Effect of TGF-β1 on chemotactic response to SDF-1 of CD34⁺ cells preexposed to SDF-1. (A) CB CD34⁺ cells were expanded for 4 days as described in “Materials and methods.” Expanded CD34⁺ cells were first pretreated with indicated doses of SDF-1 for 24 hours, and then after washing the cells their chemotactic activity toward SDF-1 (200 ng/mL) was determined. *P < .05 compared with cells cultured in cytokine cocktail. (B) CD34⁺ cells were cultured in a cytokine cocktail (SCF, TPO, and Flt-3 ligand) alone, or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 24 hours. Cells were washed, and their chemotactic activity to SDF-1 (200 ng/mL) was determined in migration assay. (C) CD34⁺ cells were pretreated with SDF-1 (200 ng/mL) in the presence of various doses of TGF-β1 for 24 hours, and their chemotactic response to SDF-1 (200 ng/mL) activity was determined. *P < .05 compared with cells pretreated with SDF-1. (D) Cytokine-expanded CD34⁺ cells were coincubated with SDF-1 and TGF-β1 or SDF-1 alone for the indicated time periods, and SDF-1 (200 ng/mL) directed chemotactic activity of the cells was assessed. *P < .05 compared with cells pretreated with SDF-1 alone. (E) Freshly isolated CD34⁺ cells were cultured in medium alone or along with TGF-β1, SDF-1, or SDF-1 and TGF-β1 for 20 hours. Cells were washed, and their chemotactic activity to SDF-1 (200 ng/mL) was determined in migration assay. (A-D) Data are presented as mean ± SD of 4 separate experiments. (E) Data are presented as mean ± SEM for 3 separate experiments. Of 3 experiments shown, 2 were conducted by pooling cells from different CB collections (after pretreatments), and in 1 experiment cells from a single CB collection were used. *P < .05 compared with cells cultured in medium alone and **P < .05 compared with SDF-1–pretreated cells.