Efficient retrovirus-mediated gene transfer of dendritic cells generated from CD34(+) cord blood cells under serum-free conditions

A retroviral-vector encoding the low affinity nerve growth factor receptor (LNGFR) was used to transduce dendritic cells (DCs) generated from CD34(+) cord blood (CB) progenitor cells under serum-free conditions. Transduction efficiency was monitored by flow cytometry (FACS) using a specific monoclonal antibody. Prior to retroviral infections, CD34(+) CE cells mere stimulated for 60 h in a serum-free medium containing a DC differentiation inducing cytokine cocktail: stem cell factor (SCF), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF-beta 1). Addition of flt3-ligand (FL) to the aforementioned growth factors significantly enhanced cell expansion (41.7 +/- 11.5 fold vs. 22.5 +/- 4.7 fold without FL) and generation of CD1a(+) DCs (mean 45.7 +/- 9.8% vs. 28 +/- 6.5% without FL, n = 4, p = 0.01). Furthermore, FL significantly increased the proportion of CD1a+(L)NGFR(+) cells (mean 10% +/- 4.4% vs. 6% +/- 2.4 without FL n = 4, p = 0.03). When serum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combination, approximately one-third of the CD1a(+) DCs generated co-expressed the LNGFR gene. Moreover, the transduced gene was also identified in more mature CD1a(+)CD80(+) and CD1a(+)CD86(+) DCs after 12-14 days of culture. In addition, transduced CD1a(+) DCs maintained their functional properties, stimulating allogeneic T cells with similar efficiency as nontransduced CD1a(+) DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a(+) DCs derived from CD34(+) progenitor cells and may be very useful for future therapeutic applications of DCs.