Title: Granulocyte–Colony Stimulating Factor Promotes Liver Repair and Induces Oval Cell Migration and Proliferation in Rats
Abstract: Background & Aims: Hepatic regeneration is a heterogeneous phenomenon involving several cell populations. Oval cells are considered liver stem cells, a portion of which derive from bone marrow (BM). Recent studies have shown that granulocyte–colony stimulating factor (G-CSF) may be effective in facilitating liver repair. However, it remains unclear if G-CSF acts by mobilizing BM cells, or if it acts locally within the liver microenvironment to facilitate the endogenous restoration program. In the present study, we assessed the involvement of G-CSF during oval cell activation. Methods: Dipeptidyl-peptidase-IV–deficient female rats received BM transplants from wild-type male donors. Four weeks later, rats were subjected to the 2-acetylaminofluorene/partial hepatectomy model of oval cell–mediated liver regeneration, followed by administration of either nonpegylated G-CSF or pegylated G-CSF. Control animals did not receive further treatments after surgery. The magnitude of oval cell reaction, the entity of BM contribution to liver repopulation, as well as the G-CSF/G-CSF–receptor expression levels were evaluated. In addition, in vitro proliferation and migration assays were performed on freshly isolated oval cells. Results: Oval cells were found to express G-CSF receptor and G-CSF was produced within the regenerating liver. G-CSF administration significantly increased both the magnitude of the oval cell reaction, and the contribution of BM to liver repair. Finally, G-CSF acted as a chemoattractant and a mitogen for oval cells in vitro. Conclusions: We have shown that G-CSF facilitates hepatic regeneration by increasing the migration of BM-derived progenitors to the liver, as well as enhancing the endogenous oval cell reaction. Background & Aims: Hepatic regeneration is a heterogeneous phenomenon involving several cell populations. Oval cells are considered liver stem cells, a portion of which derive from bone marrow (BM). Recent studies have shown that granulocyte–colony stimulating factor (G-CSF) may be effective in facilitating liver repair. However, it remains unclear if G-CSF acts by mobilizing BM cells, or if it acts locally within the liver microenvironment to facilitate the endogenous restoration program. In the present study, we assessed the involvement of G-CSF during oval cell activation. Methods: Dipeptidyl-peptidase-IV–deficient female rats received BM transplants from wild-type male donors. Four weeks later, rats were subjected to the 2-acetylaminofluorene/partial hepatectomy model of oval cell–mediated liver regeneration, followed by administration of either nonpegylated G-CSF or pegylated G-CSF. Control animals did not receive further treatments after surgery. The magnitude of oval cell reaction, the entity of BM contribution to liver repopulation, as well as the G-CSF/G-CSF–receptor expression levels were evaluated. In addition, in vitro proliferation and migration assays were performed on freshly isolated oval cells. Results: Oval cells were found to express G-CSF receptor and G-CSF was produced within the regenerating liver. G-CSF administration significantly increased both the magnitude of the oval cell reaction, and the contribution of BM to liver repair. Finally, G-CSF acted as a chemoattractant and a mitogen for oval cells in vitro. Conclusions: We have shown that G-CSF facilitates hepatic regeneration by increasing the migration of BM-derived progenitors to the liver, as well as enhancing the endogenous oval cell reaction. Liver regeneration is a heterogeneous phenomenon, involving at least 3 levels of proliferating cells: mature hepatocytes, ductular progenitors, and oval cells.1Shackel N.A. Rockey D.C. Stem cells and liver disease: promise laced with confusion and intrigue.Gastroenterology. 2004; 127: 346-348Google Scholar The term oval cells (OCs) defines small proliferating cells with an oval-shaped nucleus and a high nuclear to cytoplasm ratio, which appear within the liver after certain models of injury and carcinogenesis.2Lowes K.N. Croager E.J. Olynyk J.K. Abraham L.J. Yeoh G.C. Oval cell-mediated liver regeneration: role of cytokines and growth factors.J Gastroenterol Hepatol. 2003; 18: 4-12Google Scholar OCs are bipotential, sharing the ability to differentiate into hepatocytes and biliary epithelial cells, and, therefore, represent putative liver stem cells.2Lowes K.N. Croager E.J. Olynyk J.K. Abraham L.J. Yeoh G.C. Oval cell-mediated liver regeneration: role of cytokines and growth factors.J Gastroenterol Hepatol. 2003; 18: 4-12Google Scholar In OC-mediated liver regeneration, OCs arise from the portal tract periphery and migrate into the lobular parenchyma. Trafficking, mobilization, and homing of OCs are regulated by several factors, including adhesion molecules, cytokines, and chemotactic molecules.3Libbrecht L. Desmet V. Van Damme B. Roskams T. Deep intralobular extension of human hepatic progenitor cells correlates with parenchymal inflammation in chronic viral hepatitis: can progenitor cells migrate?.J Pathol. 2000; 192: 373-378Google Scholar As for their origin, some believe that OCs derive from ductular cells of the canals of Hering, whereas others speculate that they arise from liver-committed circulating stem cells of extrahepatic origin.4Crosbie O.M. Reynolds M. McEntee G. Traynor O. Hegarty J.E. O’Farrelly C. In vitro evidence for the presence of hematopoietic stem cells in the adult human liver.Hepatology. 1999; 29: 1193-1198Google Scholar Numerous studies conducted on animal models and human beings have suggested that bone marrow cells (BMCs) may give rise to OCs.5Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. Boggs S.S. Greenberger J.S. Goff J.P. Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Google Scholar, 6Alison M.R. Poulsom R. Jeffery R. Dhillon A.P. Quaglia A. Jacob J. Novelli M. Prentice G. Williamson J. Wright N.A. Hepatocytes from non-hepatic adult stem cells.Nature. 2000; 406: 257Google Scholar, 7Theise N.D. Nimmakayalu M. Gardner R. Illei P.B. Morgan G. Teperman L. Henegariu O. Krause D.S. Liver from bone marrow in humans.Hepatology. 2000; 32: 11-16Google Scholar, 8Piscaglia A.C. Di Campli C. Zocco M.A. Di Gioacchino G. Novi M. Rutella S. Bonanno G. Monego G. Vecchio F.M. Michetti F. Mancuso S. Leone G. Gasbarrini G. Pola P. Gasbarrini A. Human cordonal stem cell intraperitoneal injection can represent a rescue therapy after an acute hepatic damage in immunocompetent rats.Transplant Proc. 2005; 37: 2711-2714Google Scholar, 9Piscaglia A.C. Zocco M.A. Di Campli C. Sparano L. Rutella S. Monego G. Bonanno G. Michetti F. Mancuso S. Pola P. Leone G. Gasbarrini G. Gasbarrini A. How does human stem cell therapy influence gene expression after liver injury? Microarray evaluation on a rat model.Dig Liver Dis. 2005; 37: 952-963Google Scholar In vitro studies also have shown that a subpopulation of BMCs expresses hepatic markers and, conversely, that OCs express hematopoietic antigens such as CD34, c-kit, and Thy-1.10Petersen B.E. Goff J.P. Greenberger J.S. Michalopoulos G.K. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Google Scholar, 11Petersen B.E. Grossbard B. Hatch H. Pi L. Deng J. Scott E.W. Mouse A6-positive hepatic oval cells also express several hematopoietic stem cell markers.Hepatology. 2003; 37: 632-640Google ScholarMobilization of BMCs into the circulation can be induced by a wide variety of molecules such as cytoreductive drugs, chemokines, and hematopoietic cytokines.12Szumilas P. Barcew K. Baskiewicz-Masiuk M. Wiszniewska B. Ratajczak M.Z. Machalinski B. Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice.Cell Prolif. 2005; 38: 47-61Google Scholar Granulocyte–colony stimulating factor (G-CSF) is among the most commonly used BMC mobilizing agents because of its potency and lack of toxicity.13Thomas J. Liu F. Link D.C. Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor.Curr Opin Hematol. 2002; 9: 183-189Google Scholar The biological effects of G-CSF are mediated predominantly through the G-CSF receptor (G-CSFR), and partly through trans-activating signals within the BM microenvironment.14Imamura R. Miyamoto T. Yoshimoto G. Kamezaki K. Ishikawa F. Henzan H. Kato K. Takase K. Numata A. Nagafuji K. Okamura T. Sata M. Harada M. Inaba S. Mobilization of human lymphoid progenitors after treatment with granulocyte colony-stimulating factor.J Immunol. 2005; 175: 2647-2654Google Scholar Recent studies have suggested that G-CSF may be effective in mobilizing cells that contribute to liver repair after damage.15Ratajczak M.Z. Kucia M. Reca R. Majka M. Janowska-Wieczorek A. Ratajczak J. Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells hide out in the bone marrow.Leukemia. 2004; 18: 29-40Google Scholar, 16Yannaki E. Athanasiou E. Xagorari A. Constantinou V. Batsis I. Kaloyannidis P. Proya E. Anagnostopoulos A. Fassas A. G-CSF-primed hematopoietic stem cells or G-CSF per se accelerate recovery and improve survival after liver injury, predominantly by promoting endogenous repair programs.Exp Hematol. 2005; 33: 108-119Google Scholar, 17Quintana-Bustamante O. Alvarez-Barrientos A. Kofman A.V. Fabregat I. Bueren J.A. Theise N.D. Segovia J.C. Hematopoietic mobilization in mice increases the presence of bone marrow-derived hepatocytes via in vivo cell fusion.Hepatology. 2006; 43: 108-116Google Scholar, 18Huiling X. Inagaki M. Arikura J. Ozaki A. Onodera K. Ogawa K. Kasai S. Hepatocytes derived from peripheral blood stem cells of granulocyte-colony stimulating factor treated F344 rats in analbuminemic rat livers.J Surg Res. 2004; 122: 75-82Google Scholar However, it remains unclear if G-CSF acts mainly by recruiting BMCs, or if it acts locally within the liver microenvironment to facilitate the endogenous hepatic restoration program. The potential trophic effects of G-CSF on liver stem cells could have a dramatic clinical impact because G-CSF is one of the few growth factors approved for use in patients.13Thomas J. Liu F. Link D.C. Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor.Curr Opin Hematol. 2002; 9: 183-189Google ScholarIn the present study, we sought to assess the role of G-CSF during OC-mediated liver regeneration. We used the well-established model of OC activation in rats, involving the administration of 2-acetylaminofluorene (2AAF) before partial hepatectomy (PH).19Petersen B.E. Zajac V.F. Michalopoulos G.K. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats.Hepatology. 1998; 27: 1030-1038Google Scholar 2AAF is metabolized selectively by hepatocytes to an N-hydroxyl derivative, which interferes with the cyclin-D1 pathway. Therefore, the administration of 2AAF before PH inhibits hepatocyte proliferation and forces OC recruitment to mediate liver regeneration. This procedure results in a robust OC response after PH (peaking between days 9 and 11), and within 14 days OCs begin to differentiate into hepatocytes.20Alison M.R. Golding M. Sarraf C.E. Edwards R.J. Lalani E.N. Liver damage in the rat induces hepatocyte stem cells from biliary epithelial cells.Gastroenterology. 1996; 110: 1182-1190Google Scholar In addition, we have investigated the effects of G-CSF administration after 2AAF/PH in terms of magnitude of the OC response, as well as the degree of BMC contribution to the regenerative process. To assess the BM contribution to liver repopulation, we used the dipeptidyl-peptidase-IV (DPPIV)-deficient rat model.5Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. Boggs S.S. Greenberger J.S. Goff J.P. Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Google Scholar DPPIV is an exopeptidase expressed by many cell types, including BM, blood, and hepatic cells. After BM transplantation (BMTx) from wild-type rats into DPPIV− animals, the expression of DPPIV can be used to detect donor-derived cells in the chimeric recipients. Finally, we have evaluated the effects of G-CSF on OCs through in vitro assays. We have shown that G-CSF enhances hepatic regeneration after BMTx/2AAF/PH in rats by mobilizing liver committed BMCs, as well as by promoting migration and proliferation of endogenous OCs.Materials and MethodsAnimal TreatmentsAnimalsDPPIV− F344 female rats (age, 8–10 wk) were in-house bred and maintained on standard laboratory chow and daily cycles, alternating 12 hours of light and dark. Wild-type F344 male rats (age, 8–10 wk) were purchased from Charles River Laboratories (Wilmington, MA). All procedures were performed with the approval of the University of Florida Institutional Animal Care and Usage Committee. The experimental design is summarized in Figure 1.BMC isolation and transplantationBefore BMTx, DPPIV− female rats were exposed to total body γ-irradiation (137Cs, JL Shepherd Mark-I; J.L. Shepherd and Associates, San Fernando, CA), administered in 2 doses of 450 rads each, 3 hours apart. BMCs were isolated from the long bones of donor rats. Cells were passed through a 130-μm cell strainer, collected by centrifugation at 220 × g, and resuspended in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO, Grand Island, NY). BMCs were transplanted into recipient rats via tail vein injection after irradiation (5 × 107 cells/rat). Three weeks later, the donor contribution to BM reconstitution was assessed through analysis of the presence of Y-chromosome and DPPIV in blood cells.OC activation model and G-CSF administrationRecombinant methionyl human G-CSF (Filgrastim) and its pegylated counterpart (peg-Filgrastim, Peg–G-CSF) were kindly provided by Amgen Inc. (Thousand Oaks, CA). The 2AAF/PH regimen for OC induction was performed as previously described.19Petersen B.E. Zajac V.F. Michalopoulos G.K. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats.Hepatology. 1998; 27: 1030-1038Google Scholar Briefly, 4 weeks after BMTx, chimeric animals were implanted intraperitoneally with a time-released 2AAF pellet (70 mg/28-day release; Innovative Research of America, Sarasota, FL). Seven days later, rats underwent PH, as described elsewhere.21Higgins G.M. Anderson R.M. Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial surgical removal.Arch Pathol. 1931; 12: 186-202Google Scholar Animals then were administered subcutaneously Peg–G-CSF (10 mg/kg in a single dose; group A), or nonpegylated G-CSF (250 μg/kg/day for 5 days; group B). Control rats did not receive any treatment after 2AAF/PH (group C). Postsurgery, animals were placed in general housing until death at 1, 3, 5, 7, 11, 15, and 28 days after PH (21 animals in each group, 3 rats/time point). Samples of liver tissue were collected separately in optical cut temperature embedding medium (Sakura Finetek USA, Inc, Torrance, CA), snap-frozen in liquid nitrogen, and embedded in paraffin after overnight fixation in 10% formalin.Histology, Immunohistochemistry, and ImmunofluorescenceFor morphology studies, 5-μm paraffin sections were stained with H&E. Immunohistochemistry and immunofluorescence were performed either on 5-μm paraffin-embedded or OCT frozen sections using standard staining protocols. Immunophenotyping of liver samples used 1:100 mouse anti-Ki67 (proliferation index; BD Biosciences Pharmingen, San Diego, CA); 1:100 mouse anti-OV6 (oval and ductular cell marker; a generous gift from Dr Sell, Albany, NY); 1:100 sheep anti–α-fetoprotein (anti-AFP) (OCs and progenitor cell marker; Nordic Immunological Lab., Tilburg, The Netherlands); 1:100 mouse anti-CD45 (common leukocyte antigen; BD Biosciences Pharmingen); 1:100 rabbit anti–G-CSFR and 1:100 goat anti–G-CSF (both from Santa Cruz Biotechnologies, Santa Cruz, CA). Negative controls and isotype controls (Vector Laboratories, Burlingame, CA) showed negligible autofluorescence and nonspecific binding of primary antibodies to cells/tissue. Vector ABC-kit (Vector Laboratories) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Dakocytomation, Carpinteria, CA) were used in the immunoperoxidase detection procedure. For immunofluorescence staining, Vectastain kit with DAPI, Texas-red, and fluorescein-conjugated secondary antibodies (Vector Laboratories) were used. The enzymatic DPPIV staining procedure was performed as previously described.22Oh S.E. Witek R.P. Bae S.H. Zheng D. Jung Y. Piscaglia A.C. Petersen B.E. Bone-marrow derived hepatic oval cells differentiate into hepatocytes in 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration.Gastroenterology. 2007; 132: 1077-1087Abstract Full Text Full Text PDF Scopus (145) Google Scholar To confirm the epithelial nature of donor-derived cells within the liver, double immunofluorescence for OV6 and CD26 was performed, using 1:100 goat anti-CD26 (anti-DPPIV, Santa Cruz Biotechnologies). Quantification of Ki67, OV6, and DPPIV+ cells was obtained through the analysis of 5–8 fields selected randomly from each specimen (objective magnification, 20×). The samples were photographed using an Olympus microscope and an Optronics digital camera (Olympus, Melville, NY). Selected slides also were analyzed by confocal microscopy (Spectra Confocal Microscope TCS-SP2-AOBS, equipped with Software V.2.61; Leica Microsystems Inc., Bannockburn, IL).DNA Polymerase Chain Reaction, Reverse-Transcription Polymerase Chain Reaction, and Western BlottingTo assess the donor contribution to BM reconstitution, DNA polymerase chain reaction (PCR) analysis for the sexual region of the Y chromosome was performed 3 weeks after BMTx on DNA extracted from blood cells, as previously described.5Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. Boggs S.S. Greenberger J.S. Goff J.P. Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Google Scholar Reverse-transcription (RT)-PCR analysis for G-CSFR was performed on RNA isolated from normal liver, freshly isolated OCs, and cultured OCs by using the RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized from 5 μg of total RNA. RT-PCR was performed as described elsewhere.5Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. Boggs S.S. Greenberger J.S. Goff J.P. Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Google Scholar The resulting RT-PCR products were analyzed on 1.5% agarose gels stained with ethidium bromide. For Western blot analysis, frozen liver samples were thawed and total proteins were extracted from homogenates in RIPA buffer, separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene-difluoride membranes. Immunoblotting was performed using 1:500 anti–G-CSF, 1:500 anti–G-CSFR, and 1:5000 anti–β-actin (Abcam Inc., Cambridge, MA). Immunocomplexes were detected with horseradish-peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnologies). Detection was performed using the ECL plus kit (Amersham Life Science, Piscataway, NJ).In Vitro AssaysCytospins and in vitro assays were performed on OCs isolated from rats subjected to 2AAF/PH, as described elsewhere.10Petersen B.E. Goff J.P. Greenberger J.S. Michalopoulos G.K. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Google Scholar, 23Jung Y. Oh S.H. Zheng D. Shupe T.D. Witek R.P. Petersen B.E. A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells.Lab Invest. 2006; 86: 477-489Google Scholar Briefly, isolation was achieved by using a standard 2-step collagenase perfusion. Cells were centrifuged at 55 × g to separate the hepatocyte fraction from nonparenchymal cells, the latter were collected at 220 × g. Nonparenchymal cells were incubated with Thy-1–fluorescein isothiocyanate–conjugated antibody (BD Biosciences Pharmingen), followed by incubation with anti–fluorescein isothiocyanate microbeads. Thy-1+ OCs subsequently were selected using immunomagnetic sorting (MACS; Miltenyi Biotec Inc., Auburn, CA). Cell viability was determined to be greater than 90% by Trypan-blue dye exclusion.CytospinsCytocentrifugation was performed on collected Thy-1+ cells at 41 × g (Cytospin-4; Thermo-Shandon, Cheshire, England). Cytospin preparations (105 cells/slide) were stained for Thy-1, OV6, CD45, G-CSFR, and G-CSF as described earlier. For in vitro proliferation and migration assays 3 different doses of G-CSF were tested: 10 ng/mL, 100 ng/mL, and 500 ng/mL. All assays were performed in triplicate to ensure statistical significance. Antibiotic-antimycotic solutions were added to each buffer.Proliferation assayCells (2 × 105) were seeded in 6-well plates in IMDM supplemented with 10% fetal bovine serum, 1% insulin, and were incubated at 37°C, 5% CO2, overnight. The next day, medium was removed and the cells were cultured in different buffers: IMDM with 0.5% bovine serum albumin (negative control), IMDM with 10% fetal bovine serum (positive control), and IMDM with 0.5% bovine serum albumin and various doses of G-CSF (experimental groups). Cell counts were performed on trypsinized cells immediately before the test (day 1), and every subsequent 48 hours.Migration assayCell motility was assessed in transwells, as previously described.23Jung Y. Oh S.H. Zheng D. Shupe T.D. Witek R.P. Petersen B.E. A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells.Lab Invest. 2006; 86: 477-489Google Scholar Briefly, transwell culture dishes (Coring, Inc., Costar, NY) with 5-μm pore filters were precoated overnight with 0.006% rat-tail collagen. Cells (1 × 105) were suspended in migration buffer (IMDM, 10% fetal bovine serum, and 1% insulin), and allowed to attach overnight. Nonadherent cells were removed from the top of the transwell chambers and the transwells then were transferred to new wells containing various doses of G-CSF in migration buffer. Plates were incubated at 37°C, 5% CO2, for either 4 or 6 hours. As controls, G-CSF either was excluded from the lower chamber (migration control) or added to both the lower and upper chambers (chemokinetic control). At the end of the assay, cells that had migrated to the bottom of the transwell filter were fixed, stained, and counted. Data were normalized for each independent experiment with respect to the migration control, and expressed as the relative chemotactic index.Statistical AnalysisValues presented are expressed as mean ± SD. After acquiring all data for histologic parameters and in vitro assays, the Student t test was applied to determine statistical significance. A P value of less than .05 was considered significant. Data analysis was performed by Microsoft Excel software (Microsoft, Redmond, WA).ResultsG-CSF Is Produced Within the Liver During OC-Mediated Regeneration and Acts as Both a Paracrine and Autocrine Factor on Hepatic OCsMorphologic analysis of liver samples from animals subjected to BMTx/2AAF/PH (group C) confirmed the typical features of OC-mediated liver regeneration.10Petersen B.E. Goff J.P. Greenberger J.S. Michalopoulos G.K. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat.Hepatology. 1998; 27: 433-445Google Scholar Specifically, in H&E-stained liver sections, oval-like cells, appearing as dark blue areas because of their large nuclei and scant cytoplasm, were seen surrounding the portal tracts after PH, and their number increased progressively, reaching a peak at day 11. These cells were positive for both OV6 and Ki67, indicating that they were proliferating OCs. Subsequently, the number and proliferation activity of OCs progressively decreased.BM-derived cells were detected within the liver by their expression of DPPIV. Scattered patches of DPPIV+, oval-like cells, and hepatocytes were detected. These patches ranged in size from single cells to small clusters. As for the magnitude of BM contribution to liver repopulation, in agreement with published data,24Thorgeirsson S.S. Grisham J.W. Hematopoietic cells as hepatocyte stem cells: a critical review of the evidence.Hepatology. 2006; 43: 2-8Google Scholar our study showed that the BM contribution to liver repopulation was a relatively rare event, resulting in a total contribution (DPPIV+ OCs and hepatocytes within the recipients’ livers) of 0.47% ± 0.12% at day 11 and 0.68% ± 0.21% at day 28 after PH. At day 11, the majority of the DPPIV+ cells had an oval-like morphology and were concentrated within the periportal OV6+ areas, whereas at day 28 the majority of the DPPIV+ cells were hepatocyte-like.To assess whether G-CSF may act directly on OCs, we studied the expression of G-CSFR in liver samples. G-CSFR was not expressed in normal liver, whereas it was induced during OC-mediated liver regeneration. Immunofluorescence revealed that G-CSFR was expressed by ductular and periductular cells after OC activation, colocalizing with the OC marker AFP (Figure 2A–C). G-CSF expression was detected in periportal cells and colocalized with G-CSFR within the OC population (Figure 2D–F). Confocal microscopy was used to confirm these findings on representative sections (Figure 2G–I). Western blot analysis revealed that G-CSFR was not produced by normal liver, whereas its expression was induced after OC activation. G-CSF production also was increased after 2AAF/PH, peaking between days 5 and 7 after surgery (Figure 3A). Finally, hepatic expression of G-CSFR mRNA was established through RT-PCR, performed on freshly isolated OCs, cultured OCs, and normal liver tissue (Figure 3B).Figure 2(A–C) Double-immunofluorescence staining of livers from group C (11 days after 2AAF/PH) showed expression of (A) G-CSFR (green), (B) AFP (red), and (C) co-expression (yellow) by many periportal cells. A few cells expressed AFP alone (small arrows, 2B and 2C), or G-CSFR alone (large arrows, 2A and 2C). The inserts in A and B represent isotype controls for G-CSFR and AFP, respectively. Cell nuclei were stained with DAPI (blue). Original magnification, 40× objective. (D–F) Double-immunofluorescence staining of livers from group C (11 days after 2AAF/PH) detected cells expressing (D) G-CSFR (green), (E) G-CSF (red), and (F) co-expression (yellow) in many periportal cells. A few cells expressed G-CSFR alone (large arrows, 2D and 2F) or G-CSF alone (small arrows, 2E and 2F). The inserts in D and E represent isotype controls for G-CSFR and G-CSF, respectively. Cell nuclei were stained with DAPI (blue). Original magnification, 40× objective. (G–I) Confocal microscopy was used on representative sections from the same animals (group C). Immunofluorescence for (G) G-CSFR (green) and (H) G-CSF (red) shows (I) co-expression (merge) within many periportal cells. The presence of dual markers (yellow) is evident in most cells shown. Both G-CSFR and G-CSF also were seen as distinct colors in separate cellular domains, denoting differential distribution within the cell (large arrows and small arrows, respectively). Cell nuclei were stained with DAPI (blue). Original magnification, 63× objective.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3(A) Western blot analysis of liver homogenates confirmed the expression of both G-CSF and G-CSFR after 2AAF/PH in rats (at days 1, 3, 5, 7, 11, and 15 after surgery) vs normal liver (nl). G-CSFR was not produced by NL, whereas its expression was induced after OC activation, with a peak at days 3–7 after PH. G-CSF production also was increased after 2AAF/PH, peaking at days 5–7 after PH. (B) RT-PCR amplification of G-CSFR mRNA in normal liver, freshly isolated OCs, and cultured OCs provide further proof of the expression of G-CSFR by hepatic OCs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hepatotrophic Effects of G-CSF Administration During OC-Mediated Liver RegenerationTo assess the effects of exogenous G-CSF during OC-mediated liver regeneration, BMTx/2AAF/PH pretreated rats were subjected to G-CSF administration after surgery. We analyzed several parameters, such as the proliferation index (Ki67+) and the magnitude of the OC reaction (OV6+) in G-CSF–treated rats vs untreated rats. We compared 2 different molecular forms of G-CSF: recombinant methionyl human G-CSF (group B) and its pegylated counterpart (Peg–G-CSF, group A). An augmented OC reaction, relative to controls, was observed in both of the G-CSF–treated groups (Figure 4, Figure 5). Particularly at day 11 after 2AAF/PH, the magnitude of the OC reaction was increased up to 5 times in animals treated with Peg–G-CSF and up to 2 times after G-CSF injection, as compared with controls (P < 0.05) (Figure 4A–C). At day 28, when only a few OCs still were present in 2AAF/PH controls, the number of liver OCs remained significantly higher in G-CSF– and Peg–G-CSF–treated rats (up to a 5- and 9-fold increase, respectively; P < .05). The highest OC response was measured in rats injected with Peg–G-CSF, although this increase was not statistically significant when compared with nonpegylated–G-CSF–treated animals (Figure 5A). The proliferation index also was increased significantly in animals treated with G-CSF or Peg–G-CSF (data not shown).Figure 4H&E staining of liver sections 11 days after 2AAF/PH in