Interleukin 3 (il 3)

CD34⁺ HSPCs were isolated using a magnetic bead-based positive selection kit (StemCell Technologies, Vancouver, BC, Canada) from the PBMNCs of a hematopoietic stem cell donor. The purified CD34⁺ cells were cultured in HSPC expansion medium composed of StemPro-34 SFM (Thermo Fisher Scientific, Grand Island, NY, USA) containing 100 ng/mL SCF, 100 ng/mL FLT3-Ligand (FLT3-L) (ImmunoTools GmbH), 20 ng/mL IL-6 (ImmunoTools GmbH), 20 ng/mL IL-3, 100 U/mL penicillin, and 100 μg/mL streptomycin. Two days after initiating the culture, the cells were transduced with TRE-HEE-UbC-hKO1-rtTA lentiviruses by spinfection at 2250 rpm for 1.5 h. After four days, the cells were transferred to EPM containing 1 µg/mL of dox. Throughout the culture, the cells were maintained at a density of 5–6 × 10⁵ cells per mL of the medium at 37 °C, 5% CO_2, with complete medium change on alternative days.

PBMNCs were isolated from normal healthy donors using Ficoll-Paque (GE Healthcare, Uppsala, Sweden). For the expansion of EPCs, PBMNCs were cultured for 24 h in the erythroid progenitor medium (EPM) containing StemSpan SFEM-II (StemCell Technologies, Vancouver, BC, Canada) supplemented with 3 U/mL erythropoietin (Epo) (Peprotech Inc., Rocky Hill, NJ, USA), 50 ng/mL stem cell factor (SCF) (ImmunoTools GmbH, Friesoythe, Lower Saxony, Germany), 10 ng/mL interleukin-3 (IL-3) (ImmunoTools GmbH), 40 ng/mL insulin growth factor-1 (IGF-1) (ImmunoTools GmbH), 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. About two million cells were transduced with TRE-HEE-UbC-hKO1-rtTA lentiviruses by spinfection at 2250 rpm for 1.5 h and were cultured in EPM, with half medium change on every alternative day. From the fourth day, the cells were supplemented with 1 μg/mL doxycycline (dox) (Sigma-Aldrich, St. Louis, MO, USA). Throughout the culture, the cells were maintained at a density of 5–6 × 10⁵ cells/mL at 37 °C, 5% CO₂ with complete medium change on alternative days.

For induction of the erythroid differentiation of iEPCs, a previously described protocol was used with minor modifications [33 (link)]. Briefly, iEPCs cultured in EPM were seeded at a density of 2 × 10⁵ cells/mL in erythroid differentiation medium I (EDM I) for two days. EDM I consisted of Iscove’s Modified Dulbecco’s Medium (IMDM) containing Glutamax (ThermoFisher Scientific, Grand Island, NY, USA), 3% human AB serum (MP Biomedicals, Solon, OH, USA), 2% Fetal Bovine Serum (ThermoFisher Scientific, Grand Island, NY, USA), 200 µg/mL holotransferrin (Sigma-Aldrich, St. Louis, MO, USA), 3 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), 10 μg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 3 U/mL Epo (Peprotech Inc.), 10 ng/mL SCF (Immunotools GmbH), 1 ng/mL IL-3 (Immunotools GmbH), 1 µg/mL dox, 100 U/mL penicillin, and 100 μg/mL streptomycin. On day 2, the cells were reseeded at a density of 3 × 10⁵ cells/mL in EDM-I and cultured for two days. On day 4, the cells were seeded at a density of 5 × 10⁵ cells/mL in EDM-II (EDM I without dox) and cultured for two days. On day 6, the cells were seeded at a density of 1 × 10⁶ cells/mL in EDM-III (EDM II with 500 µg/mL holotransferrin) for two days, and on day 8, the cells were reseeded at 1 × 10⁶ cells/mL in EDM-IV (EDM III without SCF and IL-3) until the end of differentiation with medium change on every alternative day.