Iron saturated transferrin

In addition to the generated ES cell lines described above, E14, Bry/GFP-reporter25 (link), Eng^−/−10 (link) and iEng ES cells7 (link) were used in this study. ES cell lines were cultured on irradiated mouse embryonic fibroblasts in DMEM (Gibco) supplemented with 1,000 U ml⁻¹ LIF (Millipore), 15% inactivated fetal bovine serum (Gibco), 0.1 mM non-essential amino acids (Gibco) and 0.1 mM of β-mercaptoethanol (Sigma). For EB differentiation, ES cells were preplated for 30 min to remove mouse embryonic fibroblasts and then plated in hanging drops (100 cells per 10 μl drop) in EB differentiation medium, IMDM (Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco), 4.5 mM monothioglycerol (Sigma), 100 μg ml⁻¹ ascorbic acid (Sigma) and 200 μg ml⁻¹ iron-saturated transferrin (Sigma) in 150 mm Petri dishes. After 48 h, EBs were collected and resuspended into 10 cm Petri dishes in 10 ml of EB differentiation medium. These dishes were cultured on a slowly swirling table rotator (80 r.p.m.). To induce appropriate Eng expression during EB differentiation, doxycycline (Sigma) was added to the cultures, at 1 μg ml⁻¹, beginning at day 2, except for time window studies. In the studies involving iSmad1 ES cell lines, 3 μM of CHIR99021 or dimethylsulphoxide (vehicle) was added to EB cultures at day 3.5 and 24 h later, cells were collected for EryP CFC assay.