Adenovirus-Mediated CTLA4Ig Gene Transfer Improves the Survival of Grafted Human Hepatic Progenitors in Mouse Liver

Adenovirus-Mediated CTLA4Ig Gene Transfer Improves the Survival of Grafted Human Hepatic Progenitors in Mouse Liver F. Lan, X. Ma, Y. Liu, and I. Shen ABSTRACT The invasive nature of surgery and limited numbers of donor livers for end-stage patients has prompted the search for alternative cell therapies for intractable hepatic disease. Hepatocyte ransplantations have been performed for a variety of indications, but sustained benefits have not been observed in most cases. Rat fetal liver epithelial cells (liver stem cells) have demonstrated self-renewal in vivo and functional repopulation of the liver. We have previously isolated and expanded epithelial progenitor cells (EPC) from the human fetal liver to investigate their differentiation potential. In this study, we applied suppression of immunorejection by adenoviral CTLA4Ig gene delivery mediated to examine the survival and differentiation of human fetal EPC transplanted into normal mouse liver. The grafted EPC showed extensive proliferation at both 1 and 2 months after transplantation compared with controls. Moreover, most EPC differentiated into hepatocytes, while a small fraction became bile ductular cells. This finding suggested that human fetal EPC may be a ideal source of cell-based therapy for various liver diseases. HE WORLDWIDE SHORTAGE of donor livers for end-stage liver disease patients has prompted a search for alternative cell therapies to treat intractable liver disease. Thus, attention has been focused on hepatocyte transplantation as an alternative to liver transplantation. Although proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury, under conditions in which hepatocyte proliferation is blocked, adult hepatocytes have only a limited ability to proliferate, replacing no more than 1% to 2% of liver mass.1 Stem cells are a promising source for liver repopulation after cell transplantation. Undifferentiated epithelial cells in the periportal areas of adult liver, the “oval cells,” can proliferate, differentiate into hepatocytes, and restore liver mass.2 However, these cells do not repopulate the normal liver after transplantation. In contrast, epithelial cells isolated from early fetal liver can effectively repopulate normal livers.3 We have previously described expansion and in vitro and in vivo characterization of epithelial progenitor cells (EPC) from the human fetal liver. CTLA4-Ig, a recombinant immunoglobulin fusion protein that specifically blocks the ligands for CD28, suppresses allograft and xenograft rejection by interrupting the costimulatory pathway. Previous investigations have demonstrated that adenovirusmediated gene transfer into the donor liver with the

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CTLA4-Ig gene can suppress rejection in a rat liver transplant model.3 We therefore applied adenoviral vectors containing CTLA4-Ig to investigate repopulation of human fetal EPC transplanted into normal mice livers. MATERIALS AND METHODS Cell Culture Isolation and primary culture of human fetal liver EPC was described previously.4 The culture medium was Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, Calif) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah), 20 ng/mL epidermal growth factor (EGP; Gibco), 5 mg/mL insulin (Sigma, St. Louis, Mo), 5 mmol/L hydrocortisone (Sigma), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco). The 293 cells of the E1-containing human kidney cell line were maintained in DMEM with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin, and 1.0% L-glutamate.

Generation of Recombinant Adenovirus Vectors The recombinant adenovirus vectors used in this study were generated by the Adeasy system.5 In brief, the cDNA encoding From the Peking University Health Science Center, Peking, People’s Republic of China. Address reprint requests to Professor Li Shen, PhD, Peking University Health Science Center, Department of Cell Biology, Xue Yuan Road #38, Beijing 100191, People’s Republic of China. E-mail: [email protected]

0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.01.103

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ADENOVIRUS-MEDIATED CTLA4IG GENE TRANSFER CTLA4Ig was subcloned into a shuttle vector, pAdTracK-CMV. The resultant pAdTrack-CTLA4Ig was used next to generate adenoviral recombinants through homologous recombination with the adenoviral backbone vector, pAdEasy-1, in BJ5183 bacterial cells. After linearized with Pac I, the adenoviral recombinants were used to produce adenoviruses in HEK 293 packaging cells, resulting in an AdCTLA4Ig adenoviral vector that contained a built-in enhanced green fluorescent proteins (EGFP) expression cassette. The virus were purified by Adenopure adenovirus purification kit (Puresyn, Malvern, Pa). The virus titer was determined by plaque assay on 293 cells. The EGFP-expressing adenovirus (AdGFP) generated from the empty pAdTracK-CMV serve as the control vector.

Liver Transplantation of EPC and Immunofluorescence Twenty-four hours before transplantation, EPC were infected with eitherAdCTLA4Ig or AdEGFP; 5 ⫻ 105 EPC were transplanted via intrahepatic injection into 4-week-old Institute of Cancer Research (ICR) mice (n ⫽ 5). At 2 months after transplantation, the mice were sacrificed and the liver tissue cryosectioned. To quantify transplanted EPC, immunohistochemical detection was performed with a primary monoclonal anti-human nuclei (hNu) antibody (Chemicon, Temecula, Calif) according to the manufacturer’s instructions. To determine whether the EPC could differentiate into hepatic cells in vivo, we used immunofluorescence to detect the hepatocyte-specific markers alpha fetoprotein (AFP) and CK19, a general ductular cell marker. The secondary antibod-

1863 ies were either fluorescein (FITC)-conjugated goat antirabbit immunoglobulin G (IgG) (1:200) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG (1:200) antibodies for different primary antibodies. Quantification of hepatocytes or ductal elements followed the previously described method.3 All surgical interventions and animal care were provided in accordance with the Guide for the Care and Use of Laboratory Animals.

RESULTS

The phenotype of human fetal liver EPC was characterized as previously described. Briefly, real-time polymerase chain reaction analysis suggested that EPC expressed liver epithelial markers (cytokeratin CK-8 and CK-18) and biliaryspecific markers (CK-7 and CK-19). Fluorescence activated cell sorting (FACS) analysis indicated that these cells were positive for CD117, CD147, CD90, CD44, human leukocyte antigen class I, and CD71, but negative for CD34 and CD45. When subjected to conditions of hepatic differentiation, EPC were induced to become hepatocyte-like cells, which expressed albumin, AFP, and CK18 proteins. In vivo analysis also showed that the grafted EPC were integrated into the liver of immunodefective nude mice. The EPC at the 15th passage were analyzed by immunofluorescence before transplantation.4 The vast majority of the EPC cell

Fig 1. Phenotypic characterization of epithelial progenitor cell (EPC) by immunofluorescence: most EPC are AFP positive (B) after overlay with DAPI staining (A, C); a small fraction of EPC were CK-19 immunoreactive (E) when overlaid with DAPI staining (D, F). Bar ⫽ 20 ␮m.

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treatment improved the survival of EPC without modifying their differentiation potential. DISCUSSION

Fig 2. (A) Survival of human nucleus-positive cell number was quantified by counting the 25th section of 50 serial sections, with five mice each group. **P ⬍ .01 versus group, n ⫽ 5. (B) The proportions of hepatocytic, bile duct, and bipotential before and 2 months after cell transplantation, as determined by histological analysis. AFP, alpha fetoprotein; AdGFP, EGFP-expressing adenovirus.

fraction used for transplantation were unipotent, 88% hepatocytes (Fig 1B) and 8% bile ductular elements (Fig 1E) with only 4% bipotential (Fig 2B). The effect of AdCTLA4Ig on transplanted EPC survival was tested by counting the number of hNU⫹. As shown in Fig 2A, The mean surviving cell number of the two control groups (AdGFP and no treatment) were 210 ⫾ 63 and, 230 ⫾ 67 after 1 month, and, 150 ⫾ 52 and, 180 ⫾ 61 after 2 months, respectively. In contrast, the mean survival cell number of AdCTLA4Ig was 610 ⫾ 120 after 1 month and 1250 ⫾ 210 after 2 months, which were both significantly increased compared with controls. These data suggested that AdCTLA4Ig promoted the survival of grafted EPC, and that EPC proliferated when immunorejection was suppressed. Furthermore, to analyze the fate of transplanted EPC, cells with either AFP⫹/hNu⫹ or CK-19⫹/hNu⫹ were counted as shown in Fig 2B. Two months after transplantation, the proportion of these two phenotypes in the three groups were equivalent and similar to EPC before transplantation. This observation indicated that AdCTLA4Ig

The main objectives of this study were to examine the proliferation and differentiation of human fetal EPC in normal mouse liver with AdCTLA4Ig-induced suppression of immunorejection. In the present study, there was significant proliferation of EPC in AdCTLA4Ig-treated normal mouse livers, and more importantly, these cells showed differentiation potential. A number of investigators have isolated, cultured, and/or passaged human fetal liver epithelial cells with bipotent properties. Several of these as well as our own studies have demonstrated differentiation into hepatocytes after transplantation into Severe combined immunodeficiency disease (SCID) or nude mice.6,7 However, all of these cells and cell lines have shown only limited repopulation of the normal liver except for rat fetal liver stem/progenitor cells that produce substantial long-term replacement and function. To advance the field of liver cell therapy, it will be necessary to find cells that can be expanded in culture to successfully repopulate the liver under clinically acceptable conditions. This study suggested that human fetal liver EPC might possess the potential for in vivo self-renewal and long-term repopulation of the liver. ACKNOWLEDGMENT The authors acknowledge the financial support from the Beijing Natural Science Foundation (grant no. 7062041) and National Natural Science Foundation of China (grant no. 30471638).

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