Human erythropoietin

The c-Kit‒positive hCSCs were cultured in F12 Ham’s Medium (HyClone, #SH30026.01, GE Health care, Chicago IL, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin/streptomycin (P/S), 2 mM glutathione (Sigma–Aldrich, St. Louis, CA, USA), 10 ng/mL recombinant human basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, USA), and 0.005 unit/mL human erythropoietin (R&D Systems, Minneapolis, MN, USA) (designated hCSC medium). The culture was maintained at 37 °C in a humidified incubator with an atmosphere of 5% CO₂, and the medium was replaced every 2 days. To characterize hCSCs, cells were probed with human c-Kit, CD29, CD90, CD105, CD44, CD166, CD34, and CD45 antibodies (BD Biosciences, NJ, USA), and the expression of the relevant markers was analyzed employing a BD Accuri flow cytometer (BD Bioscience).

LCLs were cultured in upright non-adherent T25 tissue culture flasks (Nunc) at 37 °C and 5% CO₂. LCL media consisted of RPMI 1640 (Thermo Fisher, Waltham, MA, USA), supplemented with 1× GlutaMAX™ (Thermo Fisher), and 20% fetal bovine serum (Thermo Fisher). Erythroblasts were enriched in mixed PBL cultures following a previously published protocol [33 (link)]. Briefly, PBLs were cultured in erythroblast media containing StemSpan™ SFEM (Stem Cell Technologies, Vancouver, BC, Canada), 50 ng/mL recombinant human stem cell factor (R&D Systems, Minneapolis, MN, USA), 1 μM Dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL IGF1 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 ng/mL Interleukin 3 (Miltenyi Biotech), 2 U/mL human erythropoietin (R&D Systems), and 10 μg/mL Gentamicin (Thermo Fisher). Magnetic beads labelled with a CD71 antibody were then used to enrich for erythroblasts, following the manufacturer’s instructions (Miltenyi Biotec).

hCPCs were cultured in Ham’s F12 medium (Hyclone, GE Healthcare, Chicago, IL, USA) comprising 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin–streptomycin (Welgene, Daegu, Republic of Korea), 5 µg of recombinant human basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, USA), 2.5 U of human erythropoietin (R&D Systems, Minneapolis, MN, USA), and 2 mM glutathione (Sigma-Aldrich). Rapamycin (Sigma-Aldrich, St. Louis, MO, USA) treatment typically started at passage 7 for the experiments. Various concentrations (1 nM, 10 nM, and 100 nM) of Rapamycin were added to the hCPC medium and the medium was replaced every 2 days. A similar amount of dimethyl sulfoxide (DMSO) that was utilized to treat hCPCs was used as a control.