In vitro generation of human CD86⁺dendritic cells from CD34⁺haematopoietic progenitors by PMA and in serum-free medium

Abstract

The cytokine requirements to differentiate CD34 + progenitor cells from different origins either cord blood (CB) or peripheral blood (PB) into dendritic cells (DC) are known to be different. In addition to DC, macrophages and neutrophils are generated. On the other hand, phorbol esters such as PMA induce primary human CD34 + bone marrow (BM) progenitor cells to differentiate into functional DC and no other lineages are generated. In addition, FCS is used as culture supplement in most of the protocols described which contains additional foreign antigens potentially skewing the resulting immune response. Therefore, we evaluated the ability to differentiate CB- and PB-CD34 + progenitor cells into DC with PMA and under serum-free conditions. In this study, we delineate the maturation of cultured human blood DC by analysis of expression co-stimulatory molecule B7-2 (CD86). Human mature DC with typical morphology and surface antigen phenotype (CD1a - , CD83 + and CD86 + ) were obtained from CB- and PB-CD34 + progenitor cells after 1 week of culture in serum-free medium upon stimulation with PMA alone. The same result was obtained from ex vivo-expanded BM-CD34 + cells. CD86 + yield was increased by PMA compared to cytokine cocktails (28.0% ± 7.0 versus 15.3% ± 5.6 for CB and 44.6% ± 7.5 versus 28.1% ± 7.5 for PB, respectively). CD86 was most up-regulated in the presence of the calcium ionophore ionomycin. However, the number of viable cells after differentiation was decreased by PMA plus ionomycin (P < 0.05) or plus TNF-alpha (P > 0.05) as compared with that in PMA alone. We conclude that PMA is a potent activator to differentiate human CD34 + cells into mature DC in serum-free medium. This may be used for in vitro studies of primed or genetically modified DC against infectious and tumour-associated antigens.