Increased secondary CFU-Cs and cobblestone formation by AML1-ETO–expressing cells.

(A) (B) Transduced CD34⁺ cells were plated over MS-5 stromal cells 2 days after transduction. Media and cells were demidepopulated weekly. After 5 weeks, cobblestone areas (minimum of 8 phase-dark cells) were counted. (C) (D) Transduced CD34⁺cells were plated over MS-5 stromal cells 6 days after transduction. Triplicate flasks were examined for the number of cobblestone areas, and these cobblestones were evaluated microscopically for GFP expression. Total cobblestone counts are shown. (E) (F) Nonadherent (suspension) and adherent cells (from the same experiment as panels C and D) were collected from the MS-5 stromal cultures after 6 weeks, counted, and plated in methylcellulose at 5 × 10⁴ cells per dish and 20 × 10⁴ cells per dish, respectively. Two weeks later, colonies were counted and GFP expression of colonies was evaluated microscopically. The average of triplicate plates is indicated. Two independent experiments yielded essentially the same results; a representative experiment is shown.