Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090117
ISSN1525-2191
AutoresDhivya Haridass, Qinggong Yuan, Pablo D. Becker, Tobias Cantz, Marcus Iken, Michael Rothe, Nidhi Narain, Michael Bock, Miriam Nörder, Nicolas Legrand, Heiner Wedemeyer, Kees Weijer, Hergen Spits, Michael P. Manns, Jun Cai, Hongkui Deng, James P. Di Santo, Carlos A. Guzmán, Michael Ott,
Tópico(s)Pluripotent Stem Cells Research
ResumoFetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared with adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC, and ES-HPC into a new immunodeficient mouse strain (Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 ± 8.0%, 1 × 106 transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception embryos generated only 12.1 ± 3.0% and 5.1 ± 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 ± 3.9% and 2.7 ± 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. We conclude that, in contrast to expectations, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies. Fetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared with adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC, and ES-HPC into a new immunodeficient mouse strain (Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 ± 8.0%, 1 × 106 transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception embryos generated only 12.1 ± 3.0% and 5.1 ± 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 ± 3.9% and 2.7 ± 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. We conclude that, in contrast to expectations, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies. Transplanted adult hepatocytes (HC) engraft in a recipient liver and morphologically as well as functionally connect with the surrounding cells.1Gupta S Rajvanshi P Lee CD Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase IV-deficient rats.Proc Natl Acad Sci USA. 1995; 92: 5860-5864Crossref PubMed Scopus (117) Google Scholar, 2Gupta S Rajvanshi P Sokhi R Slehria S Yam A Kerr A Novikoff PM Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium.Hepatology. 1999; 29: 509-519Crossref PubMed Scopus (227) Google Scholar In animal models with liver injury and/or selective growth advantage engrafted cells respond to growth stimuli and repopulate recipient livers.3Weglarz TC Degen JL Sandgren EP Hepatocyte transplantation into diseased mouse liver: kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes.Am J Pathol. 2000; 157: 1963-1974Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Grompe M Principles of therapeutic liver repopulation.J Inherit Metab Dis. 2009; 29: 421-425Crossref Scopus (52) Google Scholar Elucidation of the molecular pathways of liver regeneration and extensive preclinical cell transplantation experiments in animals have led to the application of HC transplantation in a limited number of patients with hereditary metabolic liver disease and acute liver failure.5Fox IJ Chowdhury JR Kaufman SS Goertzen TC Chowdhury NR Warkentin PI Dorko K Sauter BV Strom SC Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation.N Engl J Med. 1998; 338: 1422-1426Crossref PubMed Scopus (906) Google Scholar, 6Fisher RA Strom SC Human hepatocyte transplantation: worldwide results.Transplantation. 2006; 82: 441-449Crossref PubMed Scopus (366) Google Scholar, 7Schneider A Attaran M Meier PN Strassburg C Manns MP Ott M Barthold M Arseniev L Becker T Panning B Hepatocyte transplantation in an acute liver failure due to mushroom poisoning.Transplantation. 2006; 82: 1115-1116Crossref PubMed Scopus (78) Google Scholar, 8Meyburg J Das AM Hoerster F Lindner M Kriegbaum H Engelmann G Schmidt J Ott M Pettenazzo A Luecke T Bertram H Hoffmann GF Burlina A One liver for four children: first clinical series of liver cell transplantation for severe neonatal urea cycle defects.Transplantation. 2009; 87: 636-641Crossref PubMed Scopus (121) Google Scholar However, the shortage of donor organs and the difficulties of cryopreservation and long-term culturing of mature HC have limited the clinical application of cell-based therapies. Stem cells have attracted considerable interest for cell replacement therapy, because they expand in cell culture or can be easily harvested from patients.9Cantz T Manns MP Ott M Stem cells in liver regeneration and therapy.Cell Tissue Res. 2008; 331: 271-282Crossref PubMed Scopus (91) Google Scholar, 10Oertel M Shafritz DA Stem cells, cell transplantation and liver repopulation.Biochim Biophys Acta. 2008; 1782: 61-74Crossref PubMed Scopus (149) Google Scholar Adult, fetal, and embryonic stem cell (ESC) sources have been studied as a potential substitute for primary adult HC in liver cell therapy. The generation of HC has been reported in recipient livers of animals, which have been transplanted with adult hematopoietic and mesenchymal stem cells.11Theise ND Nimmakayalu M Gardner R Illei PB Morgan G Teperman L Henegariu O Krause DS Liver from bone marrow in humans.Hepatology. 2000; 32: 11-16Crossref PubMed Scopus (1153) Google Scholar, 12Theise ND Badve S Saxena R Henegariu O Sell S Crawford JM Krause DS Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.Hepatology. 2000; 31: 235-240Crossref PubMed Scopus (896) Google Scholar, 13Herzog EL Chai L Krause DS Plasticity of marrow-derived stem cells.Blood. 2003; 102: 3483-3493Crossref PubMed Scopus (676) Google Scholar, 14Schwartz RE Reyes M Koodie L Jiang Y Blackstad M Lund T Lenvik T Johnson S Hu WS Verfaillie CM Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.J Clin Invest. 2002; 109: 1291-1302Crossref PubMed Scopus (1103) Google Scholar More recent studies, however, have not convincingly shown formation of HC in therapeutically relevant numbers in mouse liver repopulation or toxic injury models.15Kanazawa Y Verma IM Little evidence of bone marrow-derived hepatocytes in the replacement of injured liver.Proc Natl Acad Sci USA. 2003; 100: 11850-11853Crossref PubMed Scopus (139) Google Scholar, 16Wagers AJ Sherwood RI Christensen JL Weissman IL Little evidence for developmental plasticity of adult hematopoietic stem cells.Science. 2002; 297: 2256-2259Crossref PubMed Scopus (1299) Google Scholar, 17Cantz T Sharma AD Jochheim-Richter A Arseniev L Klein C Manns MP Ott M Reevaluation of bone marrow-derived cells as a source for hepatocyte regeneration.Cell Transplant. 2004; 13: 659-666Crossref PubMed Scopus (76) Google Scholar In one study in fumarylacetoacetate hydrolase (Fah)(−/−) deficient mice, liver tissue formation from transplanted bone marrow cells was found to be the result of monocyte fusion with recipient liver cells.18Willenbring H Bailey AS Foster M Akkari Y Dorrell C Olson S Finegold M Fleming WH Grompe M Myelomonocytic cells are sufficient for therapeutic cell fusion in liver.Nat Med. 2004; 10: 744-748Crossref PubMed Scopus (363) Google Scholar In contrast to adult stem cells, fetal liver progenitor cells (FLPC) already express an induced immature hepatic phenotype and can be isolated, cultured, and expanded in vitro. Transplantation experiments in several laboratories have demonstrated engraftment of FLPC and subsequent liver tissue formation.19Strick-Marchand H Morosan S Charneau P Kremsdorf D Weiss MC Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes.Proc Natl Acad Sci USA. 2004; 101: 8360-8365Crossref PubMed Scopus (103) Google Scholar, 20Sandhu JS Petkov PM Dabeva MD Shafritz DA Stem cell properties and repopulation of the rat liver by fetal liver epithelial progenitor cells.Am J Pathol. 2001; 159: 1323-1334Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Transplanted FLPC, which were isolated from murine fetal liver tissue, were shown to acquire the adult HC phenotype over a period of 6 to 8 weeks after transplantation.21Cantz T Zuckerman DM Burda MR Dandri M Göricke B Thalhammer S Heckl WM Manns MP Petersen J Ott M Quantitative gene expression analysis reveals transition of fetal liver progenitor cells to mature hepatocytes after transplantation in uPA/RAG-2 mice.Am J Pathol. 2003; 162: 37-45Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Although FLPC can be expanded in cell culture, the availability of donated fetal tissues restricts the clinical application of this cell source. With their unlimited potential to grow in vitro and to develop into virtually any cell type, ESCs, and more recently, induced pluripotent stem cells, might be the ideal source of donor liver cells for cell therapies in the future.22Rathjen J Rathjen PD Mouse ES cells: experimental exploitation of pluripotent differentiation potential.Curr Opin Genet Dev. 2001; 11: 587-594Crossref PubMed Scopus (91) Google Scholar, 23Takahashi K Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18677) Google Scholar, 24Nakagawa M Koyanagi M Tanabe K Takahashi K Ichisaka T Aoi T Okita K Mochiduki Y Takizawa N Yamanaka S Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.Nature Biotechnol. 2008; 26: 101-106Crossref Scopus (2182) Google Scholar We and others have generated hepatic precursor cells from human and mouse ESC lines.25Kania G Blyszczuk P Jochheim A Ott M Wobus AM Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells.Biol Chem. 2004; 385: 943-953Crossref PubMed Scopus (61) Google Scholar, 26Hamazaki T Iiboshi Y Oka M Papst PJ Meacham AM Zon LI Terada N Hepatic maturation in differentiating embryonic stem cells in vitro.FEBS Lett. 2001; 497: 15-19Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 27Agarwal S Holton KL Lanza R Efficient differentiation of functional hepatocytes from human embryonic stem cells.Stem Cells. 2008; 26: 1117-1127Crossref PubMed Scopus (316) Google Scholar, 28Cai J Zhao Y Liu Y Ye F Song Z Qin H Meng S Chen Y Zhou R Song X Guo Y Ding M Deng H Directed differentiation of human embryonic stem cells into functional hepatic cells.Hepatology. 2007; 45: 1229-1239Crossref PubMed Scopus (514) Google Scholar With the existing differentiation protocols a primitive hepatic phenotype with fetal gene expression patterns can be induced in the majority of the ESCs.28Cai J Zhao Y Liu Y Ye F Song Z Qin H Meng S Chen Y Zhou R Song X Guo Y Ding M Deng H Directed differentiation of human embryonic stem cells into functional hepatic cells.Hepatology. 2007; 45: 1229-1239Crossref PubMed Scopus (514) Google Scholar, 29Jochheim A Hillemann T Kania G Scharf J Attaran M Manns MP Wobus AM Ott M Quantitative gene expression profiling reveals a fetal hepatic phenotype of murine ES-derived hepatocytes.Int J Dev Biol. 2004; 48: 23-29Crossref PubMed Scopus (46) Google Scholar Transplantation of these cells, however, have so far resulted only in scattered formation of HC or were reported to form small HC clusters in major urinary protein promoter driven urokinase-type plasminogen activator (uPA) mice30Heo J Factor VM Uren T Takahama Y Lee JS Major M Feinstone SM Thorgeirsson SS Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver.Hepatology. 2006; 44: 1478-1486Crossref PubMed Scopus (127) Google Scholar and Fah(−/−) mice.31Gouon-Evans V Boussemart L Gadue P Nierhoff D Koehler CI Kubo A Shafritz DA Keller G BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm.Nature Biotechnol. 2006; 24: 1402-1411Crossref Scopus (346) Google Scholar Multiple progenitor cell types have been studied extensively in transplantation experiments in animals with normal liver, in toxic liver injury models, and in liver repopulation models such as the albumin promoter/enhancer (Alb) directed uPA transgenic or Fah(−/−) mice. Although the potential of transplanted stem cell derived hepatic precursor and progenitor cells to generate HC has been clearly demonstrated, a comparative analysis of the individual capacity to form liver tissue is not available. In our present study we aimed to establish and validate an animal model, which would allow us to compare side-by-side the degree of liver repopulation of various human and murine cell types in a recipient liver. To this end, we performed standardized transplantation experiments in immunodeficient heterozygous Alb-uPA mice. In this animal model the transgene is expressed under transcriptional control of the albumin promoter/enhancer sequence exclusively in HC, which causes postnatal toxic liver injury.32Heckel JL Sandgren EP Degen JL Palmiter RD Brinster RL Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.Cell. 1990; 62: 447-456Abstract Full Text PDF PubMed Scopus (180) Google Scholar Homozygous mice die from liver failure, but can be rescued by the transplantation of HC. In heterozygous mice, endogenous HC delete the transgene and regenerate the liver. Transplanted cells thus compete with endogenous HC to regenerate the liver. The capacity of a given cell type to repopulate a recipient liver organ after transplantation in this animal model is determined by its engraftment properties, the in vivo differentiation potential, and the proliferation capacity in a recipient liver. We generated a new immunodeficient xenograft mouse model by crossing Alb-uPA transgenic (tg) mice onto the Rag2(−/−)γc(−/−) background (Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mice). This new model was then transplanted with various primary human and mouse cells with hepatic phenotype and liver tissues of the transplanted animals were harvested 3 months after transplantation and analyzed for the presence of HC derived from transplanted cells. Our data indicate that immature hepatic cell types of both human and mouse origin are unexpectedly less competitive compared with adult HC in repopulation of the Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mouse liver. Additionally, the overall repopulation rates observed after transplantation of human fetal and adult cells were significantly lower compared with similar transplantations performed with respective mouse cells. C57BL/6 and enhanced green fluorescent protein (EGFP)-transgenic mice (C57BL/6-TgN(ACTbEGFP)1Osb) were purchased from the Jackson Laboratory (Bar Harbor, ME). Alb-uPAtg(+/−)Rag2(−/−)γc(−/−) mice were generated by breeding of Alb-uPA transgenic mice32Heckel JL Sandgren EP Degen JL Palmiter RD Brinster RL Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.Cell. 1990; 62: 447-456Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 33Sandgren 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 (321) Google Scholar on the severe combined immunodeficiency background34Morosan S Hez-Deroubaix S Lunel F Renia L Giannini C Van Rooijen N Battaglia S Blanc C Eling W Sauerwein R Hannoun L Belghiti J Brechot C Kremsdorf D Druilhe P Liver-stage development of Plasmodium falciparum, in a humanized mouse model.J Infect Dis. 2006; 193: 996-1004Crossref PubMed Scopus (87) Google Scholar with Rag2(−/−)γc(−/−) mice35Silva-Barbosa SD Butler-Browne GS Di Santo JP Mouly V Comparative analysis of genetically engineered immunodeficient mouse strains as recipients for human myoblast transplantation.Cell Transplant. 2005; 14: 457-467Crossref PubMed Scopus (35) Google Scholar on the nonobese diabetic background (J.P. Di Santo, unpublished). All animals were maintained and handled in accordance with institutional guidelines of the Hannover Medical School and the Helmholtz Center for Infection Research. Human adult HC were isolated as described previously by a modified three-step collagenase perfusion from surgical resectates, which have been obtained from patients with informed consent.36Alexandrova K Griesel C Barthold M Heuft HG Ott M Winkler M Schrem H Manns MP Bredehorn T Net M Vidal MM Kafert-Kasting S Arseniev L Large-scale isolation of human hepatocytes for therapeutic application.Cell Transplant. 2005; 14: 845-853Crossref PubMed Scopus (53) Google Scholar Perfusion solutions were introduced into the tissue through catheters placed into the portal or hepatic vein branches. After the digestion phase, the liver tissue was manually disrupted with sterile scissors and scalpels. To separate undigested tissue pieces, the suspended HC were passed through 750 and 500 μm filters into 50 ml Falcon tubes. The cell suspensions were centrifuged at 50 g for 10 minutes and the cell pellet was resuspended in an ice cold buffer. An aliquot of the cell preparation was separated for cell count and viability analysis (light microscopy and trypan blue exclusion test). In all transplantation experiments, suspensions with >85% of viable HC were used. The non-HC fraction (leukocytes, nonparenchymal liver cells) in the cell suspension was less than 5% based on microscopic analysis. Plating efficacy of the HC on collagen-coated cell culture surfaces exceeded 80% in all preparations. Human fetal livers (14 to 17 weeks of gestation) were obtained after termination of pregnancy with the informed consent of the mothers. The procedure and the transplantation protocols were approved by the ethical committee of the Hannover Medical School. The tissues were mechanically disrupted and the resulting fragments were treated with 0.1 to 1% collagenase D (Roche, Mannheim, Germany) at 37°C. The resulting FLPC suspensions were washed with William’s E media containing fetal calf serum (FCS) and centrifuged at 50 g. The cell pellet was resuspended with red blood cell lysis buffer (Sigma-Aldrich, München, Germany), centrifuged and resuspended in William’s E medium. Viability (as evaluated by trypan blue exclusion) always exceeded 85% in transplanted samples. Human ESC line H1 was obtained from WiCell research institute (Madison, WI). The passage number of the H1 cells used in the transplantation experiment was 43. The culture and hepatic differentiation of human ESCs were performed as previously described in the lab of Hongkui Deng.28Cai J Zhao Y Liu Y Ye F Song Z Qin H Meng S Chen Y Zhou R Song X Guo Y Ding M Deng H Directed differentiation of human embryonic stem cells into functional hepatic cells.Hepatology. 2007; 45: 1229-1239Crossref PubMed Scopus (514) Google Scholar After 18 days of differentiation, the cells were dissociated with Accutase (Sigma) and then transplanted. Adult HC from C57BL/6 and EGFP-transgenic C57BL/6 mice were isolated by a two-step collagenase perfusion method originally described by Seglen et al37Seglen PO Hepatocyte suspensions and cultures as tools in experimental carcinogenesis.J Toxicol Environ Health. 1979; 5: 551-560Crossref PubMed Scopus (113) Google Scholar with minor modifications. For the generation of mouse FLPC suspensions, the livers from mouse embryos (embryonic day [ED] 11.5 and 13.5 post conception [p.c.]) were removed under the binocular microscope. Cells were isolated by collagenase/dispase (Roche, Mannheim, Germany) digestion for 20 minutes at 37°C. The cells were treated with red blood cell lysis buffer (Sigma), washed twice in cold Dulbecco’s modified Eagle’s medium with 10% FCS and resuspended in PBS and stored on ice until transplantation. ESCs derived from Rosa26 mice were cultured on mouse embryonic fibroblasts on gelatinized dishes (Falcon BD) in culture medium I (Knock-out Dulbecco’s modified Eagle’s medium, Invitrogen, Karlsruhe, Germany) supplemented with 15% FCS (selected batches) and the following additives: β-mercaptoethanol (5 × 10−5 M); l-glutamine (2 mmol/L); nonessential amino acids; penicillin-streptomycin (all Invitrogen); and 10 ng/ml recombinant human leukemia inhibitory factor (Chemicon, Millipore, Billerica, MA), as previously described.38Sharma AD Cantz T Vogel A Schambach A Haridass D Iken M Bleidissel M Manns MP Schöler HR Ott M Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation.Cell Transplant. 2008; 17: 313-323Crossref PubMed Scopus (43) Google Scholar For hepatic differentiation, 600 ESCs were cultivated in hanging drops (20 μl), as described previously to form embryoid bodies. On day 5, embryoid bodies were plated onto gelatinized dishes in culture medium II (IMDM; Invitrogen) supplemented with 20% FCS, 450 μmol/L α-monothioglycerol (Sigma) and all additives except leukemia inhibitory factor. On days 5 and 9, embryoid bodies outgrowths were trypsinized, replated onto collagen type I-coated dishes in differentiation medium (HC culture medium, Cambrex, East Rutherford, NJ), and cultivated until days 5, 9, and 20. For hepatic phenotype selection, hepatic precursor cells derived from embryonic stem cells (ES-HPC) were transduced on day 31 (day 5 + 9 + 17) of differentiation with the lentivirus vector RRL.PPT. Alb.GFPpre (MOI ∼10) and analyzed for EGFP fluorescence after 3 days with a fluorescence microscope. After visual analysis, the cells were trypsinized and sorted for EGFP in a MoFlow cytometer. Cells expressing Dlk-1, a member of the δ-like family of cell surface transmembrane proteins, have been shown in rats to contain the full liver repopulation potential present in fetal liver cell suspensions.39Oertel M Menthena A Chen YQ Teisner B Jensen CH Shafritz DA Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver.Gastroenterology. 2008; 134: 823-832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar The surface marker was analyzed by flow cytometry by using 2 μg of a rabbit polyclonal antibody (Abcam, Cambridge, MA) in our cell preparations. Stainings with a rabbit antibody against green fluorescent protein (Invitrogen) at identical concentrations served as isotype controls. Cells were washed once with PBS before applying the fluorescein isothiocyanate-conjugated goat anti-rabbit antibody in a 1:200 dilution. All stainings were performed on ice for 30 minutes in a total volume of 100 μl PBS. Cells were washed again with PBS and analyzed with the LSR II flow cytometer (BD Biosciences, Heidelberg, Germany) and the FlowJo software (Tree Star, Ashland, Oregon). Intrasplenic transplantations of the various cell types were performed under sterile conditions. Briefly, recipient mice were anesthetized via isoflurane inhalation with an appropriate vaporator. A lateral abdominal incision was performed, the spleen was localized, and cells in a total volume of 50 μl of medium containing no FCS were injected intrasplenically. Sutured spleens were returned carefully, and the skin was closed. ES-HPC, FLPC suspensions (ED 11.5 and 13.5), and primary adult HC were incubated with the PKH26 fluorescent dye (Sigma), as previously described.38Sharma AD Cantz T Vogel A Schambach A Haridass D Iken M Bleidissel M Manns MP Schöler HR Ott M Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation.Cell Transplant. 2008; 17: 313-323Crossref PubMed Scopus (43) Google Scholar Cells, 5 × 105, (viability >85% by trypan blue exclusion) were transplanted via the intrasplenic route into the liver of C57BL/6 mice. After 24 hours, 48 hours, 14 days, and 28 days, the livers (n = 4 for each time point) were harvested and native fluorescence microscopy was performed on cryosections (10 μm) to detect and count PKH26 positive cells. Autofluorescence was excluded by parallel examination of the red (617 nm) and green emission (528 nm). The numbers of PKH-fluorescent cells were assessed in 20 high power fields (×200) of 10 representative tissue sections. Liver tissues were harvested and fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) and embedded in paraffin. For H&E staining, sections were immersed in hematoxylin solution for 3 minutes and in 0.5% eosin solution for an additional 3 minutes. Immunofluorescence staining of the EGFP antigen was performed in paraformaldehyde-fixed 5-μm permeabilized (in 0.25% Triton-X PBS) sections by using an anti-green fluorescent protein-Alexa 594 antibody (Molecular Probes, Invitrogen) in a 1:200 dilution. Human albumin and human cytokeratin (CK) 18 staining was performed in 6 μm sections. After dewaxing and rehydration endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 minutes, followed by antigen retrieval in target retrieval solution (Dako, Glostrup, Denmark) for 20 minutes in a water bath at 98°C. Primary monoclonal anti-human albumin (1:100) (Bethyl Laboratories, Montgomery, TX) and monoclonal anti-human CK 18 (1:200) (Dako) antibodies were incubated overnight. Biotinylated secondary antibody was incubated for 1 hour, followed by incubation with avidin-coupled peroxidase for 1 hour (Vector Laboratories, Peterborough, UK). Three-amino-9-ethylcarbazole was used as a chromogen (Dako) and Gill’s No. 3 hematoxylin for the counter stain. Ten formalin fixed and paraffin embedded tissue slides from all liver lobes of recipient animals were chosen for analysis. The number of HC derived from transplanted cells was counted in ten view fields/section (original magnification, ×400) in individual mice 3 months after transplantation and expressed as percentage of the total number of HC. Data were compared for all individual groups of animals with the Student’s t-test. A P value of <0.05 was considered as statistically significant. In the first experiment, groups of animals (n = 5) were transplanted with 1 × 105, 5 × 105, 1 × 106, and 2 × 106 EGFP–mouse HC and analyzed for the presence of fluorescent HC at 3 months after transplantation (Figure 1, A and B). Mice from group 1, which were transplanted with 1 × 105 cells, repopulated the recipient liver with 44.0 ± 6.5% of the HC expressing the EGFP. In groups 2, 3, and 4 (receiving 5 × 105, 1 × 106, and 2 × 106 cells), 46.6 ± 8.0%, 47.5 ± 10.7%, and 42.0 ± 14.0% of recipient livers were repopulated by the transplanted cells, respectively (Figure 1C). These results, which did not show statistically significant differences, indicate a saturation effect of intrasplenic liver cell transplantation and a limited effect on the overall repopulation in this particular animal model at numbers exceeding 1 × 105 cells. In contrast to the homogenous adult HC suspensions (< 5% of nonhepatic cells) murine FLPC suspensions derived from ED 11.5 and 13.5 fetal livers contain hepatoblasts and cells of hematopoietic origin. In previous studies the Dlk-1 surface marker has been shown to be exclusively expressed by liver repopulating hepatoblasts.38Sharma AD Cantz T Vogel A Schambach A Haridass D Iken M Bleidissel M Manns MP Schöler HR Ott M Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation.Cell Transplant. 2008; 17: 313-323Crossref PubMed Scopus (43) Google Scholar After red blood cell lysis, an average of 17.8 ± 4% (n = 3) of the ED 13.5 p.c. liver cell suspensions expressed the Dlk-1 protein as determined by flow cytometric analysis (Figure 2). In transplantation, experiments freshly isolated those murine FLPC suspensions and were less efficient for subsequent repopulation of recipient livers compared with the respective adult HC transplantations (Figure 3, A and B). EGFP–transgenic cells, 5 × 105 and 1 × 106, (containing ∼1 × 105 and 2 × 105 Dlk-1+ hepatoblasts) isolated from ED 13.5 p.c. fetal liver resulted in reduced repopulation rates of the recipient liver organ (12.1 ± 3.0% of the total number of HC) compared with the adult mouse HC transplantations. A further decrease in repopulation efficacy was noted after transplantation of cells isolated from ED 11.5 p.c. fetal liver (5.1 ± 1.1%, P < 0.05) (Figure 3C). The reduced repopulation rate in animals transplanted with mouse FLPC was the result of smaller numbers of cell clusters and smaller sizes of individual groups of cells. Nonsorted mouse ES-HPC did not result in liver tissue formation, but formed teratoma tissue in the liver and spleen of most of the transplanted animals. To facilitate normal liver tissue and HC formation from ES-HPC, a selection strategy based on albumin driven EGFP expression for cells with a hepatic phenotyp
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