Erzeugung eines humanisierten Maus-Leber unter Einsatz von menschlichem Leberstammzellen
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 growing human liver cells inside mice4-6; these models exhibit drug responses similar to those observed in the human liver. The primary mouse models currently used for humanized liver generation include uroplasminogen activator (uPA+/+) mice4,7, fumarylacetoacetate hydrolase (Fah−/−) mice6, and the recently reported thymidine kinase (TK-NOG) mice.
However, previous reports have shown that transplanted human immature cells or stem cells are less competitive than adult human hepatocytes in Alb-uPA tg(+/−)Rag2(−/−) mouse livers8-10. Moreover, Fah−/− mice provide a growth advantage only for differentiated hepatocytes and not for immature liver progenitor cells11. The transplantation of human hepatic stem cells (HpSCs) into TK-NOG mice in the lab has been unsuccessful. Hence, no useful mouse model for the efficient engraftment of human immature liver cells currently exists.
Thus, we developed a novel Alb-TRECK/SCID mouse model that could be efficiently repopulated with human immature hepatocytes. This transgenic mouse model expresses human heparin-binding EGF-like growth factor (HB-EGF) receptors under the control of a liver cell-specific albumin promoter. Following the administration of diphtheria toxin (DT), these mice develop fulminant hepatitis due to conditional ablation of hepatocytes, enabling donor cell residency and proliferation12. Although mouse hepatocytes have been successfully transplanted into Alb-TRECK/SCID mice in previous studies13,14, the generation of a humanized liver using Alb-TRECK/SCID mice has yet to be reported.
In this study, humanized livers were generated in Alb-TRECK/SCID mice via transplantation of HpSCs. This humanized liver provides an in vivo environment for universal stem cell differentiation and the ability to predict human drug metabolism patterns and drug-drug interactions.
Protocol
Representative Results
Discussion
Jüngste Studien haben gezeigt , dass die Maus Leber kann mit humanen Hepatozyten neu besiedelt werden, einschließlich Erwachsenen Hepatozyten und proliferative hepatische Stammzellen 17. Diese repopulated Lebern wurden als präklinische Versuchsmodelle für Arzneimittelmetabolismus Test und Arzneimittelentdeckung und -entwicklung 18 verwendet wird ; Darüber hinaus haben sie eine in vivo Umgebung für die Zellreifung und Differenzierung 19 versehen. Das Hauptziel der vorliegen…
Disclosures
The authors have nothing to disclose.
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 planning project (BE2015669); and by grants to H.T. for Strategic Promotion of Innovative Research and Development (S-innovation, 62890004) from the Japan Science and Technology Agency.
Materials
| 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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