Erythroid Differentiation of CD34+ HSPCs

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.