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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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 technology....

Materials

NameCompanyCatalog NumberComments
Human albuminSigmaA6684MouseIgG2a
Human CK19DakoM088801MouseIgG1
Human nucleiMilliporeMAB1281MouseIgG1
Human CK8/18ProgenGP11Guinea pigPolyclonal
CDCP1Biolegend324006MouseIgG2b
CD90BD559869MouseIgG1
CD66BD551479MouseIgG2a
GOT/AST-PIIIFujifilm14A2X10004000009
DMEM/F-12Gibco11320-033
FBSBiowestS1520
0.05% Trypsin-EDTA Gibco25300-054
Diphtheria ToxinSigmaD0564-1MG
Human Albumin ELISA KitBethyl LaboratoriesE88-129
Syringe (1ml)TerumoSS-01T
32G 1/2" needleTSKPRE-32013
O.C.T.Compound(118ml)Sakura Finetek Japan4583
MoFlo high-speed cell sorterBeckman CoulterB25982
DRI-CHEM 7000Fujifilm14B2X10002000046

References

  1. Muruganandan, S., Sinal, C. Mice as clinically relevant models for the study of cytochrome P450-dependent metabolism. Clin. Pharmacol. Ther. 83, 818-828 (2008).
  2. Nishimura, T., et al.

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Humanized Mouse LiverHuman Hepatic Stem CellsCell TransplantationLiver ReconstitutionDiphtheria ToxinCell SortingCell CultureLiver InjuryCell DifferentiationDrug Metabolism

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