Human stem cell factor scf

Differentiation of hESCs into HPS was carried out using an EB-based method, as previously described [24 (link)]. hESCs were seeded in a 96-well round-bottom plate (SPL Life Sciences, Republic of Korea) at a density of 1 × 10⁴ cells per well using APELL II (Stemcell Technologies, Canada) containing 40 ng/mL human Stem Cell Factor (SCF; Miltenyi Biotec, Cologne, Germany), 20 ng/mL human Vascular Endothelial Growth Factor (VEGF; R&D systems, Minneapolis, MN, USA), 20 ng/mL human Bone Morphogenetic Protein 4 (BMP-4; Miltenyi Biotec, Germany), and 10 μM Y-27632 for 11 days. The medium was replaced with fresh differentiation medium without Y-27632 and incubated for 18 h.

CD34⁺ progenitor cells were isolated by positive selection kit (MiltenyiBiotec) using immunomagnetic beads cell‐sorting system (AutoMACS; MiltenyiBiotec) from healthy leukapheresis samples (from allogenic stem cell transplantation donors), in agreement with our institutional ethics committee. CD34⁺ cells were cultivated as recently described²³ from day (D)1 to D6 in Iscove's modified Dulbecco's medium (IMDM) (Biochrom) supplemented with glutamine (Eurobio), containing 2% human AB serum (H2B), 100 IU/ml penicillin (Eurobio), 100 μg/ml streptomycin (Eurobio), 500 μg/ml human holo‐transferrin (Sigma‐Aldrich), 10 μg/ml recombinant human insulin (Sigma‐Aldrich), 2 IU/ml heparin (Braun), 100 ng/ml human stem cell factor (SCF) (MiltenyiBiotec) and 3 IU/ml erythropoietin (EPO) (Roche). From D7 to D20, cells were culture in the same medium but after SCF removal. UT7/EPO cells were maintained in α‐minimum essential medium (MEM) (Dominique Dutscher) supplemented with 10% FCS (Eurobio) and 2 IU/ml EPO as recently described.²⁴