The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L

This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34(+)-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34(+) enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(IV)(7-3) fractional factorial design. Expansion of Mk committed progenitors (CD41(+)) and primitive precursors (CD61(+) CD34(+)) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM- CSF (p < 0.05). Flt3- L inhibited the production of total CD61(+) cells (p < 0.05), CD61(+) CD34(+) cells (p < 0.03), and total CD41a(+) cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk (+) colonies from cells expanded in the presence of Flt3- L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3- L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.