Published online Sep 26, 2017. doi: 10.4252/wjsc.v9.i9.159
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.
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.