Ex vivo expansion of canine dendritic cells from CD34⁺ bone marrow progenitor cells

Background. The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. Methods. CD34⁺-selected cells and CD34⁺-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-α 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. Results. Two main cell populations were identified, DR⁺⁺(bright)/CD14^-- and DR⁺(dim)/CD14⁺. Ex vivo expanded CD34⁺-selected cells showed increased allostimulatory activity compared to both cultured CD34⁺-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34⁺-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34⁺-selected bone marrow to enrich for DR⁺⁺/CD14^-- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR⁺/CD14⁺ cells (P=0.02). Conclusions. Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34⁺-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.