Figure 1: Three phenotypes of CSCs: self-renew, multipotentiality, and tumor-initiating capacity. Self-renewal: single-cell culture sphere-forming assay. Differentiation: flow cytometric analysis of differentiation markers and loss of stem cell markers. Tumor-initiating properties were investigated by injection of CSCs in NOD/SCID mice. The most crucial standard for a CSC is its ability to reinitiate a serially transplantable tumor that recapitulates the original heterogeneity and tumor histology. Using this standard, many so-called CSCs reported so far are based on correlative studied rather than being true CSCs.