Background and objective: The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors.
Design and methods: Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF).
Results: The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture.
Interpretation and conclusions: These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.