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).
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.
authors.affiliation: University of Chicago, Department of Medicine, IL 60637, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Smith
authors.forename: J G
authors.initials: JG
authors.lastname: Bender
authors.forename: C
authors.initials: C
authors.lastname: Berger
authors.forename: W J
authors.initials: WJ
authors.lastname: Lee
authors.forename: M
authors.initials: M
authors.lastname: Loudovaris
authors.forename: J A
authors.initials: JA
authors.lastname: Martinson
authors.forename: J D
authors.initials: JD
authors.lastname: Opotowsky
authors.forename: X
authors.initials: X
authors.lastname: Qiao
authors.forename: M
authors.initials: M
authors.lastname: Schneidkraut
authors.forename: P
authors.initials: P
authors.lastname: Sweeney
authors.forename: K L
authors.initials: KL
authors.lastname: Unverzagt
authors.forename: D E
authors.initials: DE
authors.lastname: Van Epps
authors.forename: D E
authors.initials: DE
authors.lastname: Williams
authors.forename: S F
authors.initials: SF
authors.lastname: Williams
authors.forename: T M
authors.initials: TM
authors.lastname: Zimmerman
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis* / Q000097:blood; D001853:Bone Marrow / Q000187:drug effects* / Q000276:immunology; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000276:immunology; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012016:Reference Values
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D007377:Interleukin-3; C078524:PIXY321 fusion protein, recombinant; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor