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Abstract

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Medicine

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Published: August 29th, 2016

DOI:

10.3791/54167

1Department of Regenerative Medicine, Graduate School of Medicine, Yokohama City University, 2Department of Advanced Gastroenterological Surgical Science and Technology, Faculty of Medicine, University of Tsukuba, 3Regenerative Medicine Research Center, Jiangsu University Hospital
* These authors contributed equally

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Mice are commonly used for pharmaceutical testing since biomedical research in humans is restricted1; however, these models are not always useful since they may inaccurately simulate the effects observed in humans. Most drugs in current medical use are metabolized primarily in the liver. However, the same drug can be metabolized into different metabolites in mouse and human livers because of inter-species differences. Thus, it is often difficult to determine during development whether a potential drug poses any risks for clinical applications2,3.

To address this problem, "humanized" mouse livers have been developed by....

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All animal experimental procedures were performed according to the Animal Protection Guidelines of Yokohama City University.

1. Generation of the Acute Liver Injury Mouse Model

  1. Add 1 ml of phenolized 0.85% NaCl solution (0.6 g phenol in 100 ml of 0.85% NaCl solution) to 1 mg diphtheria toxin (DT) to make a 1 mg/ml DT stock solution. Note: DT at a concentration of 1 mg/ml can be stored for approximately 2 years at 3 °C to 8 °C.
  2. Serially dilute the 1 mg/ml DT stoc.......

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Alb-TRECK/SCID mice hepatocytes express the human DT receptor HB-EGF gene under the control of an albumin promoter and exhibit cytotoxic effects following DT administration12. To evaluate the effects of DT treatment on liver injury, DT doses of 1.5 μg/kg were injected into 8-week-old Alb-TRECK/SCID mice and the pathological changes in the liver 48 hr post DT administration were histologically assessed. Compared with control mice (not treated with DT), the DT-treated mice e.......

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Recent studies have shown that the mouse liver can be repopulated with human hepatocytes, including adult hepatocytes and proliferative hepatic stem cells17. These repopulated livers have been used as preclinical experimental models for drug metabolism testing and drug discovery and development18; in addition, they have provided an in vivo environment for cell maturation and differentiation19. The major aim of the present study was to generate a novel liver disease mouse model th.......

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We wish to thank the Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, for providing the mice. We also thank S. Aoyama and Y. Adachi of the ADME (Absorption, Distribution, Metabolism, Excretion) & Toxicology Research Institute, Sekisui Medical Company Ltd., Japan, and K. Kozakai and Y. Yamada for assistance with LC-MS/MS analysis. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan to Y-W.Z. (18591421, 20591531, and 23591872); by the Jiangsu innovative and entrepreneurial project for the introduction of high-level talent and the Jiangsu science and techno....

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Name Company Catalog Number Comments
Human albumin Sigma A6684 Mouse IgG2a
Human CK19 Dako M088801 Mouse IgG1
Human nuclei Millipore MAB1281 Mouse IgG1
Human CK8/18 Progen GP11 Guinea pig Polyclonal
CDCP1 Biolegend 324006 Mouse IgG2b
CD90 BD 559869 Mouse IgG1
CD66 BD 551479 Mouse IgG2a
GOT/AST-PIII Fujifilm 14A2X10004000009
DMEM/F-12 Gibco 11320-033
FBS Biowest S1520
0.05% Trypsin-EDTA  Gibco 25300-054
Diphtheria Toxin Sigma D0564-1MG
Human Albumin ELISA Kit Bethyl Laboratories E88-129
Syringe (1ml) Terumo SS-01T
32G 1/2" needle TSK PRE-32013
O.C.T.Compound(118ml) Sakura Finetek Japan 4583
MoFlo high-speed cell sorter Beckman Coulter B25982
DRI-CHEM 7000 Fujifilm 14B2X10002000046

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