Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma

Background Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) c ell selection, in terms of the expansion achieved Our goal was to develop p rocedures for consistent generation of large numbers of hematopoietic proge nitor and post-progenitor cells from unselected PBMC. Methods Unselected PBMC, collected from cancer patients undergoing apheresi s prior to high-nose chemotherapy and autologous stem cell rescue, were exp anded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days Results The addition of 2% autologous plasma to this cytokine combination e nhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01 ), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8 .1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold ver sus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given spe cimen was inversely related to the frequency of CD34(+) cells in the sample . CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivale nt to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cult ures were supplemented with autologous plasma. In plasma-free cultures, BFU -E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than w ith Flt3L, PIXY321, G-CSF and GM-CSF In the presence of autologous plasma, however; BFU-E expansion was higher in the Flt3L-containing media. in compa rison studies, autologous plasma suppressed BFU-E expansion in SCF-containi ng cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplemen tation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Discussion Unselected PBMC from cancer patients call be effectively expande d ex vivo in Flt3L, PIXY321, G-CSF and GM-CSF, supplemented with autologous plasma, yielding high numbers of myeloid and erythroid progenitors.