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Transplantation of Engineered Chimeric Liver with Autologous ORIGINAL ARTICLE Transplantation of Engineered Chimeric Liver With Autologous Hepatocytes and Xenobiotic Scaffold Toshiyuki Hata, MD,∗† Shinji Uemoto, MD, PhD,∗ Yasuhiro Fujimoto, MD, PhD,∗ Takashi Murakami, MD, PhD,‡ Chise Tateno, PhD,§ Katsutoshi Yoshizato, PhD,§ and Eiji Kobayashi, MD, PhD|| iver transplantation is currently regarded as the most effective Objective: Generation of human livers in pigs might improve the serious treatment for end-stage liver diseases. Because the worldwide shortage of grafts for human liver transplantation, and enable liver trans- L graft shortage remains unresolved,1,2 engineered organs are antici- plantation without the need for deceased or living donors. We developed a pated as alternative grafts to fill the scarcity and ultimately replace chimeric liver (CL) by repopulation of rat hepatocytes in a mouse and suc- those from deceased and living donors. Although regenerative tech- cessfully transplanted it into a rat recipient with vessel reconstruction. This nology has already developed various kinds of regenerative cells and study was designed to investigate the feasibility of CL for supporting the tissues,3,4 they are still insufficient to cure patients with end-stage recipient after auxiliary liver grafting. liver diseases because of the absence of complicated functions and Methods: Hepatocytes from luciferase transgenic or luciferase/LacZ double- limited volume. Therefore, a regenerative liver graft for use as an “or- transgenic rats were transplanted into 20- to 30-day-old urokinase-type gan” is required. To achieve an appropriate 3-dimensional structure plasminogen activator/severe-combined immunodeficiency (uPA/SCID) mice and differentiation to specific tissues in a single organ, application of (n = 40) to create CLs with rat-origin hepatocytes. After replacement of mouse native organs as scaffolds has been reported as a possible solution.5–7 hepatocytes with those from rats, the CLs were transplanted into wild-type Recent advances in genetic manipulation of animals such as pigs have Lewis (n = 30) and analbuminemia (n = 10) rats, followed by immunosup- produced transgenic animals with a lower risk of xenorejection.8,9 pression using tacrolimus (TAC) with/without cyclophosphamide (CPA) or Thus, using their native organogenetic potentials, development of no immunosuppression. Organ viability was traced by in vivo bioimaging and engineered liver “organs” is expected.10 In such a protocol, human Doppler ultrasonography in the recipient rats for 4 to 6 months. Rat albu- hepatocytes are transplanted to a transgenic pig to replace its native min production was also evaluated in the analbuminemia rats for 4 months. hepatocytes, resulting in development of a chimeric liver (CL) with In addition, histological analyses including Ki67 proliferation staining were human parenchyma and swine nonparenchymal components, includ- performed in some recipients. ing vessels, bile ducts, and other connecting tissues. However, it is Results: Both immunosuppressive protocols significantly improved graft sur- unclear whether such engineered CLs can be transplanted into recipi- vival and histological rejection of CLs as compared to the nonimmunosup- ents, or whether they can maintain their organ structures and functions pressed group. Although rat albumin production was maintained in the re- after transplantation. In this study, we developed a rodent model of cipients for 4 months after transplantation, ultrasonography revealed patent chimeric liver transplantation to investigate its feasibility (Fig. 1A). circulation in the grafts for 6 months. Ki67 staining analysis also revealed We used mice and rats as substitutes for transgenic pigs and humans, the regenerative potential of CLs after a hepatectomy of the host native liver, respectively, and created CLs in transgenic mice using hepatocytes whereas immune reactions still remained in the mouse-origin structures. derived from transgenic rats. After chimeric liver transplantation into Conclusions: This is the first report showing that engineered CLs have po- rat recipients with vessel reconstruction, we examined the long-term tential as alternative grafts to replace the use of grafts from human donors. viability and functions of the transplanted CL grafts. Keywords: alternative organ graft, auxiliary liver transplantation, chimeric liver, engineered organ, liver transplantation MATERIALS AND METHODS (Ann Surg 2013;257: 542–547) Animals All mice and rats were housed in a temperature- controlled environment under a 12-hour light-dark cycle with free access to water and standard rodent chow diet. Male and fe- From the ∗Division of Hepato-Biliary-Pancreatic and Transplant Surgery, De- partment of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, male albumin enhancer-/promoter-driven urokinase-type plasmino- Japan; †Division of Transplantation Surgery, Mayo Clinic Florida, Jacksonville; gen activator/severe-combined immunodeficiency (uPA/SCID) mice ‡Department of Pharmacy, Takasaki University of Health and Welfare, Gunma, (Phoenixbio Co, Ltd, Japan) (n = 40), which express and accumulate Japan; §PhoenixBio Co Ltd, Higashi-Hiroshima, Japan; and ||Division of Devel- urokinase-type plasminogen specifically in native hepatocytes, result- opment of Advanced Treatment, Center for Development of Advanced Medical 11,12 Technology, Jichi Medical University, Tochigi, Japan. ing in liver disease, were used as the scaffolds for CL regenera- Disclosure: Supported by a Grant-in-Aid for Scientific Research (No 20249058) tion. Male luciferase transgenic Lewis (Luc-LEW) (MHC haplotype; from the Japan Society for the Promotion of Science, the “Strategic Research RT1l)13 (n = 2), and female luciferase and LacZ double-transgenic Platform” for Private Universities, a matching fund subsidy from the Ministry of Lewis (Luc/LacZ-LEW) (RT1l)rats14 (n = 1) were used as hepato- Education, Culture, Sports, Science and Technology of Japan (2008), the COE cyte donors. Male wild-LEW rats (RT1l) (Charles River Japan, Japan) program from MEXT (2008), and the Kyoto University Foundation (2011). a 15 E.K. is a chief scientific advisor for Otsuka Pharmaceutical Factory, Inc. This (n = 30) and male Nagase analbuminemia rats (RT1 ) (Japan SLC, work was not supported by any funding from the National Institute of Health, Japan) (n = 10) were used as the recipients of chimeric liver transplan- Wellcome Trust, or Howard Hughes Medical Institute. tation. All experiments in this study were performed in accordance Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of with the Jichi Medical University Guide for Laboratory Animals after this article on the journal’s Web site (www.annalsofsurgery.com). approval by the ethics committee of PhoenixBio Co, Ltd. Reprints: Eiji Kobayashi, MD, PhD, 3311-1 Yakushiji, Shimotsuke, Tochigi 329- 0498, Japan.E-mail: [email protected]. Generation of CLs Copyright C 2013 by Lippincott Williams & Wilkins ISSN: 0003-4932/13/25703-0542 Isolated hepatocytes were obtained from 10-week-old Luc- DOI: 10.1097/SLA.0b013e31825c5349 LEW and Luc/LacZ LEW rats using a standard 2-step collagenase r 542 | www.annalsofsurgery.com Annals of Surgery Volume 257, Number 3, March 2013 Copyright © 2013 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. r Annals of Surgery Volume 257, Number 3, March 2013 Transplantation of Chimeric Liver FIGURE 1. A, Experimental protocol used for Chimeric liver transplantation. B, Schema of engraftment of CL into recipient rat. perfusion method.16 Collagenase (75 mg/body) (COLLAGENASE S- injected intramuscularly into the recipient rats at a dose of 0.64 mg/kg 1; Nitta-gelatin, Japan) was perfused through the portal vein, then 5.0 before and every day after transplantation. In TAC (+)CPA(+), can- × 105 hepatocytes were transplanted into 20- to 30-day-old uPA/SCID didate recipient rats were prepared with an intraperitoneal injection of mice, as previously reported.12 These mice were kept for 3 months to cyclophosphamide (Endoxan, Shionogi, Japan) at a dose of 60 mg/kg obtain CLs with satisfactory size (mean ± SEM, 1.69 ± 0.04 g) and 10 days before transplantation (dose of cyclophosphamide modified chimerism. from previous report18). For Nagase analbuminemia rat recipients (n = 8), tacrolimus was given every day intramuscularly at a dose of Harvesting of CLs 0.32 mg/kg. The recipient rats were observed daily and those in very The CLs were harvested from uPA/SCID mice along with poor condition were euthanized. the hepatic artery with the aortic cuff and infrahepatic inferior vena cava. The portal vein trunk, common bile duct, and suprahepatic inferior vena cava were ligated. After systemic injection of 300 U Evaluation of Luminescence From CLs of heparin sodium (Novo-Heparin; Mochida, Japan), the graft was In vivo luciferase imaging of CLs was performed in both harvested and cryo-preserved in 0.9% saline. A 10-mm plastic tube uPA/SCID mice for 3 months after hepatocyte transplantation and (22G SurfloR, IV catheter; Terumo, Japan) was inserted into the recipient rats throughout their survival after chimeric liver transplan- gallbladder, and a plastic cuff (18G SurfloR) was installed onto the tation using a noninvasive bioimaging system (IVIS, Xenogen, CA), end of the infrahepatic inferior vena cava. and the images were analyzed using a software package (Igor; Wave- Metrics, OR, and IVIS Living Image; Xenogen). Before imaging,
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