Erythropoietin epo

All experiments proceeded under authorization from the Institutional Review Board for Human Research at the CHA University (1044308‐202204‐LR‐023‐02) for CHA52 and Konkuk University (KUH1280081 for H1 and H9). Undifferentiated human PSCs (CHA52, H1, H9 embryonic stem cell line, PSCs) were seeded onto Matrigel®‐coated plates with mTeSR™1 medium (85850; StemCell Technologies Inc.) to differentiate HE from PSCs. The cells were incubated with APEL™ 2 medium supplemented with 3 nM − 1.5 μM CHIR‐99021 (S2924, Selleck Chemicals), 20 ng/ml of vascular endothelial growth factor (VEGF)₁₆₅ (100‐20; PeproTech Inc.), and 25 ng/ml HumanKine® BMP‐4 (HZ‐1045; Proteintech Group Inc.) for 3 days to induce mesoderm.²¹ Based on previous, to induce and expand the HE, cells were then incubated with APEL™ 2 medium supplemented with 25 ng/ml HumanKine® BMP‐4 (HZ‐1045, Proteintech), and 250 ng/ml stem cell factor (SCF; 300‐07), 20 ng/ml VEGF₁₆₅ (100‐20), 200 ng/ml FMS‐like tyrosine kinase 3 (Flt3)‐Ligand (300‐19), 100 ng/ml Thrombopoietin (TPO) (300‐18) and 20 ng/ml erythropoietin (EPO) (100‐64; all from PeproTech) as described previous papers.¹², ¹⁷, ¹⁸, ²², ²³, ²⁴, ²⁵, ²⁶ We isolated CD34⁺ cells from PSC‐derived HE by magnetic‐activated cell sorting (MACS) using CD34 MicroBead Kits (Miltenyi Biotec) as described by the manufacturer.²⁷

CD34⁺ HSPCs were expanded for 5 days in HEM and were subsequently differentiated to the erythroid lineage using the erythroid progenitor medium (EPM) containing StemSpan SFEM-II medium supplemented with 50 ng/ml SCF, 3 U/ml erythropoietin (Epo), 20 ng/ml IL-3, and 40 ng/ml insulin growth factor-1 (IGF-1) (all the cytokines and growth factors are purchased from Peprotech, Rocky Hill, NJ, USA), for 10–12 days. A previously described protocol was used with minor modifications to induce terminal erythroid differentiation^{28 (link)}. Briefly, cultured erythroid cells were seeded at a density of 5 × 10⁵ cells/ml in erythroid differentiation medium-I (CD34-EDM-I) consisting of IMDM with Glutamax (ThermoFisher Scientific), 5% human AB serum (Sigma-Aldrich), 2 IU/ml heparin (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), 5 U/ml Epo (Peprotech), 500 μg/ml holotransferrin (Sigma-Aldrich) and 1 μM mifepristone (Sigma-Aldrich). On day 4, the cells were seeded at a density of 1 × 10⁶ cells/ml in CD34-EDM-II, which consisted of CD34-EDM-I without SCF, and were cultured with the medium change every other day until the end of differentiation. Total bone marrow (BM) cells isolated from NSG and NBSGW mice were also cultured using this four-step protocol described above for HSPC expansion and erythroid differentiation.