Construction and evaluation of a highly effective culture system for erythroid differentiation of umbilical cord blood-derived hematopoietic stem and progenitor cells

Objective: To construct and evaluate a highly effective culture system for the erythroid differentiation of umbilical cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs). Methods: Magnetic-activated cell sorting (MACS) was applied to separate CD34(+) HSPCs from umbilical cord blood. Cells were cultured in 3 culture systems (1, 2, or 3), which have the three cell factors including SCF, IL3 and EPO, for 18 days using a 3-stage method to induce erythroid differentiation after in vitro proliferation for 4 days. Both cell proliferation ability and cell surface CD235a(+)CD71(+) expression were detected at each differentiation stage. The percentage of burst-forming unit-erythroid (BFU-E) colonies in each system were analyzed on day 12. Results: Cell proliferation in all culture systems peaked between day 10 and day 12, followed by a decreasing trend. Nevertheless, cell proliferation remained higher after day 12 in system 1 than in the other two systems (P<0.05). The expression of the erythroid surface markers CD71 and CD235a reached the highest level on day 18 in culture systems 1, 2, and 3 (89.1%, 79.4% and 88.0%, respectively). Statistical analyses revealed no significant differences between systems 1 and 3, which had the highest percentages of CD71(+)CD235a(+) cells among the three culture systems (P=0.44). Progenitor cells were formed in all culture systems on day 12 of differentiation. The percentage of BFU-E colonies was higher in system 1 than in systems 2 and 3, with approximate percentages of 87.5%, 73.5% and 83.5%, respectively (P<0.05). Conclusion: We successfully constructed and evaluated a high-efficiency erythroid differentiation culture system for umbilical cord blood-derived CD34+ HSPCs based on continuous cytokine supplementation during all stages of erythroid differentiation.