Effect of glycosylation of recombinant human granulocytic colony-stimulating factor on expansion cultures of umbilical cord blood CD34+ cells.
OBJECTIVE
Granulocytic colony-stimulating factor (G-CSF) is a cytokine widely used for several purposes such as stem cell mobilization, treatment of neutropenia or in vitro cultures. Recombinant human G-CSF (rh-G-CSF) is available in two forms: a non-glycosylated (E. Coli-derived), and a glycosylated CHO-derived rhG-CSF. As previously shown, glycosylation gives a higher degree of homology between the recombinant and the wild human G-CSF molecule. This study analyses the role of glycosylation in expansion cultures comparing the biological effects of the two forms of G-CSF.


METHODS
CD34+ cells from nine cord blood samples were positively selected (median purity 84%) and cultured in the presence of 50 ng/mL of stem cell factor and 1, 10 or 100 ng/mL of glycosylated rh-G-CSF (GG) or a deglycosylated form (DG). After 5 days of a static, serum-dependent culture fed on day 0, nucleated cells (NC), CD34+ cells and colony-forming units were evaluated and compared using the paired Student's t-test.


RESULTS
For all concentrations tested, GG was able to generate more NC and progenitors than DG was able to (p<0.05). This effect was mainly observed in CFU-GM colonies, and in CFU-Mix, and indeed no influence was detected in terms of BFU-E expansion. The presence of GG in culture causes the generation of more mature granulocytic cells, assessed by the expression of CD11b/CD15 on CD13+ population, than the presence of DG. In order to check the role of the molecule's stability in this difference, the effect of daily supplementation was tested. Continuous presence of cytokines using either form of G-CSF (daily feeding) significantly increased the rate of expansion, but again GG produced higher generation than its DG counterpart.


CONCLUSIONS
Our results suggest that the stability of the G-CSF molecule has a predominant effect on the higher biological activity found. A glycosylated form of G-CSF is recommended for in vitro cultures using serum-dependent conditions.
Querol
S
S
Cryobiology and Cell Therapy Department and Barcelona Cord Blood Bank, Institut de Recerca Oncològica (I.R.O.), Hospital Universitari Sant Joan de Déu, Barcelona, Spain. squerol@iro.es
Cancelas
J A
JA
Amat
L
L
Capmany
G
G
Garcia
J
J
D018952:Antigens, CD34 / Q000097:blood*; D002470:Cell Survival; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004305:Dose-Response Relationship, Drug; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology*; D005434:Flow Cytometry; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D010641:Phenotype; D011247:Pregnancy; D011994:Recombinant Proteins / Q000378:metabolism; D014471:Umbilical Veins
D018952:Antigens, CD34; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor