Ex Vivo Generation of RBCs from CD34+ Cells in Human Umbilical Cord Blood and Expression Profile Analysis Using Microarray

Background :In this study, we attempted to generate RBCs from CD34+ cells in cord blood using a 3-step culture protocol and also evaluated a change in immunophenotypic characteristics and expression profile according to erythropoietin (EPO) concentrations and culture duration. Method :Using mini-MACS columns, CD34+ cells were isolated from cord blood. The culture procedure comprised three steps. For each step, cells were cultured sequentially for 7 days in a serum free liquid medium with a specific combination of growth factors for 21 days. [1st step: Flt3-ligand (Flt3-L), thrombopoietin and stem cell factor (SCF); 2nd step: IGF-1, SCF and EPO; and 3rd step: IGF-1 and EPO] To evaluate the effect of EPO on proliferation and differentiation, cells were cultured with different EPO concentrations (0, 3, 10 & 20 U/mL). Cell count and morphology were monitored during the culture. For phenotyping, antibodies to CD34, CD38, CD45 and glycophorin A (GPA) were used. The expression profile of cultured cells was analyzed by 17,000-gene microarray analysis. Result :As EPO concentration increased, cell expansion was also increased, showing a maximum expansion at 20 U/mL. The cell population showed a gradual decrease in expression of CD34 and CD45, whereas the expression of GPA was not prominent in any conditions. However, we observed increased expression in some genes associated with erythropoiesis (e.g. glycophorin A, rhesus blood group CcEe antigens). Conclusion :This study shows that erythropoietin enhances the proliferation of hematopoietic progenitor cells. Our culture system did not achieve pure production of RBCs, but induced expression changes that indicated erythroid differentiation.ope