529 GH enhances proliferation of human hepatocytes grafted into immunodeficient mice with damaged liver Norio Masumoto1,2, Chise Tateno1,3, Asato Tachibana1,4, Rie Utoh1, Yoshio Morikawa4, Takashi Shimada4, Hiroyuki Momisako1,2, Toshiyuki Itamoto2, Toshimasa Asahara2,3 and Katsutoshi Yoshizato1,3,5 1Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan 2Division of Frontier Medical Science, Department of Surgery, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan 3Hiroshima University Liver Project Research Center, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan 4PhoenixBio Co. Ltd, 3-4-1 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan 5Developmental Biology Laboratory and Hiroshima University 21st Century COE Program for Advanced Radiation Casualty Medicine, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8526, Japan (Correspondence should be addressed to K Yoshizato; Email: [email protected]) (C Tateno and K Yoshizato are now at PhoenixBio Co. Ltd, 3-4-1 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan) (R Utoh is now at Institute of Advanced Life and Medical Sciences, Tokyo Women’s Medical College, Kawada-chou 8-6, Shinjuku-ku, Tokyo 162-8666, Japan) Abstract We investigated effects of human (h) GH on the proliferation and cell cycle regulatory genes such as human forkhead box of h-hepatocytes that had been engrafted in the liver of M1, human cell division cycle 25A, and human cyclin D1. To albumin enhancer/promoter driven-urokinase plasminogen confirm the reproducibility of these effects of rhGH, similar activator transgenic/severe combined immunodeficiency experiments were run using h-hepatocytes from a 46-year disease (uPA/SCID) mice (chimeric mice). The h-hepato- (46Y)-old donor. rhGH similarly enhanced their repopula- cytes therein were considered to be deficient in GH, because tion speed and up-regulated the expression of the above- hGH receptor (hGHR) is unresponsive to mouse GH. tested genes, especially hIGF-1 and hSTAT1. The extent of Actually, hIGF-1 was undetectable in chimeric mouse sera. the enhancement by rhGH was much less than that in 6Y- The uPA/SCID mice were transplanted with h-hepatocytes hepatocyte-chimeric mice most probably due to the from a 6-year (6Y)-old donor, and were injected with difference in GHR expression levels between the two donors. recombinant hGH (rhGH). rhGH stimulated the repopulation In conclusion, this study clearly demonstrated that rhGH speed of h-hepatocytes; and up-regulated hIGF-1, human stimulates the proliferation of h-hepatocytes in vivo. signal transducers and activators of transcription (hSTAT) 3, Journal of Endocrinology (2007) 194, 529–537 Introduction albumin enhancer/promoter driven-urokinase plasminogen activator transgenic/severe combined immunodeficiency Using rodents as experimental animals, it has been shown that disease (uPA/SCID) mice. The transplanted cells were differentiated hepatocytes can re-enter into the cell cycle engrafted in the liver, continuously replicated, and repopu- when stimulated by growth factors, cytokines, and hormones lated in the liver. The extent of repopulation was calculated as (Taub 1996, Michalopoulos & DeFrances 1997, Fausto 2000). the ratio (replacement index, RI) of the h-hepatocyte- Hepatocytes in culture usually lose their replication ability repopulated area to the total examined area. Recent and normal phenotypes and, thus, have had limited usability technological improvements enabled us to yield children’s in testing the effects of these factors on hepatocytes. In hepatocyte-chimeric mice with RIO96%. The chimeric addition, their effects on human (h)-hepatocytes in vivo have mice have been proven to be a useful animal model to not been studied due to the lack of a suitable animal model. examine biological and pathological features of h-hepatocytes Previously, we developed a method to yield humanized in vivo (Tateno et al. 2004, Tsuge et al. 2005). mice (chimeric mice) whose liver is mostly replaced with The regeneration capacity of rat liver decreases with age h-hepatocytes (Tateno et al. 2004). The h-hepatocytes were (Bucher et al. 1964, Stocker & Heine 1971), which is transplanted into immunodeficient mice with diseased liver, coincident with the fact that serum concentrations of growth Journal of Endocrinology (2007) 194, 529–537 DOI: 10.1677/JOE-07-0126 0022–0795/07/0194–529 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 01:33:12AM via free access 530 N MASUMOTO and others . h GH and liver-deficient immunodeficient mice hormone (GH) and insulin-like growth factor 1 (IGF-1) rhGH treatment diminish with age (Kelijman 1991, Corpas et al. 1993), The 6YG- and 46YM-hepatocyte-chimeric mice were divided suggesting the association of GH with liver regeneration. into two groups at 1 day post-transplantation; recombinant hGH Actually, evidence has been accumulating that GH is involved (rhGH; Wako Pure Chemical Industries Ltd, Osaka, Japan)- in liver regeneration and accounts for an aspect of age- C K treated (rhGH , experimental) and -untreated (rhGH , dependent regenerative response of the liver in rodents control) groups. rhGH was dissolved in water and used for (Krupczak-Hollis et al. 2003). However, the effects of GH animal injection. Animals of experimental groups were daily on the growth of h-hepatocytes have not been studied in vivo at administered from day 1 after transplantation to 1 day before the all yet. hGH is capable of stimulating rodent cells, whereas day of killing with rhGH by subcutaneous injection at rodent GH cannot stimulate human cells because of its 2.5 mg/10 ml per g body weight. disability to bind to hGH receptors (hGHRs; Souza et al. 1995). Furthermore, it should be noted that hGH is not circulating in h-hepatocyte-chimeric mice, which indicates that h-hepato- Measurement of human albumin (hAlb) and human IGF-1 cytes in chimeric mice are in GH-deficient conditions. These concentrations in mouse blood or sera facts and considerations strongly suggest that a chimeric mouse hAlb concentration in blood of a chimeric mouse is correlated will provide an opportunity to examine the effects of hGH on with RI of transplanted hepatocytes (Tateno et al. 2004). growth of h-hepatocytes in vivo. Blood (2 ml) was collected from the tail vein of h-hepatocyte- In this study, we examined the effects of hGH on chimeric mice. The blood hAlb concentrations were the proliferation of h-hepatocytes using chimeric mice. determined with a latex agglutination assay (Eiken Immuno- The treatment of chimeric mice with hGH increased the chemicalLaboratory,Tokyo,Japan)orahAlb ELISA repopulation speed and RI of transplanted h-hepatocytes, and quantitation kit (Bethyl Laboratories Inc., Montgomery, up-regulated the GH-related signaling molecules. The TX, USA). As a measure of GH/IGF-1 signaling in the present study shows that a h-hepatocyte-chimeric uPA/SCID chimeric mice, serum human IGF-1 (hIGF-1) concentrations mouse is a useful in vivo model to examine the effects of were determined using a hIGF-I ELISA kit (R&D Systems growth factors, cytokines, and hormones on h-hepatocytes. Inc., Minneapolis, MN, USA). Immunohistochemistry and measurement of RI Materials and Methods Frozen sections were prepared from chimeric livers, fixed in K20 8C acetone for 5 min and incubated with anti-human Animals cytokeratin 8 and 18 (hCK8/18) antibodies (dilution, 1:25; MP . Biomedicals, Aurora, OH, USA). The hCK8/18 antibodies The uPA/SCID mice weighing 6 3–10 0 g were produced as reacted with h-hepatocytes but not with mouse (m)-hepatocytes. previously described (Tateno et al. 2004). The zygosity of the Formalin-fixed paraffin sections of chimeric livers were uPA transgene was determined by a multiplex PCR as incubated with mouse anti-BrdU antibodies (dilution, 1:10; previously described (Meuleman et al. 2003). Homozygous DakoCytomation, Glostrup, Denmark) and goat anti-hAlb uPA/SCID mice were used as hosts throughout this study. antibodies (dilution, 1:1000; Bethyl Laboratories). The primary antibodies were visualized with a Vectastain ABC kit (Vector Transplantation of hepatocytes and bromodeoxyuridine (BrdU)- Laboratories, Burlingame, CA, USA) or peroxidase- and labeling dextran-conjugated anti-mouse immunoglobulins (Dako Envi- sion C; DakoCytomation) with 3, 30-diaminobenzidine Cryopreserved h-hepatocytes from a 6-year-old Caucasian girl (Sigma) as substrates. The sections were counterstained with (6YG) and 46-year-old Caucasian man (46YM) respectively Mayer’s hematoxylin. RI was calculated as the ratio of area were purchased from In vitro Technologies (Baltimore, MD, occupied by hCK8/18-positive hepatocytes to the entire area USA) and thawed as previously described (Tateno et al. 2004). examined on immunohistochemical sections of six lobes (Tateno Trypan blue-exclusion test showed that the viability of 6YG- et al. 2004). The ratios of BrdU-positive nuclei to hAlb-positive and 46YM-hepatocytes was 71.5G4.3% (nZ3) and 72.2G h-hepatocytes were determined by counting at least 1000 cells in 5 2.3% (nZ3) respectively. The h-hepatocytes (7.5or10.0!10 10 to 15 randomly selected vision fields in sections. viable cells) were transplanted into the inferior splenic pole of uPA/SCID mice at 20–30 days after birth, through a small left- flank incision (Tateno et al. 2004). BrdU (Sigma Chemical Co.) Quantification of mRNA in the livers of chimeric mice was intraperitoneally injected into chimeric mice at a dose of Total RNAs were purified from liver tissues by an RNeasy 50 mg/kg body weight at 1 h before killing. Histological mini kit (Qiagen). Using 1 mg total RNA by PowerScript sections were prepared from the liver and stained with anti- reverse transcriptase (Clontech Inc.) and Random Primer BrdU antibodies as described below.