NEUTROPHIL MATURATION OF CD34-BLOOD AND BONE-MARROW IN SERUM-FREE CULTURE-MEDIUM WITH PIXY321 AND GRANULOCYTE-COLONY-STIMULATING FACTOR (G-CSF)( CELLS FROM PERIPHERAL)

Bone marrow (BM) or peripheral blood (PB) CD34+ cells were cultured fo r 12 days in serum-free culture medium containing PIXY321 (1L-3/GM-CSF fusion protein) with or without periodic supplements of granulocyte-c olony stimulating factor (G-CSF). The cultures were evaluated at day 1 2 for total cell proliferation (fold increase from day 0), neutrophil differentiation by flow cytometry, using dual staining with CD15-FITC and CD11b-PE, and morphology using Wright-Giemsa and granule staining. In cultures containing PIXY321 where 6000 U/ml of G-CSF was added on days 0 and 6, there was no significant difference (p less than or equa l to 0.05) in cell proliferation or the percent of CD1S+/CD11b+ cells when compared with cultures with PIXY321 alone. ELISA analysis showed G-CSF levels had declined by 90% after 3 days of culture. Further stud ies were performed to assess the benefit of supplementing lower concen trations of G-CSF (600 U/ml) at more frequent intervals. A significant increase (p less than or equal to 0.05) in cell proliferation and per cent CD15+/CD11b+ was observed when G-CSF was added on days 0, 3, 6, a nd 9 (every 3 days) as compared with those cultures with PIXY321 alone . CD34+ cell proliferation without G-CSF was 19.6 +/- 4.8-fold, with G -CSF added on days 0 and 6 was 28.7 +/- 6.4-fold, and with G-CSF added on days 0, 3, 6, and 9 was 45.9 +/- 10.6-fold. Percent of CD15+/CD11b + cells was 19.0 +/- 4.6%, 38.2 +/- 7.3%, and 58.5 +/- 6.5%, respectiv ely, in these cultures. We observed more CD15+/CD11b+ cells, myelocyte s/metamyelocytes, and secondary granule staining in cultures with G-CS F added on day, 0, 3, 6, and 9 as compared with cultures with G-CSF ad ded on days 0 and 6 or no G-CSF added. We conclude that PIXY321 and G- CSF act synergistically on the in vitro proliferation and neutrophil d ifferentiation of BM and PB CD34+ cells and that frequent supplements of G-CSF facilitate neutrophil differentiation.