Dmem with high glucose

HEK293 cells were cultivated in DMEM with high glucose (PAA Laboratories, Cölbe, Germany) supplemented with 10% FBS (Life Technologies, Darmstadt, Germany), 2 mM L-glutamine (PAA Laboratories), and 1% penicillin/streptomycin (PAA Laboratories). BJ human foreskin fibroblasts (Stemgent, Cambridge, USA) were cultivated in DMEM with high glucose containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, and 30 mM HEPES (Life Technologies, Darmstadt, Germany). Human endothelial cells (ECs) were isolated as described previously [12 (link)] and cultivated in flasks precoated with 0.1% gelatin in Vasculife® EnGS EC culture medium (CellSystems, Troisdorf, Germany) containing VascuLife EnGS LifeFactors Kit, 50 mg/ml gentamicin, and 0.05 mg/ml amphotericin B (PAA Laboratories). Cells were kept at 37°C with 5% CO₂ and media was changed every 3 days. Cells were passaged using trypsin/EDTA (0.04%/0.03%, PromoCell, Heidelberg, Germany). BJ fibroblasts at passage 7-9, endothelial cells at passage 3-5 were used for all experiments.

H9 ESCs were obtained from WiCell as per agreement 15-W0341. H9 ESCs were cultured on Matrigel coated plates (corning #354277) in mTeSR 1 media (STEMCELL Technologies # 05857), washed with DMEM/F12 (GE Healthcare # SH3002302) and passaged with accutase (MP Biomedicals # 091000449) and ROCK Inhibitor Y-27632 (BD # 562822). For ACTIVIN A treatment, cells were grown to about 75–80% confluency. The cells were washed with PBS and 10 ml of RPMI-1640 (GE Healthcare # SH30027.01) was added with 1X B-27 supplement (Life Technologies # 17504–044) along with ACTIVIN A (Sigma # SRP3003) to a final concentration of 50 ng/ml. For CX-5461 treatment the cells were washed with PBS and received RPMI with B-27 as above with CX-5461 (Selleckchem # 52684) to a final concentration of 1 μM. The media was replaced daily with fresh reagents until cells were harvested. HEK293T cells were maintained in DMEM with high glucose (GE Healthcare # SH30243.01) with 10% FBS (GE Healthcare # SH30070.03) and passaged with 0.25% trypsin-EDTA (Gibco # 25200–056).