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+ 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 (TNFalpha), and transforming growth factor beta 1 (TGF-beta1). 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.
Bello-Fernandez
C
C
Vienna International Research Cooperation Center at Novartis Forschungsinstitut, University of Vienna, Austria.
Matyash
M
M
Strobl
H
H
Pickl
W F
WF
Majdic
O
O
Lyman
S D
SD
Knapp
W
W
D018949:Antigens, CD1 / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology; D003713:Dendritic Cells / Q000737:chemistry; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells; D006801:Humans; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000494:pharmacology; D017475:Receptors, Nerve Growth Factor / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology
D018949:Antigens, CD1; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016207:Cytokines; D008565:Membrane Proteins; D017475:Receptors, Nerve Growth Factor; C084881:flt3 ligand protein