Biphenotypic B220⁺GR-1/MAC-1⁺ cells sustain CFU-GM progenitors at high frequencies and require cytokines for sustained proliferation. (A) At 24 days after transduction, GFP⁺GR-1/MAC-1⁺ cells from control-transduced cultures and GFP(PAX5)⁺B220⁺GR-1/MAC-1⁺ from PAX5-transduced cultures were purified by FACS and plated into MethoCult as described in “Materials and methods.” Colony numbers were counted at day 14. Data are mean (SD) values from 3 control and 2 PAX5 experiments with 3 to 5 replicates in each experiment. *P < .05. (B) Appearance of typical myeloid colonies (CFU-GMs) generated in methylcellulose from GFP(PAX5)⁺B220⁺GR-1/MAC-1⁺ cells, with corresponding May-Grünwald and Giemsa morphology of myeloid cells in colonies. (C) At 24 days after transduction, 1 × 10⁶ cells (stapled line) from PAX5-transduced culture were washed and seeded in medium alone or in medium supplemented with either GM-CSF or IL-3. Thereafter, cellular expansion was evaluated at different time points as indicated. One of 2 experiments with similar results is shown. (D) GFP(PAX5)⁺B220⁺GR-1/MAC-1⁺ cells from 24 days of culture were sorted by FACS and 1 × 10⁴ GFP(PAX5)⁺B220⁺GR-1/MAC-1⁺ cells were seeded in SCM supplemented with either IL-3 or GM-CSF. Total number of cells was assessed 2 weeks later. Mean (SD) values of 3 experiments. *P < .05.