A reliable method for the sorting and identification of ALDHhigh cancer stem cells by flow cytometry

Cancer stem cells (CSCs) are a rare tumorigenic population of cells found in multiple types of cancer. It has been suggested that CSCs are responsible for cancer drug resistance, metastasis and recurrence. Therefore, it is important to develop techniques to correctly sort and identify CSCs. In the current study, the sorting and identification of aldehyde dehydrogenase high (ALDHhigh) CSCs was performed using flow cytometry. Cells from three colon cancer cell lines were cultured in serum-free medium to obtain CSCs-enriched spheroid cells. Subsequently, two subpopulations of ALDHhigh CSCs were isolated by flow cytometry either with the use of propidium iodide (PI) or not, respectively. The two subpopulations of ALDHhigh CSCs exhibited distinct characteristics, including stem cell related gene expression, self-renewal capacity and tumorigenicity in vitro and in vivo. Key regulators of the epithelial-mesenchymal transition (EMT), including vimentin, snail and slug were highly expressed in ALDHhigh CSCs. Therefore, the current study indicates that PI staining prior to the sorting of ALDHhigh CSCs by flow cytometry is an appropriate system for the study of CSCs. The current study also demonstrated that there was partial overlap between the transcriptional programs underlying the EMT and CSCs.