EFFICIENT RETROVIRUS-MEDIATED GENE-TRANSFER OF DENDRITIC CELLS GENERATED FROM CD34(-BLOOD CELLS UNDER SERUM-FREE CONDITIONS() CORD)

A retroviral-vector encoding the low affinity nerve growth factor rece ptor (LNGFR) was used to transduce dendritic cells (DCs) generated fro m 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 (SC F), granulocyte/macrophage-colony stimulating factor (GM-CSF), tumor n ecrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF-beta 1). Addition of flt3-ligand (FL) to the aforementioned gro wth 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). Furthermor e, 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 se rum-free viral supernatants were used to infect DCs progenitors under entirely serum-free conditions and with the most potent cytokine combi nation, approximately one-third of the CD1a(+) DCs generated co-expres sed 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 funct ional properties, stimulating allogeneic T cells with similar efficien cy as nontransduced CD1a(+) DCs. Thus, the serum-free system described allows efficient generation and transduction of CD1a(+) DCs derived f rom CD34(+) progenitor cells and may be very useful for future therape utic applications of DCs.