Efficient Retrovirus-Mediated Gene Transfer of Dendritic Cells Generated from CD34 + Cord Blood Cells under Serum-Free Conditions

Abstract

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⁺ CB cells were 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 (TNFα), and transforming growth factor beta 1 (TGF-β1). 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⁺LNGFR⁺ 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.

Overview summary

Substantial numbers of DCs can be generated from hematopoietic progenitor cells or from peripheral blood monocytes. Taking advantage of a serum-free system described previously, the feasibility of using an entirely serum-free system to efficiently generate and transduce CD1a⁺ DCs derived from CD34⁺ CB cells is reported. Furthermore, addition of the flt3-ligand to the standard DC inducing cytokine cocktail results in higher-cell expansion and greater generation of transduced CD1a⁺ DCs under serum-free conditions. Transduced CD1a⁺ cells are still capable of stimulating allogeneic T cells.