Summary of experiments
| . | % CD34+ Prior to Initiating Transduction . | Transduction Conditions . | % G418-Resistant CFU-GM (BFU-E) Following Transduction . |
|---|---|---|---|
| 1 | 20 | IL-3, IL-6, SCF* | 8.7 |
| 2 | 20.5 | IL-3, IL-6, SCF | 66 |
| 3 | 89.9 | IL-3, IL-6, SCF | 17.5 |
| 4a | 88.2 | IL-3, IL-6, SCF | 20.2 |
| 4b | 88.2 | IL-3, IL-6, Flt3L, SCF, αTGFβ† | 30 (25) |
| 5 | 65 | IL-3, IL-6, SCF | 10 (18.6) |
| 6a | 79.3 | IL-3, IL-6, Flt3L, SCF, αTGFβ | 22 |
| 6b | 79.3 | IL-3, IL-6, SCF, Flt3L‡ | 15 (14.5) |
| 7 | 93.2 | IL-3, IL-6, Flt3L, SCF, αTGFβ | 20 (8.8) |
| . | % CD34+ Prior to Initiating Transduction . | Transduction Conditions . | % G418-Resistant CFU-GM (BFU-E) Following Transduction . |
|---|---|---|---|
| 1 | 20 | IL-3, IL-6, SCF* | 8.7 |
| 2 | 20.5 | IL-3, IL-6, SCF | 66 |
| 3 | 89.9 | IL-3, IL-6, SCF | 17.5 |
| 4a | 88.2 | IL-3, IL-6, SCF | 20.2 |
| 4b | 88.2 | IL-3, IL-6, Flt3L, SCF, αTGFβ† | 30 (25) |
| 5 | 65 | IL-3, IL-6, SCF | 10 (18.6) |
| 6a | 79.3 | IL-3, IL-6, Flt3L, SCF, αTGFβ | 22 |
| 6b | 79.3 | IL-3, IL-6, SCF, Flt3L‡ | 15 (14.5) |
| 7 | 93.2 | IL-3, IL-6, Flt3L, SCF, αTGFβ | 20 (8.8) |
Experiments 1, 2, 3, 4a, and 5 used a transduction method that entailed incubation of retroviral supernatant with CD34+ cord blood cells in DMEM with 15% FBS and hIL-6, 100 ng/mL; hIL-3, 50 ng/mL; and rhSCF, 50 ng/mL. The media were replaced with fresh viral supernatant and cytokines at 24-hour intervals for three days.
Experiments 4b, 6a, and 7 used a transduction method adapted from Dao and Nolta.12 CD34−-selected cord blood cells were incubated for 12 hours in serum-free media (ExVivo 15, BioWhittaker) in the presence of IL-3, IL-6, Flt3, and SCF (50 ng/mL each). Following prestimulation, an equivalent volume of retroviral supernatant was added, and the cells were cultured for an additional 12 hours.
The transduction procedure for experiment 6b was equivalent to that described for experiments 1, 2, 3, 4a, and 5 except for the addition of a 24-hour prestimulation step that was performed in the presence of IL-3, IL-6, Flt3L, and SCF (50 ng/mL each).