Ex vivo generation of functional dendritic cells from mobilized CD34⁺ hematopoietic stem cells

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34⁺ cells collected from healthy donors. CD34⁺ cells purified from leukapheresis product were seeded at 1 x 10⁴ cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 ± 5.4-fold compared to the starting number of CD34⁺ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 ± 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a ⁺CD14 ^-HLA-DR⁺, and CD80⁺CD86⁺HLA-DR⁺, constituted 49.0% ± 7.5%, and 38.9% ± 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a⁺HLA-DR⁺ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.