Ex vivo expansion and differentiation of unselected peripheral blood progenitor cells in serum-free media

The ability to expand and differentiate unselected PBPC was investigated. C ells were grown in serum-free media containing stem cell factor, GCSF and m egakaryocyte growth and development factor (pegylated PEG-rHuMGDF) with or without supplemental serum. Optimal proliferation occurred when PBPC were c ultured without prior Ficoll-Paque separation in serum-free media. Cell yie lds after 17 days of culture were proportional to the percentage of CD34+ c ells in the starting population and were 1170 +/- 302-fold higher than the starting numbers of CD34+ cells. Granulocyte-macrophage colony-forming unit s increased over 12 days of culture, whereas the numbers of erythroid colon y-forming cells peaked between 4 and 7 days. Elimination of PEG-rHuMGDF fro m cell cultures resulted in significantly lower yields of myeloid and eryth roid colony-forming cells and total cell numbers. Cell differentiation into neutrophils was indicated by progressive increases in CD11b, CD15, and CD6 6b expression. Expanded neutrophils phagocytosed and killed bacteria as eff iciently as neutrophils from normal donors. Large-scale expansion studies y ielded similar proliferation and differentiation results as parallel small- scale cultures. Therefore, unselected PBPC can be efficiently expanded and differentiated into large numbers of functional mature neutrophils.