Copyright © 2013 Cognizant Communication Corporation DOI: 10.3727/096368913X672046 CT-1062 Accepted 08/24/2013 for publication in “Cell Transplantation”
Potential Barriers to Human Hepatocyte Transplantation in MUP-uPAtg(+/+)Rag2-/-C-/- Mice Authors: Junji Komori1, Aaron D. DeWard1, Roberto Gramignoli2, Stephen C. Strom2, Paulo Fontes3 and Eric Lagasse1
1 McGowan Institute for Regenerative Medicine Department of Pathology University of Pittsburgh School of Medicine, Pittsburgh PA. 2 Karolinska Institutet and Hospital Department of Laboratory Medicine Division of Pathology
Stockholm, 141-86, Sweden 3 McGowan Institute for Regenerative Medicine Department of Surgery University of Pittsburgh School of Medicine, Pittsburgh PA.
Address for correspondence: Eric Lagasse, PharmD, PhD. McGowan Institute for Regenerative Medicine University of Pittsburgh School of Medicine 450 Technology Drive. Room: 329. Pittsburgh, PA 15219. Phone: 412-624 5285. Fax: 412-624-5363. Email:
[email protected] CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 1
Copyright © 2013 Cognizant Communication Corporation
Abstract
Primary human fetal and adult hepatocytes have been considered feasible donor cell sources for cell transplantation. We compared the engraftment efficiencies between adult human, fetal human, and adult porcine hepatocytes after transplantation into MUPuPAtg(+/+)Rag2-/-C-/- mice. Transplantation of adult human hepatocytes yielded a thousandfold higher serum albumin level compared to transplantation of fetal human hepatocytes, while transplantation of adult porcine hepatocytes resulted in a hundred-fold higher serum albumin level than adult human hepatocytes. These results suggest that adult liver cells are superior to fetal liver cells for transplantation, and caution should be applied if porcine hepatocytes are used for preclinical studies as a proof of concept for human hepatocytes.
Key words: Liver cell transplantation, fetal human hepatocyte, adult human hepatocyte, adult porcine hepatocyte.
CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 2
Copyright © 2013 Cognizant Communication Corporation
Introduction
During development, fetal cells have abundant proliferative capacity and differentiation potential, which makes them an attractive cell source for regenerative medicine. However, transplantation of fetal liver cells is still controversial, due to the conflicting reports about their engraftment capability (2,7,12,16). In this report, we investigated and compared the engraftment efficiencies of three different donor hepatocyte sources, murine, porcine and human hepatocytes under similar condition of transplantation into a new stable immune deficient animal model of liver failure. Chimeric human-mouse livers (“humanized livers”) have been generated previously in immunodeficient mice that lack the fumarylacetoacetate hydrolase gene (Fah) or that overexpress the urokinase-type plasminogen activator (uPA) (1,24)_ENREF_1. The Fah mouse is invaluable for liver cell transplantation with one caveat; no studies can exceed 9 months due to the spontaneous generation of hepatocarcinoma in older animals (6). In Albumin-uPA mice, uPA expression is controlled by the albumin enhancer/promoter, which results in diffuse vacuolization within hepatocyte rough endoplasmic reticulum that increases in severity with age and is accompanied by sporadic hepatocyte death (20, 27). Transplantation of healthy donor hepatocytes in this environment led to high repopulation of human donor cells (24). However, these mice have increased neonatal death due to frequent hemorrhaging and require hepatocyte transplant at an early age, because the albumin promoter is activated during embryonic development (8,20,24,27). On the other hand, the major urinary protein (MUP) promoter is activated approximately 2-3 weeks after birth in the liver, which eliminates neonatal hemorrhaging and allows for postnatal transplantation that is not possible in Albumin-uPA mice (27). Therefore, we crossed MUP-uPA mice with the CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 3
Copyright © 2013 Cognizant Communication Corporation immunodeficient Rag2-/-C-/-mice, which lack B, T and NK cells, to allow cell repopulation from xenogeneic donors, to alleviate neonatal lethality and to expand the window of opportunity for hepatocyte transplant (25). Our aim was to establish and validate this new animal model for human hepatocyte transplantation and compare side-by-side the degree of liver repopulation of various hepatocyte populations.
Materials and Methods
Animals Green fluorescent protein (GFP) transgenic mice (C57BL/6-Tg(UBC-GFP)30Scha/J) were purchased from the Jackson Laboratory (Bar Harbor, ME). To generate MUPuPAtg(+/+)Rag2-/-C-/- mice, MUP-uPA mice (a kind gift from Dr. Eric Sandgren, University of Wisconsin-Madison, WI) were crossed with Rag2-/-C-/- mice (Taconic, Germantown, NY). PCR-based genotyping was carried out on 200 ng genomic DNA isolated from tail tissue as previously described (24). Sow 6 weeks of age (around 70 kg) were purchased from Animal Biotech Industries (Danboro, PA). Animals were bred and housed in the Division of Laboratory Animal Resources facility at the University of Pittsburgh Center for Biotechnology and Bioengineering. Experimental protocols followed National Institutes of Health guidelines for animal care and were approved by the Institutional Animal Care and Use Committee at University of Pittsburgh.
Preparation of primary hepatocytes from mouse, human and swine. All research protocols were reviewed and approved at the University of Pittsburgh by the Institutional Review Board for Human Research Studies and the Institutional Animal Care CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 4
Copyright © 2013 Cognizant Communication Corporation and Use Committee for murine and porcine studies. The tissue dissociation and subsequent hepatocyte isolation procedures used were developed by Seglen (21) and modified as described previously (4). For adult human liver (gender not identified), a total of 12 liver tissues were used in this study. Most of the patients (age 20 to 80) were undergoing scheduled liver resection, and residual liver tissue not needed for diagnostic purposes was transported to the laboratory from the operating rooms in cold Eagle’s minimum essential medium (EMEM) (Lonza, Atlanta, GA) within 30 min of removal. Most of these liver resections were the result of metastatic colon cancer. Several livers were obtained from nonheart- beating donors and were reported to have 15 and 40 min of warm ischemia. Fetal human liver tissues were obtained from the Tissue Bank at the Magee Women’s Hospital of UPMC. Samples were between 19-23 weeks of gestation (gender not identified). Fetal livers were placed in HBSS (Thermo Scientific, Waltham, MA) and minced into small pieces. Liver samples were incubated with EBSS (Thermo Scientific, Waltham, MA) /10mM EGTA (Sigma, St. Louis, MO) /1% HEPES (Cellgro, Manassas, VA) for 15 min at 37C and treated with 1 mg/ml Collagenase II (Life Technologies, Carlsbad, CA) + 1 mg/ml Hyaluronidase (Sigma, St. Louis, MO) + 100 g/ml of DNaseI (Roche, Basel, Switzerland) for 1-1.5 hours to obtain a cell suspension. Adult mouse hepatocytes were isolated using the classic 2-step collagenase perfusion technique described by Seglen (21) from 6 to 12 weeks old mice. Adult porcine hepatocytes were isolated using the same method and enzymes as for human adult hepatocytes. Briefly, the Landrace pigs (females, 6 weeks of age and 70kg) underwent a partial hepatectomy (left lateral resection) and the graft was immediately flushed with cold solution (4°C). A resected liver segment was transported (ice chest) to the lab in a sterile plastic bag while fully immersed at 4°C. The cell isolation was performed within 1 to 3 hours from the initial hepatic resection as described previously (4). Cell viability for each fetal or adult hepatocyte preparation was assessed by mixing an aliquot of the final cell suspension with an equal volume of 0.4% (w/v) trypan blue (Cellgro, Manassas, VA) in phosphatebuffered saline and counting only the number of viable (unstained) and dead (blue) CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 5
Copyright © 2013 Cognizant Communication Corporation hepatocytes with the aid of a hemocytometer. Viabilities are expressed as a percentage of the total hepatocyte number. Splenic injection of murine, human and porcine hepatocytes One million viable hepatocytes isolated freshly in 50 l of HBSS (Thermo Scientific, Waltham, MA) were injected into the spleen via a 28-gauge needle of an insulin syringe.
Human and porcine albumin measurement Blood collection (100 l) was performed using the submandibular bleeding technique every 2-4 weeks. Human and porcine albumin concentration was measured with the Human and Porcine Albumin ELISA Quantitation Kit respectively (Bethyl, Montgomery, Tx) according to the manufacturer’s protocol.
Immunohistochemistry Frozen sections (5m) were fixed in cold acetone (Thermo Scientific, Waltham, MA) for 5 minutes. For IHC staining, sections were washed with PBS and blocked with 5% skim milk. Sections were then incubated with primary antibody, goat anti-human or porcine albumin (Bethyl Laboratories Inc., Montgomery, TX) for 1 hour, secondary antibody, Alexa Fluor 594 anti-goat IgG (Invitrogen, Carlsbad, CA) 1 hour and a third antibody, FITC conjugated antimouse albumin (Bethyl Laboratories Inc) for 1 hour. Sections were mounted with Hoechst mounting media (Life Technologies, Carlsbad, CA). Images were captured with an Olympus IX71 inverted microscope.
Statistical analyses CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 6
Copyright © 2013 Cognizant Communication Corporation Statistical significance was determined with an unpaired two-tailed Student's-t-test and Bonferroni correction.
Results
Generation of MUP-uPAtg(+/+)Rag2-/-C-/- (uRG) mice In MUP-uPA mice, hepatocyte damage begins to increase at 4 weeks of age, peaks at 5 weeks, and returns to normal levels (no damage) by 13 weeks of age (27). 2-4 week-old recipients demonstrated high repopulation of healthy donor hepatocytes 4 weeks after transplantation (27). First, we transplanted, via splenic injection, C57BL/6 GFP+ mouse hepatocytes into immunocompetent MUP-uPA mice (C57BL/6 background). Repopulation 4 weeks after transplant in the livers of homozygous MUP-uPA transgenic (MUP-uPAtg(+/+)) mice was distinctly higher than that in heterozygous MUP-uPA transgeneic (MUP-uPAtg(+/-)) mice, due to the increased uPA expression in homozygous mice (Figure 1). This result was similar to what was observed with the Albumin-uPA mice (19,20). As a result, MUPuPAtg(+/+)Rag2-/-C-/- (uRG) mice were used for all syngeneic and xenogeneic transplantations to maximize engraftment efficiencies.
Syngeneic transplantation of murine hepatocytes into uRG mice To define a successful repopulation of hepatocytes in uRG mice, we transplanted 1x106 C57BL/6 GFP+ primary adult mouse hepatocytes by splenic injection. The syngeneic repopulation ranged from 91.2 to 99.9% GFP+ hepatocytes engrafted in the liver 4 weeks after transplantation, a level of repopulation similar to what we observed with the immunocompetent MUP-uPa (Figure 1) or previously with Fah mice (17,18). This result CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 7
Copyright © 2013 Cognizant Communication Corporation indicates that the cross of MUP-uPA mice with immunodeficient Rag2-/-c-/- mice does not affect the evolution of liver disease or the engraftment of wild type hepatocytes in this animal model.
Xenogeneic transplantation of porcine hepatocytes into uRG mice Next we transplanted 1x106 primary adult porcine hepatocytes from 6 donors into C57BL/6 mice (n=4), MUP-uPA mice (n=4) and uRG mice (n=17 mice). One uRG mouse died before first bleeding at 2 weeks after transplantation for unknown reasons. Serum porcine albumin level in uRG mice reached over 5mg/ml at 2 weeks after transplant, while as expected the levels in immunocompetent C57BL/6 and MUP-uPA control mice were undetectable 2 weeks after transplant (Figure 2A). Serum porcine albumin levels in uRG plateaued 4 weeks after transplantation and remained relatively stable for an additional 10 weeks (Figure 2B). Splenic injection of 1x106 xenogeneic porcine hepatocytes repopulated over 90% of the uRG host liver (Figure 2C-E), as expected by the high porcine albumin level detected in the mouse serum (over 10mg/ml). It is interesting to note that, although the porcine albumin concentration is high, it is still not at normal mouse serum levels. Presumably mouse albumin is still expressed from the remaining mouse hepatocytes present in the liver, as demonstrated by immunostaining in Figure 2E. The relationship of host and donor hepatocytes in the context of serum albumin concentration after xenogeneic transplantation in mice has been discussed previously (24). Strikingly, the high engraftment of xenogeneic porcine hepatocytes is at a similar level as syngeneic murine hepatocytes (Figure 1). Our results suggest no detectable xenogeneic barrier to adult porcine hepatocyte transplantation in uRG mice, although a xenogeneic barrier was present in immunocompetent animals because no transplanted adult porcine hepatocytes engrafted. CT‐1062 Cell Transplantation Epub; provisional acceptance 06/20/2013 8
Copyright © 2013 Cognizant Communication Corporation
Xenogeneic transplantation of human adult and fetal hepatocytes into uRG mice Finally we transplanted 1x106 primary adult human hepatocytes from 12 different donors (n=75 mice) and 1x106 primary fetal human hepatocytes from 5 donors (n=36 mice). Two mice transplanted with adult human hepatocytes and one mouse transplanted with fetal human hepatocytes died before first bleeding 2 weeks after transplantation for unknown reasons. Therefore, we analyzed 108 of 111 transplanted mice (97.3%). The serum albumin level of uRG mice transplanted with adult human hepatocytes was significantly higher than in mice transplanted with fetal human hepatocytes 4-5 weeks after transplant (Figure 3A). Experimental animals were necropsied at the termination of the experiment and tested for species-specific donor blood serum albumin concentration, which correlates with donor repopulation in the host liver (24). Thus fetal human hepatocytes engraft significantly lower than adult human hepatocytes. It is interesting to note that despite the variability in age or other factors concerning the source of human hepatocytes isolated (see methods), serum albumin difference is highly significant between fetal and adult human hepatocyte transplants (p
