PRODUCTION OF FUNCTIONAL MYELOID CELLS FROM CD34-SELECTED HEMATOPOIETIC PROGENITOR CELLS USING A CLINICALLY RELEVANT EX-VIVO EXPANSION SYSTEM

There is increasing clinical interest focused on ex vivo manipulation and expansion of hematopoietic cells. In this study, we demonstrate th at a simple combination of growth factors can expand progenitors to yi eld functional myeloid cells. Furthermore, this system can produce mat ure, functionally competent cells in the absence of fetal bovine serum (FBS), which will enhance the clinical utility of this approach. Hema topoietic progenitor cells obtained from normal bone marrow and from l eukapheresis products mere studied. The mononuclear fraction was enric hed for CD34 cells using the Ceprate CD34 biotin kit (CellPro #LC34-1 or LC34-2). The selected cells were expanded for two weeks in Iscove's medium supplemented with 20% FBS and various combinations of interleu kin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and interleukin-6 (IL-6) added either simultaneously or sequentially. The optimal combination of these factors identified for myeloid expansion was simultaneous addition of IL-3, SCF and G-CSF (at 50 ng/ml each), resulting in an average 773 +/- 133-fold expansion of nucleated cells (n = 5). When corrected for the purity of CD34 cells in the starting population, the mean fold expansion with IL-3, SCF and G-CSF was 2,265 +/- 729. A mean of 74.7 +/- 10.5% (n = 3) of the expa nded cells was positive for CD11b; 86-91% (n = 2) of the cells were pr omyelocytes or more mature granulocytes. Functional assays demonstrate d normal phagocytosis and intracellular killing of Staphylococcus aure us (S. aureus) by the expanded cell population. Studies performed usin g cells expanded in defined serum-free media demonstrated that fold ex pansion was decreased and that the cells pro duced were less mature an d functionally less competent than cells expanded with FBS. The decrea sed expansion could be partially reversed, and the functionality almos t completely restored by the addition of autologous plasma.