Generation of Dendritic Cells from Adherent Cells of Cord Blood by Culture with Granulocyte-Macrophage Colony-Stimulating Factor,Interleukin-4,and Tumor Necrosis Factor-α

Abstract

Although dendritic cells (DC) can be cultured from cord blood (CB) CD34⁺ progenitor cells, the generation of DC from CB monocytes has not been reported. In this paper, we explored the generation of DC from CB monocytes to establish the simplest way to obtain a substantial number of DC from CB. We isolated monocytes from CB mononuclear cells (CB-MNC) by the plastic adherence method. These adherent cells (monocyte-rich cells) were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) or in serum-free X-VIVO 15 medium (SFM) for 7 days, both of which contained 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml interleukin-4 (IL-4) with or without 10 ng/ml tumor necrosis factor-α (TNF-α) (added at day 5). In the presence of GM-CSF and IL-4, CB-adherent cells became nonadherent, acquired DC morphology, and showed increased expression of CD1a, CD80, CD86, and HLA-DR; they lost membrane CD14 and some cells with the expression of CD83 and CMRF-44 were generated. With the addition of TNF-α to these cultures and culturing for further 2 days, the proportion of CD83⁺ cells was elevated in both the FBS and SFM culture systems, compared with the culture without TNF-α. In the culture with TNF-α, cells expressing CD1a, CD80, CD86, HLA-DR, and HLA-DQ were markedly increased. TNF-α-treated cells were demonstrated to be stronger stimulators for proliferation of both allogeneic CB lymphocytes and PB lymphocytes than were cells not treated with TNF-α. The yield of CD83 + DC at day 7 of cultures was 4.9 ± 1.1 X 10⁵ or 3.0 ± 0.5 X 10⁵ per 1.2 X 10⁷ CB-MNC plated initially when cultured in FBS or SFM, respectively. These results have shown that a substantial number of mature DC could be generated from CB-adherent cells even by serum-free culture. We then compared these CB-adherent cell-derived DC (CB-DC) with peripheral blood (PB)-adherent cell-derived DC (PB-DC) in cell-surface phenotype and function. We found day 7 CB-DC have lower expression of CD80, CD1a, CD83, and CMRF-44 than day 7 PB-DC, but CB-DC have a similar capacity to stimulate the proliferation of both allo-CB lymphocytes and PB lymphocytes, compared with PB-DC. CB-DC cultured with GM-CSF and IL-4 have almost identical capacity of phagocytosis to take up fluorescein isothiocyanate (FITC)-dextran and Lucifer yellow (LY), compared with PB-DC. In summary, our findings suggest CB adherent cells, when cultured with GM-CSF, IL-4, and TNF-α, are a potent source of functional DC. Thus, CB-DC as well as PB-DC may become valuable tools for immunotherapy.