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• W2008099316 abstract "Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγcnull (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator–NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system. Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγcnull (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator–NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system. In Europe, Australia, and North America at least 25% of HIV-infected persons have a concomitant hepatitis C virus (HCV) infection, and 5% to 10% are co-infected with chronic hepatitis B virus (HBV).1Lacombe K. Rockstroh J. HIV and viral hepatitis coinfections: advances and challenges.Gut. 2012; 61: i47-i58Crossref PubMed Scopus (114) Google Scholar Although the incidence of monoinfections (HIV, HBV, and HCV) is declining because of prophylaxis, vaccination, and newly available treatments, co-infections of HIV with HBV/HCV are still problematic, and the medical care of these co-infected patients remains a difficult task.1Lacombe K. Rockstroh J. HIV and viral hepatitis coinfections: advances and challenges.Gut. 2012; 61: i47-i58Crossref PubMed Scopus (114) Google Scholar, 2Naggie S. Sulkowski M.S. Management of patients coinfected with HCV and HIV: a close look at the role for direct-acting antivirals.Gastroenterology. 2012; 142: 1324-1334 e3Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar These human-specific co-infections require a small animal model to study double infections, such as HIV/HCV or HIV/HBV, and to test new antiviral/antiretroviral therapeutics.A search is ongoing for the best strain of mice for dual reconstitution and available tissue sources. Among multiple mouse models for human hepatocyte transplantation, the most robust and effective for mouse liver cell depletion and human hepatocytes engraftment are tyrosine catabolic enzyme fumarylacetoacetate hydrolase (Fah) mutants3Azuma H. Paulk N. Ranade A. Dorrell C. Al-Dhalimy M. Ellis E. Strom S. Kay M.A. Finegold M. Grompe M. Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice.Nat Biotech. 2007; 25: 903-910Crossref PubMed Scopus (606) Google Scholar and transgenic mice with a tandem array of murine urokinase genes under the control of the albumin promoter (Alb-uPA) on a CB-17-scid-bg background.4Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. Addison W.R. Fischer K.P. Churchill T.A. Lakey J.R. Tyrrell D.L. Kneteman N.M. Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar The advantages (up to 99% of human hepatocyte reconstitution) and the disadvantages (colony maintenance, limited time window for transplantation, and mouse health problems) of these mouse models for liver repopulation with human hepatocytes have been extensively discussed.5de Jong Y.P. Rice C.M. Ploss A. New horizons for studying human hepatotropic infections.J Clin Invest. 2010; 120: 650-653Crossref PubMed Scopus (44) Google Scholar, 6Shafritz D.A. Oertel M. Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells.Int J Biochem Cell Biol. 2011; 43: 198-213Crossref PubMed Scopus (36) Google Scholar, 7Strom S.C. Davila J. Grompe M. Chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity.Methods Mol Biol. 2010; 640: 491-509Crossref PubMed Scopus (115) Google Scholar, 8Gilgenkrantz H. Rodent models of liver repopulation.Methods Mol Biol. 2010; 640: 475-490Crossref PubMed Scopus (8) Google Scholar Several models for human hematopoietic stem cell (HSC) transplantation are based on the nonobese diabetic (NOD) and BALB/c background mouse strains, known as NOG (NOD/Shi-scid/IL-2Rγcnull),9Ito M. Hiramatsu H. Kobayashi K. Suzue K. Kawahata M. Hioki K. Ueyama Y. Koyanagi Y. Sugamura K. Tsuji K. Heike T. Nakahata T. NOD/SCID/gamma (c) (null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1095) Google Scholar NSG (NOD/scid/γC−/−/SzJ),10Ishikawa F. Yasukawa M. Lyons B. Yoshida S. Miyamoto T. Yoshimoto G. Watanabe T. Akashi K. Shultz L.D. Harada M. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.Blood. 2005; 106: 1565-1573Crossref PubMed Scopus (700) Google Scholar and double knockout for Rag2+IL-2Rγc.11Traggiai E. Chicha L. Mazzucchelli L. Bronz L. Piffaretti J.C. Lanzavecchia A. Manz M.G. Development of a human adaptive immune system in cord blood cell-transplanted mice.Science. 2004; 304: 104-107Crossref PubMed Scopus (782) Google Scholar, 12Strowig T. Rongvaux A. Rathinam C. Takizawa H. Borsotti C. Philbrick W. Eynon E.E. Manz M.G. Flavell R.A. Transgenic expression of human signal regulatory protein alpha in Rag2-/-{gamma}c-/- mice improves engraftment of human hematopoietic cells in humanized mice.Proc Natl Acad Sci U S A. 2011; 108: 13218-13223Crossref PubMed Scopus (164) Google Scholar A successful co-transplantation of fetal human liver cells and HSCs has been reported in double knockout BALB/c mice with the transgenic expression of the FK506 binding protein–caspase 8 fusion gene driven by the albumin enhancer/promoter.13Washburn M.L. Bility M.T. Zhang L. Kovalev G.I. Buntzman A. Frelinger J.A. Barry W. Ploss A. Rice C.M. Su L. A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease.Gastroenterology. 2011; 140: 1334-1344Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar Despite partial success, the manipulation of newborn animals and the use of fetal tissues create technical and ethical problems.An important issue for any model that is based on human tissue is the source and type of hepatocytes that can be used for transplantation, which must be syngeneic (matched) with HSCs for immune system reconstruction in experimental animals. The fetal liver provides both types of cells for transplantation. A possible choice is fetal liver cells with a progenitor phenotype that expresses epithelial cell adhesion molecule (CD326).14Schmelzer E. Zhang L. Bruce A. Wauthier E. Ludlow J. Yao H.L. Moss N. Melhem A. McClelland R. Turner W. Kulik M. Sherwood S. Tallheden T. Cheng N. Furth M.E. Reid L.M. Human hepatic stem cells from fetal and postnatal donors.J Exp Med. 2007; 204: 1973-1987Crossref PubMed Scopus (471) Google Scholar The adult hepatocytes can be transplanted with high efficiency and can be sustained long enough6Shafritz D.A. Oertel M. Model systems and experimental conditions that lead to effective repopulation of the liver by transplanted cells.Int J Biochem Cell Biol. 2011; 43: 198-213Crossref PubMed Scopus (36) Google Scholar; however, the matched sources of HSCs are limited. These cells must be isolated from the same donor bone marrow or peripheral blood. Thus, no existing model is ideal for dual reconstitution.Here, we investigated the utility of a new urokinase-type plasminogen activator (uPA)-NOG strain15Suemizu H. Hasegawa M. Kawai K. Taniguchi K. Monnai M. Wakui M. Suematsu M. Ito M. Peltz G. Nakamura M. Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.Biochem Biophys Res Commun. 2008; 377: 248-252Crossref PubMed Scopus (48) Google Scholar of mice for dual reconstitution and compared different sources of human cells for transplantation. A homozygous line of uPA-NOG mice carries two copies of the transgene array that stably reinforces transgene expression. Perinatal bleeding, embryonic or neonatal lethality, and severe tissue pathology did not occur in homozygous uPA-NOG mice compared with Alb-uPA/scid mice. The alanine aminotransferase (ALT) levels were persistently elevated along with evidence of modest hepatic injury by 6 weeks of age in the uPA-NOG homozygotes. Compared with Alb-uPA/scid transgenic mice, which have an age-dependent decrease in uPA expression caused by deletion of the integrated transgene, a relatively low frequency of physical loss of the transgene is observed from uPA-NOG mice. The persistence of the hepatic injury marker should facilitate human hepatocyte engraftment and expansion throughout the life of the mouse.15Suemizu H. Hasegawa M. Kawai K. Taniguchi K. Monnai M. Wakui M. Suematsu M. Ito M. Peltz G. Nakamura M. Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.Biochem Biophys Res Commun. 2008; 377: 248-252Crossref PubMed Scopus (48) Google Scholar This property of uPA-NOG strain allows for the manipulation of adult animals and expands the window for human cell transplantation. The engraftment of HSCs also requires the creation of a niche in mouse bone marrow for human cells. The widely used total body irradiation increases the risks of severe bacteremia and body weight loss. In this study, we used non-myeloablative conditioning with treosulfan as a safe and well-tolerated alternative to total body irradiation for HSC transplantation.16Andersson G. Illigens B.M. Johnson K.W. Calderhead D. LeGuern C. Benichou G. White-Scharf M.E. Down J.D. Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice.Blood. 2003; 101: 4305-4312Crossref PubMed Scopus (48) Google Scholar, 17van Pel M. van Breugel D.W. Vos W. Ploemacher R.E. Boog C.J. Towards a myeloablative regimen with clinical potential, I: treosulfan conditioning and bone marrow transplantation allow induction of donor-specific tolerance for skin grafts across full MHC barriers.Bone Marrow Transplant. 2003; 32: 15-22Crossref PubMed Scopus (24) Google Scholar, 18Ploemacher R.E. Johnson K.W. Rombouts E.J. Etienne K. Westerhof G.R. Baumgart J. White-Scharf M.E. Down J.D. Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.Biol Blood Marrow Transplant. 2004; 10: 236-245Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19Stephan L. Pichavant C. Bouchentouf M. Mills P. Camirand G. Tagmouti S. Rothstein D. Tremblay J.P. Induction of tolerance across fully mismatched barriers by a nonmyeloablative treatment excluding antibodies or irradiation use.Cell Transplant. 2006; 15: 835-846Crossref PubMed Scopus (17) Google Scholar, 20Sjoo F. Hassan Z. Abedi-Valugerdi M. Griskevicius L. Nilsson C. Remberger M. Aschan J. Concha H. Gaughan U. Hassan M. Myeloablative and immunosuppressive properties of treosulfan in mice.Exp Hematol. 2006; 34: 115-121Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Human hepatocyte engraftment at the 3% to 5% level is adequate to perform important studies on intrahepatic pathogens, such as those that cause malaria and hepatitis B. Successful HCV infection was achieved with >10% of hepatocytes being of human origin. Efficient hematolymphoid repopulation in combination with partial liver repopulation is sufficient to study HIV co-infection, and uPA-NOG mice offer this possibility.Materials and MethodsAnimalsThe uPA-NOG mice were provided by Central Institute for Experimental Animals (Kanagawa, Japan)15Suemizu H. Hasegawa M. Kawai K. Taniguchi K. Monnai M. Wakui M. Suematsu M. Ito M. Peltz G. Nakamura M. Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice.Biochem Biophys Res Commun. 2008; 377: 248-252Crossref PubMed Scopus (48) Google Scholar and were bred in the animal breeding facility at the University of Nebraska Medical Center. All animal procedures were approved by the University of Nebraska Medical Center Animal Care and Use Committee and were within the guidelines for humane care of laboratory animals. Female hemizygotes (n = 5) and male homozygotes (n = 3) were obtained, and 158 pups were generated by breeding. The zygosity was determined by the degree of liver damage observed by the serum levels of ALT. Serum ALT levels from 6-week-old males were determined by VetScan VS2 (Abaxis, Union City, CA), and males with elevated ALT were selected for transplantation. Females were used for the next breeding, and we found that homozygous females were able to produce one litter before 4 to 5 months of age when these mice showed phenotypically high ALT levels. NSG mice (The Jackson Laboratory, Bar Harbor, ME; stock no. 005557) were obtained from our breeding colony, which was established in 2005.Human CellsHSCs and hepatoblasts were isolated from fetal tissue (Fhbs). Tissues were provided by the University of Washington, Laboratory of Developmental Biology, supported by the National Institutes of Health Award 5R24HD000836 and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (90–117 days of gestation). The tissues arrived 48 hours after collection and were mechanically disrupted, and the resulting fragments were treated with collagenase, hyaluronidase, and DNase at 37°C. The resulting suspensions were washed with medium that contained fetal calf serum, were centrifuged at 50 × g, and processed as described.21Haridass D. Yuan Q. Becker P.D. Cantz T. Iken M. Rothe M. Narain N. Bock M. Norder M. Legrand N. Wedemeyer H. Weijer K. Spits H. Manns M.P. Cai J. Deng H. Di Santo J.P. Guzman C.A. Ott M. 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.Am J Pathol. 2009; 175: 1483-1492Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Viability (as evaluated by trypan blue exclusion) always exceeded 80% in the transplanted samples. The fractions contained 1% to 6% asialo glycoprotein receptor-positive hepatocytes as determined by staining with anti-asialo glycoprotein receptor 1–phycoerythrin (PE) antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA).22Basma H. Soto-Gutierrez A. Yannam G.R. Liu L. Ito R. Yamamoto T. Ellis E. Carson S.D. Sato S. Chen Y. Muirhead D. Navarro-Alvarez N. Wong R.J. Roy-Chowdhury J. Platt J.L. Mercer D.F. Miller J.D. Strom S.C. Kobayashi N. Fox I.J. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.Gastroenterology. 2009; 136: 990-999Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar Collected after low-speed centrifugation, the supernatant fluid was used to isolate HSCs. CD34+ HSCs were isolated by using the CD34 MicroBead Kit (Miltenyi Biotec, Auburn, CA), and the purity of the isolated cells evaluated by fluorescence-activated cell sorting (FACS) was >90%. CD34+ cells were frozen for future transplantation. Adult hepatocytes were obtained from 6-month-old donors. The hepatocytes were cryopreserved and were 80% viable and 40% attachment efficient at the time of transplantation.Transplantation of Hepatocytes and HSCsSamples of freshly isolated Fhbs were injected intrasplenically (ispl.) at 2 × 106 cells/mouse.23Fox I.J. Schafer D.F. Yannam G.R. Finding a home for cell transplants: location, location, location.Am J Transplant. 2006; 6: 5-6Crossref PubMed Scopus (13) Google Scholar Recipient mice were anesthetized with a xylozine and ketamine combination diluted in distilled water. The left sides of the mice were disinfected with a betadine solution, and a 1.5-cm cut was made 5 mm below the lower edge of the rib cage to enter the peritoneal cavity. The spleen was located and protracted slightly with the blunt-ended forceps, and the lower pole was ligated with a suture. The injection needle of the 1-mL syringe was inserted through the ligation into the spleen, and 100 μL of the cell suspension was injected slowly into the spleen. The needle was retracted, and the ligation was tightened. The spleen was pushed back into the body cavity, and the peritoneum and skin were closed with 3-0 absorbable sutures. The conditioning of the animals for HSC transplantation was done with a non-myeloablative regimen of treosulfan (medac GmbH, Hamburg, Germany), intraperitoneally injected for 3 days at a dose of 1.5 g/kg/day. CD34+ cells were intravenously (i.v.) transplanted into mice at 0.5 to 1 × 106 cells/mouse in 100 μL of PBS via the tail vein with the use of a 28G1/2-gauge needle or with hepatoblasts in 1:1 ratio by intrasplenic injection. Figure 1, A–C, shows the schematic representation of experimental approaches. Five sets of experiments were conducted, and six donor samples and 30 animals were used for approach I. The engraftment was evaluated by the human Alb (hu-Alb) concentration in peripheral blood samples at 4 weeks after surgery. For the second approach of intrasplenic co-transplantation of hepatoblasts and HSCs two samples of donor tissues and 10 animals were used. The third approach included transplantation of cryopreserved hepatocytes after transplantation of cryopreserved mismatched HSCs (n = 8).hu-Alb Level EvaluationThe hu-Alb levels in mice that received a transplant were measured every 4 to 5 weeks by using a Human Albumin ELISA (enzyme-linked immunosorbent assay) Quantitation kit (Bethyl Laboratories, Inc., Montgomery, TX). Western blot analysis confirmed the presence of hu-Alb. Animal plasma samples were diluted 1:3 in PBS and were mixed with SDS sample buffer with 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) in 1:1 ratio. The proteins were subjected to SDS-PAGE and were transferred to Hybond-ECL membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The membranes were incubated overnight with mouse monoclonal anti-human serum albumin antibody diluted 1:2000 (Abcam Inc., Cambridge, MA; ab no. 10241) and goat anti-mouse IgG–horseradish peroxidase conjugate (Sc-2005; Santa Cruz Biotechnology Inc.) secondary diluted 1:10,000 for 30 minutes. The immunoblots were developed with the ECL Western Detection System and Hyperfilm ECL (GE Healthcare Bio-Sciences Corp.).Flow CytometryPeripheral blood samples were collected from the facial vein in EDTA-coated tubes. Six-color FACS analyses of whole blood samples were performed to monitor changes in the human cell populations. In brief, 100-μL aliquots of whole blood were incubated with respective antibodies for 30 minutes at 4°C. The red blood cells were first lyzed with FACS Lysing Solution (Becton Dickinson, San Jose, CA) and then washed twice with PBS that contained 2% fetal bovine serum. Blood leukocytes were tested for human pan-CD45, CD3, CD4, CD8, CD14, and CD19 markers by multicolor panel. Antibodies and isotype controls were obtained from BD Pharmingen (San Diego, CA), and the staining was analyzed with a FACS DIVA (BD Immunocytometry Systems, Mountain View, CA). The results were expressed as percentages of the total number of gated lymphocytes. The gating strategy was human CD45⇒CD3⇒CD4/CD8, CD45⇒CD19, and CD45⇒CD14.ImmunohistochemistryTissues were fixed with 4% paraformaldehyde overnight at 4°C and then embedded in paraffin. Five-micron sections were cut from the paraffin blocks, mounted on glass slides, and subjected to immunohistochemical staining with mouse monoclonal antibodies for HLA (human leukocyte antigen)-DQ/DP/DR (clone CR3/43, 1:100 dilution), CD45 (1:200 dilution), CD68 (1:100 dilution), and cytokeratin 18 (clone DC 10, 1:33 dilution) from Dako (Carpinteria, CA), and α-smooth muscle actin (1:50 dilution) antibodies from Abcam Inc., the M30CytoDEATH (1:10 dilution) antibody was purchased from Roche Applied Science (Indianapolis, IN), the rabbit monoclonal antibody for CD8 (1:100 dilution) antibody was purchased from Abcam Inc,, and the trichrome stain was purchased from ScyTek Laboratories, Inc. (Logan, UT). Polymer-based horseradish peroxidase–conjugated anti-mouse Dako EnVision systems were used as secondary detection reagents and were developed with 3,3′-diaminobenzidine. All paraffin-embedded sections were counterstained with Mayer’s hematoxylin. For immunofluorescent staining, secondary anti-mouse and anti-rabbit Alexa Fluor 488 and Alexa Fluor 594 and blue-fluorescent DAPI nucleic acid stain were used (Invitrogen, Eugene, OR). Bright field and immunofluorescent images were obtained with a Nikon Eclipse E800 (Nikon Instruments, Melville, NY) with the use of NIS-Elements F version 3.0 software (Nikon Instruments). Immunofluorescent images were incorporated into a spectral unmixing algorithm (Nuance version 2.10; Advanced Molecular Vision, Lincolnshire, UK) that quantitatively separated the gray-scale images that represent each spectral component.ResultsOutcomes for Liver Cell Transplantation in uPA-NOG Mice Depend on the Experimental ApproachThree experimental approaches for the construction of mice combining human immune and liver cells are presented in Figure 1, A–C. Animals for human liver cell transplantation were selected on the basis of elevated ALT activity in the peripheral blood at 6 to 8 weeks of age (Figure 2A). For the first approach, we transplanted Fhbs via intrasplenic infusion (2–3 × 106 cells/mouse). Then, the animals were conditioned with treosulfan, and the cryopreserved HSCs (106 cells/mouse isolated from the same donor tissue samples) were injected i.v. The liver repopulation by human Fhbs was monitored by the hu-Alb ELISA up to 30 weeks after surgery. At 4 weeks after Fhb transplantation, hu-Alb levels >1 μg/mL were detected in 14 animals (median, 4.0 μg/mL; range, from 1.0 to 21.2 μg/mL). Fhb engraftment was also confirmed in eight animals by Western blot analysis at week 10 (Figure 2B). By 18 weeks after surgery, the hu-Alb levels declined, and only six animals had detectable hu-Alb levels (Figure 2C). At the observational end point, the presence of single CK-18+ or human cells positive for apoptotic caspase-cleaved fragment of CK18 (M30) were sporadically found (not shown). The human hematolymphoid tissue development was assessed by blood FACS analysis for the percentage of human CD45+CD3+ (T cells), CD19+ (B cells), and CD14+ (monocytes). All animals successfully established a human immune system.Figure 2Liver damage levels and the kinetics of human albumin plasma concentration in uPA-NOG mice after fetal or adult hepatocyte transplantation. A: The ALT levels in 6- to 8-week-old mice for all three schemes were comparable. B and C: Scheme I, whereby Fhbs were transplanted first after syngeneic cryopreserved HSC intravenous injection. B: Western blot analysis detected human albumin in 5 μL of mouse plasma diluted 1:3. Lanes 2 to 9 represent individual mice at 10 weeks after Fhb transplantation. Lane 10 represents hu-Alb detected in 1 mL of pooled (AB) sera diluted 100 times. C: ELISA-based detection of hu-Alb in mouse plasma. D: Scheme II, whereby treosulfan-conditioned animals received ispl. a transplant with a combination of two types of cells: Fhbs and HSCs. Fhbs did not sustain in uPA-NOG mouse liver regardless of combination with HSCs or adenovirus expressing human hepatocyte growth factor administration. E: Scheme III, whereby adult hepatocytes were transplanted first after treosulfan-conditioning and mismatched HSC intravenous injection. Adult hepatocytes were sustained in the mouse liver in the presence of mismatched hematopoiesis and lymphoid/macrophage repopulation. Open symbols represent mice with reduced hu-Alb concentration at the end of observation. Individual measurements and medians are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To improve the engraftment of Fhbs, we used the second approach, in which two types of cells (106 cells/mouse freshly isolated Fhbs and HSCs at 1:1 ratio) were co-transplanted by intrasplenic infusion in treosulfan-conditioned mice. We expected that the dynamic interaction between HSCs and Fhbs would support differentiation/maturation into hepatocytes.24Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (357) Google Scholar At week 10 after transplantation, the concentration of hu-Alb in plasma was 10 times lower than with the first approach (median, 0.5 μg/mL; range, 0.1–1.9 μg/mL) (Figure 2D). We assumed that the migration and the engraftment of Fhbs were delayed. Fhbs did not sustain, as in scheme I, and were not detected by immunohistological evaluation of liver tissue samples. However, hematolymphoid reconstitution was successful in all animals.Finally, as an alternative third approach, the transplantation of adult cryopreserved hepatocytes (2 × 106 cells/mouse) was performed, followed by intravenous injection of major histocompatibility complex mismatched cryopreserved HSCs (0.5 × 106 cells/mouse) in eight animals (Figures 2E and 3). The infusion of mature hepatocytes ensured the stable engraftment and expansion of human cells (Figures 2E and 3, A, B, and I). The median hu-Alb concentration in the peripheral blood 15 weeks after transplantation was 43 μg/mL (range, 0.3–239 μg/mL) and continued to increase up to 30 weeks of observation (median, 111.6 μg/mL; range, 6.6–312 μg/mL). The clusters of CK18+ human hepatocytes were present at the end point of observation, as seen on two representative mice liver tissue slides (Figure 3, A–D). The areas occupied by human CK18+ cells in selected sections were 4.8% to 6.8% of mouse liver tissues. These results showed that adult hepatocytes were able to survive and expand in uPA-NOG mice. Conditioning with treosulfan did not have a negative effect on hepatocyte engraftment. For all three experimental schemes, the increase of the levels of liver damage determined by ALT was similar; however, only the transplantation of mature hepatocytes and HSCs in uPA-NOG mice appeared to be a reliable way to create dual reconstituted mice.Figure 3Liver pathomorphology in two representative dual reconstituted uPA-NOG mice. A and B: Clusters of human hepatocytes were immunostained with human-specific cytokeratine-18. C and D: Magnified view of selected regions of human hepatocytes is shown. E and F: Scattering human HLA-DR-positive lymphocytes, macrophages, and Kupffer cells in close proximity in tissue sections of panels A and B. Significant brown-stained areas are occupied by activated human macrophages/histiocytes (F). G and H: The regions with activated human macrophages were also positively immunostained for α-smooth muscle actin. I: Immunofluorescent staining of human CD8+ cells (red) and mismatched hepatocytes by cytokeratine-18 (green). J: The accumulation of human CD8+ lymphocytes (red) was not observed around mismatched human hepatocytes and was often found in areas with a significant number of human CD68+ macrophages. Represented are mouse 1561 (A, C, E, and G) and mouse 1563 (B" @default.
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• W2008099316 title "Human Hepatocytes and Hematolymphoid Dual Reconstitution in Treosulfan-Conditioned uPA-NOG Mice" @default.
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