In Vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34⁺ (10⁵ cells/mL) or light-density (10⁶ cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10^-6 M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated ≅ 1-2 × 10⁷ erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45^low/glycophorin (GPA)^neg/ CD71^low cells at day 7, 50 - 60% of which became CD45^neg/GPA⁺/CD71^high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (>90% benzidine^pos and CD45^neg/GPA⁺/CD71^medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.