Expansion and Erythroid Differentiation of CD34+ Cells

K562 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin at 37°C in 5% carbon dioxide. Umbilical cord blood (CB) and adult peripheral blood (AB) samples were obtained from the Obstetrics Department of Beijing Maternity Hospital with informed consent for inclusion in the study signed by the donors. The mononuclear cells from CB and AB were isolated using lymphocyte separation solution Ficoll-Paque™ Plus (GE Healthcare, #17144002); then, primary CB and AB CD34⁺ cells were purified using the EasySep™ Human CD34 Positive Selection Kit II (STEMCELL, #17896, #17856) according to the manufacturer's instructions. Isolated CD34⁺ cells were cultured in StemSpan™ SFEM II medium for two phases [13 (link)]. Phase 1 involved cell proliferation and expansion for 7 days, and phase 2 involved erythroid differentiation for 16 days. The HUDEP-2 cell line donated by RIKEN Tsukuba Branch, Ibaraki, Japan, was cultured and differentiated as previously described [18 (link)].