Erythroid Differentiation of CD34+ Cells

CD34⁺ cells were individually cultured in a 2-phase medium systems to drive the cellular commitment to the erythroid lineage and differentiate into mature red blood cells. Cells from each donor were cultured in Phase I medium: Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco^®, Thermo Fisher Scientific, Inc., MA, USA) containing 20% FBS (Sigma-Aldrich^®, Sigma-Aldrich, Inc., MO, USA), 300 μg/mL holo-Transferrin (holo-TF, PromoCell^®, PromoCell GmbH, Heidenberg, Germany), 50 ng/mL human Stem Cell Factor (hSCF, CellSignaling Technology^®, Cell Signaling, Inc., MA, USA), 10 ng/mL interleukine-3 (IL-3, CellSignaling Technology^®), 2 U/mL erythropoietin (EPO, EPREX^®, Janssen-Cilag, Auckland, NZ) in the presence of 100 U penicillin/streptomycin (Gibco^®). On day 5, cells were replaced with fresh Phase II medium: IMDM containing 20% FBS, 300 μg/mL holo-TF, 5 U/mL EPO and 100 U penicillin/streptomycin at 37°C under 5% CO₂ and 100% humidity. Fig 1 displays a flowchart of experimental design for cell culture, lentiviral transduction settings, and strategies for erythroid differentiation.