CD44+/CD24-/ESA+ cells are enriched for self-renewal activity in vitro. (a) Serial colony-forming units (CFUs) from 300 SUM159 sorted cells. Data are present as mean ± standard error of the mean for three separate experiments. Exhaustion of CFU is defined as the P3 colonies = P2 colonies. (b) Flow chart describing sorting scheme for reconstitution. (c) SUM149 or SUM159 sorted cells were expanded and the cellular outgrowths were reanalyzed by flow cytometry to assay reconstitution of the parental line. The parental line was run through the flow cytometer and expanded in parallel to control of the effects of fluorescence-activated cell sorting (top).(d) Representative phase-contrast brightfield images of unsorted SUM159 or SUM149, and colonies that grew out from CD44+/CD24+/ESA+, CD44+/CD24+/ESA-, CD44+/CD24-/ESA+, or CD44+/CD24-/ESA- sorted cells. ESA, epithelial-specific antigen.