepatocytes from c-kit mutant (Ws/Ws) rats,20Tsujimura T Hirota S Nomura S Niwa Y Yamazaki M Tono T Morii E Kim HM Kondo K Nishimune Y Kitamura Y Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene.Blood. 1991; 78: 1942-1946PubMed Google Scholar which were demonstrated to be almost defective in oval cell proliferation after application of hepatocarcinogenic procedures.21Matsusaka S Tsujimura T Toyosaka A Nakasho K Sugihara A Okamoto E Uematsu K Terada N Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model.Hepatology. 1999; 29: 670-676Crossref PubMed Scopus (77) Google Scholar Ws/Ws hepatocytes also showed branching morphogenesis (Figure 2C) and protein expression of bile duct-specific CK similar to those in the control rats (Figure 2D), suggesting that bile duct-like differentiation of mature hepatocytes might not be dependent on oval cell transformation. Cultured hepatocytes fairly well maintained the expression of their differentiation markers, such as albumin, transthyretin, claudin2, and CYP2B2, for at least 2 weeks, whereas the expression of CYP8B1 gradually declined (Figure 3). However, the expression of CK19 mRNA appeared soon after culture, reached a plateau after 5 days in spheroids, and was maintained thereafter within the collagen gels (Figure 3, data not shown). The gene expression of another bile duct marker, GST-P,29Lupp A Anschutz T Lindstrom-Seppa P Muller D Developmental changes in glutathione S-transferase isoforms expression and activity in intrasplenic fetal liver tissue transplants in rats.Exp Toxicol Pathol. 2003; 55: 107-119Crossref PubMed Scopus (2) Google Scholar also dramatically increased and persisted in cultured hepatocytes (Figure 3). Although DMBT1 protein has been reported to be positive in atypical ductular reaction and negative in normal hepatocytes and bile ducts,23Bisgaard HC Holmskov U Santoni-Rugiu E Nagy P Nielsen O Ott P Hage E Dalhoff K Rasmussen LJ Tygstrup N Heterogeneity of ductular reactions in adult rat and human liver revealed by novel expression of deleted in malignant brain tumor 1.Am J Pathol. 2002; 161: 1187-1198Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar its mRNA was clearly detected in normal portal tissues (Figure 3). Notably, DMBT1 gene expression emerged in cultured hepatocytes (Figure 3). The aberrant expression of the DMBT1 gene in hepatocytes also took place in monolayer culture on collagen-coated dishes (Figure 4). As shown in a previous study,30Lemire JM Fausto N Multiple alpha-fetoprotein RNAs in adult rat liver: cell type-specific expression and differential regulation.Cancer Res. 1991; 51: 4656-4664PubMed Google Scholar the major form of AFP mRNA (2.1 kb), which was undetectable in adult hepatocytes, was expressed in portal tissues containing bile ducts, although the expression level was much lower than that of the fetal liver (Figure 3). The AFP mRNA gradually increased in hepatocytes, but its expression level did not exceed that of portal tissues (Figure 3).Figure 4Effects of dexamethasone (Dex) on the morphogenesis of hepatocytic spheroids within a collagen gel matrix and gene expression of several differentiation markers. Hepatocytes were cultured in the presence of insulin (10−7 mol/L) and EGF (10 ng/ml), with or without Dex (10 μmol/L). A: Phase-contrast microscopy of spheroid morphology cultured for 7 days within the gels. Original magnification, ×25. B: RT-PCR analyses for the time course of expression of albumin, CYP2B2, CK19, DMBT1, Jagged1, an
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