Assessment of cell death and proliferation of UCB-derived CD34⁺ cells cultured in the presence of SCF, FLT3L, TPO in 2% to 3% versus 20% O₂. (A) Freshly isolated CD34⁺ progenitors were either plated directly (left and middle) or prestimulated with SCF + TPO for 2 to 4 hours (right) prior to plating on non–tissue culture–treated plates coated with PLL (white bar), or VCAM-1 (gray bar), or FN (black bar). Annexin V/PI staining was done on cultured cells after 48 hours of culture. A total of 4 to 5 separate cord blood samples was used for each experiment and statistics were done using Student t test. *P < .05. (B) Total protein levels and phosphorylated AKT and MAPK were assessed by immunoblotting from lysates for cells cultured in 20% (left) versus 2% to 3% (right) O₂ on PLL- versus FN-coated plates for 48 hours. (C) CD34⁺ progenitors harvested after culture in 20% and 2% to 3% O₂ on fibronectin were stained with Ki-67 antibody to assess the percentage of cells that was in G₀ (left). FACS analysis of CFSE-labeled CD34⁺ cells after culture for 7 days on fibronectin in 20% and 2% to 3% O₂ to measure cell division kinetics.