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).
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.
Smith
S L
SL
University of Chicago, Department of Medicine, IL 60637, USA.
Bender
J G
JG
Berger
C
C
Lee
W J
WJ
Loudovaris
M
M
Martinson
J A
JA
Opotowsky
J D
JD
Qiao
X
X
Schneidkraut
M
M
Sweeney
P
P
Unverzagt
K L
KL
Van Epps
D E
DE
Williams
D E
DE
Williams
S F
SF
Zimmerman
T M
TM
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
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