Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Sep 26, 2017; 9(9): 159-168
Published online Sep 26, 2017. doi: 10.4252/wjsc.v9.i9.159
Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity
Aparna Jayachandran, Ritu Shrestha, Bijay Dhungel, I-Tao Huang, Marianna Yumi Kawashima Vasconcelos, Brian J Morrison, Charmaine A Ramlogan-Steel, Jason C Steel
Aparna Jayachandran, Ritu Shrestha, Bijay Dhungel, I-Tao Huang, Marianna Yumi Kawashima Vasconcelos, Charmaine A Ramlogan-Steel, Jason C Steel, the University of Queensland School of Medicine and the Gallipoli Medical Research Institute, Greenslopes Private Hospital, Brisbane, QLD 4120, Australia
Brian J Morrison, Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, United States
Author contributions: Jayachandran A and Steel JC conceived and designed the experiments; Jayachandran A, Shrestha R, Dhungel B, Huang IT, Vasconcelos MYK, Morrison BJ and Ramlogan-Steel CA performed the experiments; Jayachandran A, Shrestha R, Dhungel B, Huang IT and Steel JC analysed the data; Jayachandran A, Shrestha R, Dhungel B and Steel JC wrote the paper.
Supported by The Gallipoli Medical Research Foundation, Australia, No. 016092; and the Cyril Gilbert Foundation, Australia, No. 017348.
Institutional animal care and use committee statement: No animals were used in our experiments.
Conflict-of-interest statement: The authors declare no conflict of interest. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the United States Government. Brian J Morrison is a military service member. This work was prepared as part of his official duties. Title 17 U.S.C. §105 provides that Copyright protection under this title is not available for any work of the United States Government. Title 17 U.S.C. §101 defines a United States Government work as a work prepared by a military service member or employee of the United States Government as part of that person’s official duties.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Jason C Steel, PhD, Director, Liver Cancer Unit, the University of Queensland School of Medicine and the Gallipoli Medical Research Institute, Greenslopes Private Hospital, Newdegate Street, Brisbane, QLD 4120, Australia.
Telephone: +61-7-33460611
Received: May 8, 2017
Peer-review started: May 10, 2017
First decision: June 16, 2017
Revised: June 29, 2017
Accepted: July 14, 2017
Article in press: July 16, 2017
Published online: September 26, 2017

To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT).


In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR.


Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai, Zeb and Twist family of EMT transcription factors.


Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Keywords: Hepatocellular carcinoma, Hepa 1-6, Cancer stem cells, Cancer initiating cells, Epithelial-to-mesenchymal transition, Cellular plasticity, Epithelial-to-mesenchymal transition transcription factors, AML12

Core tip: Although existing therapies can initially eliminate the bulk population of a tumor, the stem cell properties of cancer stem cells (CSCs) enable them to survive and repopulate the tumor, resulting in disease relapse. Therefore, elimination of CSCs has the potential to improve patient outcomes and survival. Isolation and characterization of liver CSCs is essential for the selective targeting of this crucial population of cells. We report that the sphere culture method is a more precise and reliable tool for the enrichment of murine stem-like cells which relies on their functional property of anchorage-independent growth.