Retroviral Gene Transfer with Triple Genetic Reporter Genes into Human Cord Blood CD133⁺ Cells.

Umbilical cord blood (CB) is used increasingly as a source of hematopoietic support in bone marrow transplant patients lacking family or unrelated donors. Little is known about human CB with respect to in vivo migration, homing, long-term viability, the organ-specific checkpoints and its differentiation. In order to study CB migration and homing, the gibbon ape leukemia virus (GALV)-pseudotyped retroviral gene transfer technique was developed to genetically modify CB CD133⁺ cells with triple reporter genes which encode enhanced green fluorescent protein (eGFP), luciferase, and herpes simplex virus type one thymidine kinase (HSV1-tk). The novelty of this approach is that it allows multimodal repetitive non-invasive imaging using fluorescence, bioluminescence (BLI), and nuclear imaging techniques, respectively. CD133⁺ cell fractions were isolated from human CB using the MidiMACS device (Miltenyi). To facilitate transduction, CB CD133⁺ cells were stimulated with 100ng/ml each of stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO) in alpha-MEM medium supplemented with 20% fetal bovine serum. After 3, 5, 7 or 14 days of stimulation, CB cells were transduced by RetroNectin (TaKaRa)-assisted gene transfer. RetroNectin, recombinant human fibronectin fragment CH-296, can provide colocalization of retroviral particles and target cells. Specifically, 6-well plates were coated with 5ug/cm² RetroNectin for 2 hours at room temperature, followed by preloading with fresh virus-containing medium (VCM) for 4 hours at 37°C. VCM was then removed and the CB cells were added in medium containing cytokines for 2 or 3 days. This approach resulted in > 90% transduction of control U87 human glioma cells and up to 36% transduction of CD133⁺ CB cells. Preliminary experiments suggested that optimal transduction appeared to be at day 7 of CB stimulation. When compared to non-RetroNectin coated plates, this approach showed a 5- to 11- fold increase in eGFP⁺ cells as determined by flow cytometry, increasing the transduction efficiency from < 5% to a maximal 36%. The addition of polybrene, a cationic polymer, to this process did not improve transduction efficiency. Studies are underway to visualize retrovirally-transduced CB CD133⁺ cells in NOD/SCID mice in vivo. The ability to trace the fate of CD133⁺ cells following transplantation may allow a better understanding of the mechanisms of migration, homing and long-term viability.