Ex Vivo Human CD34+ Erythroid Culture.

The human CD34⁺ cell erythroid differentiation method is composed of four phases over 21 d: expansion (days 0–4) and differentiation (Dif) I (days 5–9), II (days 10–13), and III (days 14–21). Cells were thawed according to the FHCRC protocol, and cultured in expansion medium (StemSpan SFEM, CC100 cytokine mixture and 2% penicillin–streptomycin) at 10⁵ cells per milliliter from days 0–4. After expansion, cells were subsequently cultured in Iscove’s modified Dulbecco’s medium (IMDM)-based erythroid differentiation medium supplemented with different cytokines in Dif I–III. The medium base for all three differentiation phases comprises: IMDM, 15% FBS, 2 mM glutamine, 1% BSA, 500 µg/mL holo human transferrin, 10 µg/mL recombinant human insulin, and 2% penicillin–streptomycin. On days 5–9, cells were cultured in Dif I medium, which contains erythroid medium base, 2 µM dexamethasone, 1 µM β-estradiol, 5 ng/mL rhIL-3, 100 ng/mL rhSCF, and 6 units/mL rhEpo. On days 10–13, cells were grown in Dif II medium containing erythroid medium base, 50 ng/mL rhSCF, and 6 units/mL rhEpo. On days 14–21, cells were cultured in fibronectin-coated plates in Dif III medium, which is erythroid medium base supplemented with 2 units/mL rhEpo. T4 or 1–850 was added in Dif I (10⁻⁶ M) or Dif III (10⁻⁶ M) when indicated. Cell numbers reseeded in the beginning of Dif I–III are 10⁵, 2 × 10⁵, and 3 × 10⁵/mL, respectively. Regular FBS was used throughout the culture unless noted.