Long-term expansion of CD34⁺ CB cells transduced with lentiviral vectors.

Twenty thousand CB CD34⁺ cells were prestimulated for 96 hours with FL, TPO, SCF, and IL6; then 1 × 10⁵CD34⁺ prestimulated cells in 100 μL were transduced overnight with 2 × 10⁶ lentiviral particles (MOI of 20) or with control medium. Cells were then washed and seeded in new 24-well plates and cultured in the presence of the same growth factors for up to 30 weeks. Aliquots of weekly demidepopulated wells were counted, analyzed for CD34/GFP expression (A), and plated in semisolid cultures for assessment of clonogenic progenitor output (B). Values represent the fold expansion, compared with input cells and colonies present in a single well (input CD34⁺/GFP⁺cells are those evaluated by FACS on the third day of liquid culture after transduction procedure; input GFP⁺ CFCs are the fluorescent colonies present in clonal cultures prepared soon after transduction). Representative experiment performed in triplicate wells.