Despite the wide popularity of laparoscopic incisional hernia repair (LIHR) in the nontransplant population, there are very few reports of LIHR available in abdominal organ transplant patients and none exclusively on kidney and/or pancreas (KP) transplant patients. We retrospectively reviewed a consecutive series of LIHR in KP transplant recipients performed over a period of 4 years and compared the results with LIHR in non-transplant patients during the same period. A total of 36 transplant patients were compared with 62 nontransplant patients. There were five patients converted to the open procedure in the transplant and four in nontransplant patients (p-NS). There were three seromas and one patient had a bowel perforation in the transplant group versus eight seromas, one bowel perforation and one small bowel obstruction noted in the nontransplant group. One patient in each group had a mesh infection requiring explant. Patients were followed up for a mean period of 2.2 years in the transplant group and 3 years in the nontransplant group. Overall there were five recurrences in the transplant group and four in the nontransplant group (p = NS). These results suggest that that LIHR is a safe and effective alternative to open repair.
Despite being pioneered by gynecologists, single-incision endosurgery has not been widely reported for the treatment of ovarian and adnexal pathology in neonates, children, and adolescents. We describe our initial experience using single-incision pediatric endosurgery (SIPES) for these indications and discuss advantages and drawbacks.All children who underwent SIPES with a preoperative diagnosis of ovarian or adnexal pathology were included in the study. Data on age, operative time, complications, length of hospital stay, and outcomes were collected.From January 2010 until January 2012, 19 girls (mean age, 11.4 years; range, 6 days-17 years; weight range, 4.0-90 kg) underwent SIPES procedures for ovarian or adnexal diagnoses, including hemorrhagic/follicular/paratubal cysts (n=8), torsion (n=7), tumor (n=3), and parauterine cyst (n=1). The operations included cyst unroofing (n=4), detorsion and oophoropexy (n=7), (salpingo)oophorectomy (n=5), marsupialization of cyst (n=2), and cyst aspiration (n=1). Median operative time was 42 ± 29 minutes; there were no conversions to conventional laparoscopy or open surgery. Fifteen patients (79%) were discharged within 24 hours after the procedure. There were no peri- or postoperative complications. Histopathology showed hemorrhagic/follicular/paratubal cyst (n=7), necrotic/calcified ovarian tissue after torsion (n=6), cystadenofibroma (n=1), granulosa cell tumor (n=1), and mature teratoma (Grade 0) (n=1).SIPES is an excellent alternative to conventional laparoscopy for the treatment of adnexal pathology. Using a single umbilical incision that can be enlarged instead of three smaller trocar sites facilitates the resection and extraction of ovarian masses without compromising cosmesis.
Single-incision pediatric endosurgery (SIPES) is gaining popularity. The aim of this study was to review the authors' experience with SIPES splenectomy and compare it with conventional laparoscopic splenectomy.After institutional review board approval, data on SIPES splenectomy in children were collected prospectively. The study group was compared with a control group of patients who were retrospectively identified as having undergone conventional laparoscopic splenectomy during the same time period.Sixteen children underwent SIPES splenectomy. Ages ranged from 1 to 15 years, with a median of 7 years, and weights were between 10 and 70 kg, with a median of 24 kg. The control group was similar in age and weight characteristics. The most common diagnoses were hereditary spherocytosis, sickle cell disease, and immune thrombocytopenic purpura. There were two conversions to open splenectomy in the SIPES group and one in the laparoscopic group. Operative times were 40-190 minutes (median, 84 minutes) in the SIPES group and 51-154 minutes (median, 99 minutes) in the conventional laparoscopic group.The SIPES technique is well suited for splenectomy. Despite instruments and camera being in-line, working angles are not compromised, and visualization is adequate. Operating time and hospital stay are comparable to those with standard laparoscopic splenectomy, but the cosmetic result may be superior.
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 (122) 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 (122) 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 (75) 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 (660) 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 (766) 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 (40) 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 (129) 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 (1177) 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 (746) 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 (822) 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 (177) 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 (247) 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 (494) 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 (40) 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 (48) 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 (20) 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 (70) 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. The 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. HSCs 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 (91) 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 (436) 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. Samples 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). The 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.). Peripheral 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. Tissues 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. Three 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. 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 (367) 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. All animals conditioned with treosulfan and which received a transplant with fetal liver-derived CD34+ HSCs successfully developed a human immune system. Human CD45+ cells were detected in the peripheral blood 23 to 30 weeks after HSC transplantation. The proportion of human CD45+ cells for all three schemes was similar (median, approximately 50%–60%) to the lymphocyte gate (Figure 4, A–D). Furthermore, the proportion of CD4 and CD8 positive T cells within the pool of CD3+ lymphocytes was also similar for animals that received a transplant either i.v. or ispl. with matched or mismatched hepatocytes. For scheme I and II, the animals received a transplant with 106 CD34+ HSCs, whereas the animals received a transplant with 0.5 × 106 cells in scheme III. The difference in the amount of transplanted HSCs can explain the different human B-cell proportions in the peripheral blood. Such different dynamics could also be donor dependent and is normally observed in NSG/NOG or double knockout strains of mice.25Gorantla S. Sneller H. Walters L. Sharp J.G. Pirruccello S.J. West J.T. Wood C. Dewhurst S. Gendelman H.E. Poluektova L. Human immunodeficiency virus type 1 pathobiology studied in humanized BALB/c-Rag2-/-gammac-/- mice.J Virol. 2007; 81: 2700-2712Crossref PubMed Scopus (110) Google Scholar, 26Chang H. Biswas S. Tallarico A.S. Sarkis P.T. Geng S. Panditrao M.M. Zhu Q. Marasco W.A. Human B-cell ontogeny in humanized NOD/SCID gammac(null) mice generates a diverse yet auto/poly- and HIV-1-reactive antibody repertoire.Genes Immun. 2012; 13: 399-410Crossref PubMed Scopus (26) Google Scholar, 27Takahashi M. Tsujimura N. Otsuka K. Yoshino T. Mori T. Matsunaga T. Nakasono S. Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice.J Biosci Bioeng. 2012; 113: 529-535Crossref PubMed Scopus (6) Google Scholar, 28Choi B. Chun E. Kim M. Kim S.Y. Kim S.T. Yoon K. Lee K.Y. Kim S.J. Human T cell development in the liver of humanized NOD/SCID/IL-2Rgamma(null)(NSG) mice generated by intrahepatic injection of CD34(+) human (h) cord blood (CB) cells.Clin Immunol. 2011; 139: 321-335Crossref PubMed Scopus (27) Google Scholar, 29Lang J. Kelly M. Freed B.M. McCarter M.D. Kedl R.M. Torres R.M. Pelanda R. Studies of lymphocyte reconstitution in a humanized mouse model reveal a requirement of T cells for human B cell maturation.J Immunol. 2013; 190: 2090-2101Crossref PubMed Scopus (79) Google Scholar, 30Lang J. Weiss N. Freed B.M. Torres R.M. Pelanda R. Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rgammanull mouse model: a multivariable optimization approach.Clin Immunol. 2011; 140: 102-116Crossref PubMed Scopus (32) Google Scholar Treosulfan conditioning was
Success in lineage-specific differentiation of embryonic stem cells and evidence for adult stem cell plasticity have enhanced interest in cell transplantation. Transplantation of a variety of cell types could revolutionize treatment of diseases ranging from heart failure to diabetes. For some disorders, such as heart failure, transplanted cells must engraft directly in the diseased tissue in order to affect repair. For other illnesses, it may be possible to correct disease by transplantation at a site distant from the failing organ. Two examples include reversal of diabetes by engrafting pancreatic islets under the kidney capsule or in the liver and correction of liver failure by engrafting hepatocytes in the spleen. Percutaneous transplantation of cells into the liver can result in laceration of the liver, and requires infusion through the portal circulation with disruption of hepatic sinusoids for engraftment. This produces transient portal hypertension and increases the risk of portal thrombosis and donor cell embolization into the lungs. Physiologically, this limits the number of cells that can be transplanted at any one time and produces inefficient engraftment. Transplantation into the spleen is similarly problematic. The peritoneal cavity and the subcutaneous space have been examined as preferable sites for engraftment as access is easier and safer (1Selden C Casbard A Themis M Hodgson HJ. Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum.Cell Transplant. 2003; 12: 569-578Crossref Scopus (11) Google Scholar,2Jirtle RL Biles C Michalopoulos G. Morphologic and histochemical analysis of hepatocytes transplanted into syngeneic hosts.Am J Pathol. 1980; 101: 115-126PubMed Google Scholar). Theoretically, either site could accommodate a large number of cells, and engrafted cells in these locations could be more readily assessed by biopsy to determine whether loss of function was a result of graft rejection or apoptosis. Subcutaneous engraftment might also be amenable to assessment by noninvasive imaging. So far, however, successful hepatocyte engraftment in these locations has been limited. In this issue of the American Journal of Transplantation and in a companion article in Hepatology, Yokoyama, Ohashi and their associates demonstrate engraftment and function of primary hepatocytes in the subcutaneous space lasting from 3 to as many as 9 months by preparing the site in advance with fibroblast growth factor (FGF) to induce neovascularization and by providing a matrix to enhance cell engraftment (3Yokoyama T Ohashi K Kuge H et al.In vivo engineering of metabolically active hepatic tissues in a neovascilarized subcutaneous cavity.Am J Transplant. 2005; 6: 50-59Abstract Full Text Full Text PDF Scopus (61) Google Scholar,4Ohashi K Waugh JM Dake MD et al.Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases.Hepatology. 2005; 41: 132-140Crossref PubMed Scopus (113) Google Scholar). Functionally important hepatocyte-specific gene expression was documented by RT-PCR and immunohistochemistry and transplanted cells produced albumin for an extended period of time in Nagase rats and reconstituted 5–10% of normal factor VIII activity and reduced bleeding time in hemophilia A mice. During a single infusion, the authors were also able to successfully transplant ten times the usual number of hepatocytes into the subcutaneous space than could safely be introduced into the liver. These results represent an important development for cell transplantation. While we do not know whether graft survival lasting months in rodents will translate into more prolonged survival in patients, even transient function by subcutaneously transplanted hepatocytes could support patients with acute liver failure until their native livers regenerate. In addition, because delivery into the subcutaneous space carries little risk, repeated hepatocyte infusions could potentially provide enough support for patients with chronic liver failure to improve disabling hepatic encephalopathy. A few caveats, however, are in order. The level of proteins generated by the subcutaneously transplanted hepatocytes appears to range from one-third to one-tenth the level produced when a similar or much smaller number of hepatocytes are transplanted in the spleen or liver (5Bumgardner GL Heininger M Li J et al.A functional model of hepatocyte transplantation for in vivo immunologic studies.Transplantation. 1998; 65: 53-61Crossref PubMed Scopus (69) Google Scholar,6Moscione AD Rozga J Chen S Naim A Scott HS Demetriou AA. Long-term correction of albumin levels in the Nagase analbuminemic rat: Repopulation of the liver by transplanted normal hepatocytes under a regeneration response.Cell Transplant. 1996; 5: 499-503Crossref PubMed Google Scholar). Thus, the hoped for benefit of more efficient engraftment and function by subcutaneous transplanted cells has yet to be realized, and some advantages are diminished by the need to implant more precious donor hepatocytes. This has significance for patients, where transplantation of several billion hepatocytes has been needed to produce a clinically demonstrable effect, even in infants (7Fox IJ Roy Chowdhury J. Hepatocyte transplantation.J Hepatol. 2004; 40: 878-886Abstract Full Text Full Text PDF PubMed Google Scholar). If the functional efficacy of donor hepatocytes implanted in the subcutaneous space were identical to the published experience for hepatocytes implanted in the liver, the cell pellet that would need to be implanted might be as large as 150 milliliters; considering the efficiency reported, the packed cell volume needed in the subcutaneous space might be more than a liter. This might be difficult to accomplish. For more long-term application, additional studies will be needed to determine whether the immunogenicity associated with implanting cells in the subcutaneous space will increase their susceptibility to rejection; whether cell survival will be adversely affected by the lack of a biliary system and the local production of bile by the engrafted cells; whether the cells can function and survive long term without the nutritional support provided by the portal circulation; or whether cells engrafted outside the liver can proliferate, as appears to be necessary, to compensate for loss of hepatocyte mass from injury or natural cell turnover. Hepatocyte transplantation has proven to be effective in the laboratory for correction of a variety of liver disorders. Innovations aimed at improving hepatocyte delivery, survival and function, such as these, may help improve the clinical experience so that it matches the expectations created by laboratory success (8Matas AJ Sutherland DER Steffes MW et al.Hepatocellular transplantation for metanolic deficiencies: Decrease of plasma bilirubin in Gunn rats.Science. 1976; 192: 892-894Crossref PubMed Scopus (283) Google Scholar). This work was supported by grants AI49472 and DK48794 from the National Institutes of Health.
