A robust culture method for maintaining tumorigenic cancer stem cells in the hepatocellular carcinoma cell line Li-7

Abstract

Cancer tissues contain small populations of highly tumorigenic cells termed cancer stem cells (CSCs). Immortalized cell lines containing CSCs are valuable and powerful experimental tools for research into the characteristics of these stem cells. We previously reported that the hepatocellular carcinoma cell line Li-7 includes abundant CD13⁺ CD166^- CSCs; however, the number of these cells decreases after long-term culture as a result of differentiation to non-CSC populations. To ensure consistent and reproducible results in experiments using Li-7 cells, it is important that the CSC population is maintained stably regardless of culture duration and passage. In the present study, we found that a commercially available culture medium for maintenance of embryonic stem cells and induced pluripotent stem cells, mTeSR1, effectively prevented spontaneous differentiation by CD13⁺ CD166^- cells to CD13^- CD166⁺ cells and therefore maintained the CSC population in Li-7 cell cultures. CD13⁺ CD166^- CSCs maintained using this culture medium retained high tumorigenicity after transplantation into mice; they also showed the ability to differentiate in vitro into non-CSC populations in RPMI-1640 with 10% FBS medium. We analyzed gene expression profiles of CSC and non-CSC populations in Li-7 cultures using an RNA sequencing method. Genes such as FGFR, NOTCH1, and JAG1, that are associated with tumorigenicity and stemness, were upregulated in the CSC population. Our results suggest that CSCs can be maintained in immortalized cancer cell lines cultured over an extended period using a medium developed for culture of embryonic/induced pluripotent stem cells.

Keywords: CD13; CD166; cell culture; mTeSR1; tumorigenicity.

Figure 1

Characteristics of Li‐7 cells cultured in RPMI‐1640 or mTeSR1. A, Flow cytometric analysis of CD13 and CD166 cells in cultures using RPMI‐1640 + 10% FBS. Li‐7 cells were obtained from a cell repository and are shown as day 0. B, The percentages of CD13⁺ CD166⁻, CD13⁺ CD166⁺, CD13⁻ CD166⁻, and CD13⁻ CD166⁺ cells in cultures using RPMI‐1640 + 10% FBS at every passage. Results shown are representative of 3 independent experiments. C, Flow cytometric analysis of cells cultured in RPMI‐1640 + 10% FBS or 4 other media for embryonic stem cells/induced pluripotent stem cells (mTeSR1, StemFit AK02N, Essential 8, and StemPartner). Cells were cultured for 15 d and then subjected to analysis. D, Flow cytometric analysis of CD13 and CD166 cells in cultures using in mTeSR1, on days 4 and 46. E, The percentages of CD13⁺ CD166⁻, CD13⁺ CD166⁺, CD13⁻ CD166⁻, and CD13⁻ CD166⁺ cells in cultures using mTeSR1 at every passage. Results shown are representative of 3 independent experiments. F, Morphologies of cells in cultures using RPMI‐1640 + 10% FBS or mTeSR1. Magnification, ×100

Figure 2

Characterization of CD13⁺ CD166⁻ cells cultured in mTeSR1. A,B, Flow cytometric analysis of cells cultured in RPMI‐1640 + 10% FBS (Li‐7 RPMI) or mTeSR1 (Li‐7 mTeSR1) for 6 wk; the cells were stained using CD13, CD166, epithelial cell adhesion molecule (EpCAM), and CD133. B, Population analysis of cells cultured in mTeSR1. CD13⁺ CD166⁻ cells are shown as dark blue dots and CD13⁺ CD166⁺ cells are shown as red dots. C, Flow cytometric analysis with SSEA‐4, TRA‐1‐60, and TRA‐1‐81. The percentages of SSEA‐4‐, TRA‐1‐60‐, and TRA‐1‐81‐positive cells in CD13⁺ CD166⁻ cells cultured in mTeSR1 or RPMI‐1640 + 10% FBS for 15 wk are shown. D, Flow cytometric analysis of intracellular aldehyde dehydrogenase enzymatic activity of CD13⁺ CD166⁻, CD13⁺ CD166⁺, and CD13⁻ CD166⁺ cell fractions in cultures using mTeSR1. Results shown are representative of 2 independent experiments. E, Spheroid formation assay. CD13⁺ CD166⁻, CD13⁺ CD166⁺, and CD13⁻ CD166⁺ cell fractions were sorted in cultures using mTeSR1. Magnification, ×40. Values are mean ± SD of 3 wells. Results shown are representative of 2 independent experiments. F, Phenotypic changes in Li‐7 cells after transfer to RPMI‐1640 + 10% FBS. CD13⁺ CD166⁻ cells were sorted from cultures using mTeSR1. Cells cultured in mTeSR1 2 months after sorting (left, mTeSR1) and cells transfer to RPMI‐1640 + 10% FBS (right, RPMI) for 84 d are shown. *P <0.05. BAA‐, BODIPY‐aminoacetate; SSC, side scatter

Figure 3

Characterization of tumors formed in immunodeficient mice. A, The histology of tumors formed in mice after transplantation of cells cultured in RPMI‐1640 + 10% FBS (RPMI) or mTeSR1. Tumor sections were stained with H&E. Magnification, ×200. B, Flow cytometric analysis using CD13 and CD166. Tumor cells derived from cells cultured in mTeSR1 were then cultured in RPMI‐1640 + 10% FBS for 35 d. Results shown are representative of 3 independent experiments

Figure 4

RNA sequence analysis. A,B, The number of genes upregulated (A) and downregulated (B) in CD13⁺ CD166⁻ cells cultured in mTeSR1 compared to CD13⁺ CD166⁻, CD13⁻ CD166⁻, or CD13⁻ CD166⁺ cells cultured in RPMI‐1640 + 10% FBS. C, Expression levels of genes encoding fibroblast growth factor receptors (FGFRs) and their downstream transcriptional factors. Expression levels are compared with the CD13⁻ CD166⁺ cells showing the lowest tumorigenicity of cells cultured in RPMI‐1640 + 10% FBS. Results from ANPEP(CD13) and ALCAM(CD166) are also shown as positive and negative controls, respectively. D, Results of RNA sequence analysis for NOTCH1,JAG1, and BMP2. Expression levels are compared with CD13⁻ CD166⁺ cells showing lowest tumorigenicity of cells cultured in RPMI‐1640 + 10% FBS. FC, fold change