FLT3 ligand preserves the uncommitted CD34(+) CD38(-) progenitor cells during cytokine prestimulation for retroviral transduction

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this stu dy, a retroviral vector carrying the genes for the enhanced version of gree n fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34(+) cells. The effect of cytokine prestimulation on tra nsduction efficiency and the population of uncommitted CD34(+)CD38(-) cells was determined. CD34(+) cells harvested from umbilical cord blood were kep t in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 p rior to transduction. Expression of the two genes was assessed by flow cyto metry and determination of neomycin-resistant colonies in a selective colon y-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there see med to be a positive correlation between expression of the two genes. The e ffect of cytokine prestimulation was therefore monitored using EGFP as mark er for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL -3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04) . However, prestimulation with SCF in combination with IL-3 and IL-6 also r educed the percentage of CD34(+) cells (p = 0.02). Then, prestimulation wit h SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34(+)CD38(-) cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytok ine combinations increased the transduction efficiency, but reduced the fra ction of CD34(+) cells. Importantly, the use of FLT3 ligand seemed to prese rve the population of uncommitted cells.