Development of Humanized Mice for the Study of Hepatitis C Virus Infection P. Turrini, R. Sasso, S. Germoni, I. Marcucci, A. Celluci, A. Di Marco, E. Marra, G. Paonessa, A. Eutropi, R. Laufer, G. Migliaccio, and J. Padron ABSTRACT The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepato- cytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of 10 5 genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development. A BSENCE OF APPROPRIATE in vitro and in vivo animal models is a major obstacle in many areas of preclinical research and development. The generation of chimeric animals harboring human or humanized tissues is a very promising approach for the development of more predictive animal models. Generation of chimeric mice harboring a humanized liver is indeed a major effort in academic and/or pharmaceutical laboratories. Such animals might represent not only a fundamental tool for the better understanding of hepatic disease such as hepatitis C virus (HCV), but also for the preclinical drug metabolism and pharmacokinetic studies in many other therapeutic areas. Though transgenic mice expressing particular human genes have been an important advance to accomplish the goal of “humanizing” mice, 1,2 the goal of our studies is an overall humanization of the mouse liver. In other words, we are interested in an extensive repopulation of the mouse liver with human hepatocytes, or human hepatocyte-like cells, and to this purpose we have been using several different experimental approaches. MATERIALS AND METHODS Materials Cryopreserved human hepatocytes were purchased from Cambrex. The human hepatoma cell line C3A was obtained from the American Type Culture Collection (ATCC) and then kept in culture for no more than five to six passages before use. Most antibodies were obtained from the following commercial sources; monoclonal hamster anti-mouse Fas antibody (Jo2) was from From the Department of Pharmacology, IRBM “P. Angeletti” Merck Research Laboratories, Pomezia, Italy. This work was supported in part by a grant from the Ministero dell’Industria dell’Universita’ e della Ricerca. Address reprint requests to Julio Padron, PhD, Department of Pharmacology, IRBM “P. Angeletti” Merck Research Laborato- ries, Via Pontina km 30.600, 00040 Pomezia (RM), Italy. E-mail: julio_padron@merck.com © 2006 by Elsevier Inc. All rights reserved. 0041-1345/06/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2006.02.149 Transplantation Proceedings, 38, 1181–1184 (2006) 1181