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

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.