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semisolid colony-forming serum-free medium (CF-SFM) containing 40% ES-Cultā¢ M3120 methylcellulose (2.5% solution in IMDM; Stem Cell Technologies, Vancouver, BC, Canada), 25% StemSpanā¢ serum-free expansion medium (SFEM; Stem Cell Technologies), 25% human endothelial serum-free medium (ESFM; Invitrogen), 10% BIT 9500 supplement (Stem Cell Technologies), GlutaMAXā¢ (1/100 dilution; Invitrogen), Ex-CyteĀ® supplement (1/1000 dilution; Millipore, Billerica, MA), 100 Ī¼M MTG, 50 Ī¼g/ml ascorbic acid and 20 ng/ml basic fibroblast growth factor (FGF2) |
human PDGF-BB (10 ng/ml), VEGF (20 ng/ml), stem cell factor (SCF; 50 ng/ml), epidermal growth factor (EGF; 20 ng/ml), insulin-like growth factor 1 (IGF1; 50 ng/ml), hepatocyte growth factor (HGF; 20 ng/ml), activin A (1 ng/ml), transforming growth factor Ī²1 (TGF-Ī²1; 1 ng/ml), bone-morphogenic protein-4 (BMP-4; 5 ng/ml), apelin-12 (100 ng/ml; Phoenix Pharmaceuticals Inc), or anti-APLNR monoclonal antibody (5 Ī¼g/ml; R&D Systems) were added to cultures |