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  • Title: 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).
    Author: Smith SL, Bender JG, Berger C, Lee WJ, Loudovaris M, Martinson JA, Opotowsky JD, Qiao X, Schneidkraut M, Sweeney P, Unverzagt KL, Van Epps DE, Williams DE, Williams SF, Zimmerman TM.
    Journal: J Hematother; 1997 Aug; 6(4):323-34. PubMed ID: 9377071.
    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 CD11b-PE, and morphology using Wright-Giemsa and granule staining. In cultures containing PIXY321 where 6000 U/ml of G-CSF was added days 0 and 6, there was no significant difference (p > or = 0.05) in cell proliferation or the percent of CD15+/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 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 < or = 0.05) in cell proliferation and percent CD15+/CD11b+ 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+/CD11b+ cells was 19.0 +/- 4.6%, 38.2 +/- 7.2%, and 58.5 +/- 6.5%, respectively, in these cultures. We observed more CD15+/CD11b+ 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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