HUDEP-2 cell culture and induced maturation

HUDEP-2 cells were maintained in culture as previously described.²⁹ For expansion, the cells were grown to 0.2 to 0.8 × 10⁶ cells/mL in StemSpan serum-free expansion medium (Stem Cell Technologies, 9650) in the presence of 50 ng/mL SCF, 3 IU/mL EPO, 1 µM dexamethasone, 1 µg/mL doxycycline, and 1% penicillin-streptomycin. To induce erythroid maturation, HUDEP-2 cells were grown to 0.7 to 1.4 × 10⁶ cells/mL in Iscove modified Dulbecco medium (Invitrogen) supplemented with 2% fetal bovine serum, 3% human serum albumin, 3 IU/mL EPO, 10 µg/mL insulin, 1000 µg/mL holotransferrin, 3 U/mL heparin, and 1 µg/mL doxycycline for days 1 to 3. Cells were grown to 1 to 2 × 10⁶ cells/mL in the same medium for days 4 and 5.