TNF indicates tumor necrosis factor; CFC, colony-forming cell; LTC, long-term culture; LTC-IC, long-term culture-initiating cell; FACS, fluorescence-activated cell sorting; FL, Flt3 ligand; SF, Steel factor; IL, interleukin; G-CSF, granulocyte colony-stimulating factor.

FACS-purified Annexin V⁻ PI⁻CD34⁺CD38⁻ cells were isolated from the light-density fraction of 3 normal human marrow samples, and 1500 of these cells per treatment group (ie, 3 × 500 μL cultures, each containing 500 cells) were incubated for either 7 or 11 days in serum-free medium supplemented with FL and SF (at 100 ng/mL each) and IL-3, IL-6, and G-CSF (at 20 ng/mL each). At the end of the first 2 days, 0.1 ng/mL TNF was added as indicated. Three or 5 days later (for the 7 day and 11 day groups, respectively), the cells in each culture were washed, resuspended in fresh medium with growth factors ± TNF at 0.1 ng/mL as indicated, and returned to the incubator. At the end of the 7 or 11 days in suspension culture, the cells were harvested and washed, and aliquots were assayed for CFC before and after further culture under LTC conditions for 3 weeks. The results of these assays are shown as the mean ± SEM for the pooled data from the 3 experiments performed. Input CFCs and 3-week LTC-IC–derived CFC numbers (per 1000 initial CD34⁺CD38⁻ cells) were 45 ± 4 and 2200 ± 1600, respectively.