Expansion Of Dendritic Cells From Normal And Chronic Myelogenous Leukemic CD34～+ Progenitor Cells

Objectives:Dendritic cells (DCs) are the most potent antigen presenting cells and play an important role in the development of anti-tumor immune response. The expansion efficiency of DCs from normal and chronic myelogenous leukemic (CML) CD34~+ progenitor cells by a two-step process was studied and the biological properties of CML-derived DCs and normal DCs were also investigated in this study.Methods:CD34~+ cells were purified by using CD34 microbeads from normal and CML bone marrow and cultured in serum-dependent medium or serum-free medium by two ways: i. Two-step culture method, primary in the presence of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin-3 (IL-3) for 14 days, and then further cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days in addition to tumor necrosis factor-α (TNF-α) for another 3-4 days (group GI), or with GM-CSF plus TNF-α for 10 or more days (group GT). ii. In SCF, FL, TPO, IL-3, GM-CSF, TNF-a for 14 days (convention culture method). Morphologies of cultured cells were examined every day under phase contrast microscopy. The viability was evaluated by Trypan blue exclusion. Cell surface markers were analysed by flow cytometry. The allostimulatory ability was examined by mixed lymphocyte culture (MLC). The fluid phase macromolecule uptake function was examined by fluorescein isothiocyanate-conjugated dextran (FITC-DX) uptake test. The expression of bcr/ab1 fusion gene of uncultured and cultured CML cells was detected by fluorescence in situ hybridization (FISH).