Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

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 applications^2,3.

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 stock solution to 0.3 μg/ml DT in phenolized 0.85% NaCl solution and prepare an aliquot of the 0.3 μg/ml DT solution as a working solution. Note....

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 exhibi.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human albumin	Sigma	A6684	Mouse
Human CK19	Dako	M088801	Mouse
Human nuclei	Millipore	MAB1281	Mouse
Human CK8/18	Progen	GP11	Guinea pig
CDCP1	Biolegend	324006	Mouse
CD90	BD	559869	Mouse
CD66	BD	551479	Mouse
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 (1 ml)	Terumo	SS-01T
32G 1/2" needle	TSK	PRE-32013
O.C.T.Compound(118 ml)	Sakura Finetek Japan	4583
MoFlo high-speed cell sorter	Beckman Coulter	B25982
DRI-CHEM 7000	Fujifilm	14B2X10002000046