CD24+/CD44+ cells are hybrid E/M cells and have increased stem-like properties.

(A) CD24/CD44 flow cytometry profile of HP_late cells. (B) Single cell qPCR analysis of 20 individual cells sorted from each of the six indicated CD24/CD44 subpopulations (from two independently passaged HP_late cell-lines). Mean expression of 10 E and 7 M genes (as in Fig 3) is visualized in the E-M state space. The crosses mark the average expression within the three different CD24/CD44 subpopulations (40 cells). Indicated p-values were determined using a cross-match test by comparing mean of E gene expression and/or M gene expression between indicated groups of single cells (S5 Table). (C) Principal component (PC) projections of the 120 individual cells as tested in (B) for the three different subpopulations colored according to their CD24/CD44 origin are shown. (D) PC loading projections of the 48 genes as tested in (B), showing the contribution of each gene to the first two PCs. E genes are colored in orange, M genes in blue, pluripotency genes in green, and housekeeping genes in black. (E) CD24/CD44 flow cytometry analysis of cells cultured under standard adhesion conditions for 10 days that had been freshly sorted from 100 cells of the three subpopulations from (A). (F) Assessment of ALDH1 activity in cultured cells derived from the three subpopulations (E) using the ALDEFLUOR assay. Gate shows percentage of ALDH1+ cells. Small insets show the negative control cells incubated with DEAB. (G) Mammosphere-forming ability per 1,000 sorted cells of the three HP_late derived cell populations (A) after two weeks in suspension.