In Vitro Proliferation and Differentiation of Erythroid Progenitors of Cord Blood

Abstract

Stem cell factor (SCF) is known to synergize with erythropoietin (EPO) for erythropoiesis in vitro. Clonogenic assay and suspension culture were used to assess the effect of EPO alone or its combination with SCF on the proliferation and differentiation of erythroid progenitors of cord blood. Colony formation, increase in cell count, and cell cycling status for the proliferation as well as expression of Glycophorin A (Gly A) and hemoglobinization as the marker of differentiation were determined with each stimulation. The cell cycle status of the cells in suspension cultures was determined using FACScan after labeling of cells with propidium iodide. Expression of Gly A and degree of hemoglobinization were determined by FACScan and spectrophotometer on the cells plucked from colonies in semisolid culture. Larger increases in cell counts in suspension culture were observed with EPO + SCF after 12 days of inoculation than with EPO alone. Mean doubling time was 14.2 h with EPO + SCF and 22.7 h with EPO alone. The proportion of cells in S and G₂ + M phase in day 14 suspension culture was 48% with EPO + SCF and 43% with EPO alone (no significant difference). Mean colony counts per 10⁵ nonadherent mononuclear cells were 76 ± 14 with EPO + SCF and 51 ± 15 with EPO at day 14 (p < 0.05). The number of macroscopic colonies with > 0.5 mm diameter was 10.7 ± 1.2 with EPO + SCF and 0.3 ± 0.5 with EPO (p < 0.05). Percent of Gly A⁺ cells was 75% for both EPO + SCF and EPO colonies at day 14. Hemoglobin concentration/10⁵ cells at day 14 was 0.70 ± 0.17 μg with EPO + SCF, and 1.16 ± 0.32 μg with EPO alone (p < 0.05). In conclusion, SCF in the combination with EPO showed a synergistic effect for erythroid proliferation in colony number as well as colony size derived from cord blood, while SCF with EPO decreased hemoglobin synthesis but not Gly A expression at day 14.