Influences of Hematopoietic Growth Factors on CFU-GM and CD34 Positive Cells of Mobilized Peripheral Blood Progenitors

Kwon, Ki Young

Abstract

BACKGROUND: Peripheral blood progenitor cells (PBPC) mobilized by hematopoietic growth factors such as G-CSF or GM-CSF are increasingly being used instead of bone marrow to allow hematopoietic reconstitution after myeloablative therapy for variety of malignancies. Ex vivo expansion of PBPC with growth factors leads marked increase in CFU-GM and CD34+ cells. To define the influence of G-CSF and stem cell factor alone and in combination on in vitro culture of PBPC, and to address the question of optimal duration of exposure with growth factors and the effects of G-CSF according to dosages, mobilized progenitors were incubated in liquid media containing autologous serum, stem cell factors and different dose of G-CSF. After 1, 7 and 10 day culture, viable cells were collected and innoculated to methylcellulose media, CFU-GM assay and evaluation of CD33 and CD34 positive cells were done. METHOD: PBPC were obtained from 10 patients by apheresis using COBE Spectra after chemotherapy with or without G-CSF. After Ficoll Hypaque separation, viable 2x106 PBPC were incubated in each 6 sets of RPMI media containing 10% autologous serum and addition of 100ng/mL of stem cell factor, 1,000U/mL of G-CSF, 5,000U/mL of G-CSF, 100ng/mL stem cell factor+1,000U/mL of G-CSF, 100ng/mL stem cell factor+5,000U/mL G-CSF in each culture flask and control group which didn' t contain any growth factor. After 1, 7 and 10 day of culture, viable cells were collected and 1x105 cells were seeded in methylcellulose media containing PHA-LCM and were cultured in duplicate. After 14 day incubation, aggregated with over 50 cells were scored as colony. And 1 day and 10 day of culture of control group and 10 day culture of stem cell factor+5,000U/mL G-CSF group, 1x105 cells were also collected for evaluation of CD33 and CD34 positive cells using flow cytometry. RESULT: CFU-GM were significantly increased even in 1 day exposure with combination of stem cell factor and G-CSF and there showed synergistic effect of stem cell factor and G-CSF. Seven day exposure with growth factor also represented similar increase in CFU-GM. In 10 day exposure of PBPC with growth factor showed significant increase in CFU-GM except 1,000ng/mL G-CSF group. The peak increase of CFU-GM was noted on 7 day culture with G-CSF+stem cell group and on 10 day culture of stem cell group. Number of CD33 & CD34 positive cells were increased in growth factor group and most of them were CD33+ CD34+ cells. There revealed significant positive correlation between CD34+ cells and day 14 CFU-GM.
CONCLUSION
G-CSF and stem cell factor act synergistically and their action on ex vivo expansion of PBPC was prominent even in 1 day exposure with stem cell factor and G-CSF. CD34+ cells were also increased under the effect of growth factors and showed good positive corelation with CFU-GM.