Three-dimensional hepatocyte culture system for the study of Echinococcus multilocularis larval development.

Li Li,Wanzhong Jia,Baoquan Fu,Bing Chen,Xingquan Zhu,Yannan Zhao,Hongbin Yan,Jianqiu Li,Jianwu Dai,Donald P Mcmanus,Zhongzi Lou,Mara Cecilia Rosenzvit

doi:10.1371/journal.pntd.0006309

Abstract

BackgroundHepatocyte-based metacestode culture is an attractive method to study alveolar echinococcosis (AE), but it is limited by the relatively short lifespan of cultured hepatocytes in maintaining their normal function.Methodology/principal findingsWe describe a three-dimensional (3D) hepatic culture system developed from co-cultured hepatocytes and mesenchymal stem cells using a collagen scaffold to study the development of Echinococcus multilocularis larvae. This 3D culture system preserved the function of hepatocytes for a longer period of time than their monolayer counterparts, with albumin secretion, 7-ethoxyresorufin O-deethylation activity, urea synthesis, CYP3A4 and CYP2D6 activity being highly preserved for 21–28 days. The expression levels of hepatocyte-specific genes including CLDN-3, Bsep, AFP, G6P, A1AT, CYP3A4 and NR1I3 were significantly higher in the 3D cultured system compared with their monolayer counterparts after 14-days in culture. Additionally, in the presence of 3D cultured hepatocytes, 81.2% of E. multilocularis protoscoleces rapidly de-differentiated into infective vesicles within eight weeks. Transcriptomic analyses revealed 807 differentially expressed genes between cultured vesicles and protoscoleces, including 119 genes uniquely expressed in protoscoleces, and 242 genes uniquely expressed in vesicles. These differentially expressed genes were mainly involved in parasite growth relating to the G-protein coupled receptor activity pathway, substrate-specific transmembrane transporter activity, cell-cell adhesion process, and potentially with neuroactive ligand-receptor interaction.Conclusions/significanceThis culture system provides a valuable advance in prolonging hepatocyte functionality, a foundation for future in-depth analysis of the host-parasite interaction in AE, and a useful model to evaluate potential therapeutic strategies to treat AE.

Highlights

Human alveolar echinococcosis (AE), is caused by the larval stage of the tapeworm Echinococcus multilocularis, one of the most lethal of the human helminthiases
We describe a three-dimensional (3D) hepatic culture system developed from co-cultured hepatocytes and mesenchymal stem cells using a collagen scaffold to study the development of Echinococcus multilocularis larvae
An in vitro hepatocyte based cultivation system for the metacestode stage of E. multilocularis has been developed to improve our understanding of AE

Summary

Introduction

Human alveolar echinococcosis (AE), is caused by the larval stage of the tapeworm Echinococcus multilocularis, one of the most lethal of the human helminthiases. Untreated AE has a fatality rate of >90% in humans, causing great public health concern in the northern hemisphere [1,2]. Human infection, following the ingestion of E. multilocularis eggs, results in the development of metacestodes in the liver with clinical signs such as an abdominal mass and/or pain, jaundice and, liver failure [3]. Understanding the biological mechanisms of E. multilocularis metacestode development and differentiation is the key for the control and treatment of AE. Hepatocyte-based metacestode culture is an attractive method to study alveolar echinococcosis (AE), but it is limited by the relatively short lifespan of cultured hepatocytes in maintaining their normal function

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Conclusion