Effect of glycosylation of recombinant human granulocytic colony-stimulating factor on expansion cultures of umbilical cord blood CD34(+) cells

Backgound and 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), an d a glycosylated CHO-derived rhG-CSF. As previously shown, glycosylation gi ves 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. Design and Methods. CD34(+) cells from nine cord blood samples were positiv ely selected (median purity 84%) and cultured in the presence of 50 ng/mL o f 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 cultu re fed on day 0, nucleated cells (NC), CD34(+) cells and colony-forming uni ts 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 CN-Mix, and indeed no influence was detected in terms of BFU-E expansion. The presence of GG in culture causes the generati on of more mature granulocytic cells, assessed by the expression of CD11b/C D15 on CD13(+) population, than the presence of DG. In order to check the r ole of the molecule's stability in this difference, the effect of daily sup ple mentation was tested. Continuous presence of cytokines using either for m of G-CSF (daily feeding) significantly increased the rate of expansion, b ut again GG produced higher generation than its DG counterpart. Interpretation and Conclusions. Our results suggest that the stability of t he G-CSF molecule has a pre dominant effect on the higher biological activi ty found. A glycosylated form of G-CSF is recommended for In vitro cultures using serum-dependent conditions. (C) 1999, Ferrata Storti Foundation.