Neutrophil Maturation of CD34+ Cells from Peripheral Blood and Bone Marrow in Serum-Free Culture Medium with PIXY321 and Granulocyte-Colony Stimulating Factor (G-CSF)

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Abstract
Bone marrow (BM) or peripheral blood (PB) CD34+ cells were cultured for 12 days in serum-free culture medium containing PIXY321 (IL-3/GM-CSF fusion protein) with or without periodic supplements of granulocyte-colony stimulating factor (G-CSF). The cultures were evaluated at day 12 for total cell proliferation (fold increase from day 0), neutrophil differentiation by flow cytometry, using dual staining with CD15-FITC and CDllb-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 ≤ 0.05) in cell proliferation or the percent of CD15+/CDllb+ cells when compared with cultures with PIXY321 alone. ELISA analysis showed G-CSF levels had declined by 90% after 3 days of culture. Further studies were performed to assess the benefit of supplementing lower concentrations of G-CSF (600 U/ml) at more frequent intervals. A significant increase (p ≤ 0.05) in cell proliferation and percent CD15+/CDllb+ was observed when G-CSF was added on days 0,3, 6, and 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+/CDllb+ cells was 19.0 ± 4.6%, 38.2 ± 7.3%, and 58.5 ± 6.5%, respectively, in these cultures. We observed more CD15+/CDllb+ cells, myelocytes/metamyelocytes, and secondary granule staining in cultures with G-CSF added on day, 0, 3, 6, and 9 as compared with cultures with G-CSF added 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 differentiation of BM and PB CD34+ cells and that frequent supplements of G-CSF facilitate neutrophil differentiation.

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