Effect of medium conditioned by CD34⁺ cells on cell survival.

(A) Effect of media conditioned by human CD34⁺ cells (2 × 10⁶/mL per 24 hours) on survival of freshly isolated CD34⁺ cells (10⁵/mL). Cells were cultured for 7 days in media conditioned by CD34⁺ cells (CM) or in a control serum-free medium (SFM). Cell viability was evaluated by 0.5% trypan blue exclusion tests. The data are representative of 4 experiments yielding similar results. (B) (C) (D) Effect of a medium conditioned by human CD34⁺ cells on survival of clonogeneic progenitors in CD34⁺ cells. Cells were cultured for 7 days in media conditioned by CD34⁺cells (CM, solid bars) or in a control serum-free medium (CFM, open bars). After 7 days, cells were plated in methylcellulose medium and stimulated to grow erythroid (B), myeloid (C), and megakaryocytic (D) colonies. (E) Detection of activated capase-3 by intracellular staining with monoclonal antibody against activated form of this enzyme. Normal human BM-derived CD34⁺ cells were cultured for 72 hours in conditioned medium harvested from CD34⁺ cells (i) or serum-free medium (ii). Data are shown in comparison with the intracellular caspase-3 staining in freshly isolated CD34⁺cells. The experiment was repeated 4 times with similar results.