A new protocol for long-term culture of a specific subpopulation of liver cancer stem cells enriched by cell surface markers.

Liver cancer stem cells (L-CSCs) are considered to be an important therapeutic target for hepatocellular carcinoma (HCC). This study provides a new in vitro long-term culture model for a specific subpopulation of L-CSCs enriched by cell surface markers. We combined CD13, CD133 and EpCAM to selectively enrich L-CSCs, which we then cultured in modified chemically defined medium (CDM). The enriched L-CSCs exhibited enhanced proliferation, self-renewal and long-term clonal maintenance ability as compared to non-CSCs. Compared with wild-type HCC, the expression of stemness surface markers, oncogenes, drug-resistance and tumorigenicity in enriched L-CSCs were significantly increased. In summary, the subpopulation of L-CSCs still maintain CSC-related phenotypes after 14 days of culture.