Human fgfb

hESC H1 cells were seeded from MEF condition into 12‐well plates and grown in Nutristem hESC XF (Biological Industries, 05‐100‐1A) on 2% Geltrex for 1–2 days until they reached 90% confluency. Some samples were transfected using Lipofectamine Stem Reagent as described above, untransfected samples were kept in fresh Nutristem. Twenty‐four hours after transfection (= Day0), endoderm differentiation was initiated. At day 0, pluripotency medium was replaced with endoderm base medium (RPMI1640 (Gibco, 11875093), 1% GlutaMAX™, 1% penicillin–streptomycin solution) supplemented with 2 μM CHIR 99021 (Reagent Direct, 27‐H76) and 100 ng/ml Activin A (home‐made). After 24 h_, medium was replaced with endoderm base medium supplemented with 100 ng/ml Activin A, 50 μg/ml Ascorbic Acid (Sigma‐Aldrich; A8960), and 5 ng/ml human FGFb (Peprotech; 100‐18B). Medium was replaced daily for 3 days using 1.5 ml per 12 well. On day 2 after endoderm induction, medium was supplemented with Doxycycline at a final concentration of 0.8 μg/ml for samples transfected with bidirectional reporter systems and some of the control samples.

As representative of mesenchymal stem cells, the human immortalized stem cell line SCP-1 was used [37 (link)]. SCP-1 cells were cultured in Minimal Essential Medium Alpha (MEM-α, HiMedia Laboratories, Thane West, Maharashtra, India), supplemented with 5% fetal calf serum (FCS, Thermo Fisher, Waltham, MA, USA). Human umbilical cord vein cells (HUVEC) were used as a proxy for endothelial cells. HUVECs were cultured in Endothelial Cell Growth Basal Medium 2 (EBM-2, Peprotech, Hamburg, Germany), supplemented with 2% FCS, 1% Antibiotic/Antimycotic (A/A, PAA Laboratories, Toronto, Canada), 0.5 ng/mL human VEGFA165, 10 ng/mL human FGF-b, 5 ng/mL human epidermal growth factor (EGF), 20 ng/mL human IGF-R3 (all Peprotech, Hamburg, Germany), 22.5 µg/mL heparin (Leo Pharma, Bellerup, Denmark), 0.2 µg/mL hydrocortisone (Pfizer, New York, NY, USA), and 1 µg/mL L-ascorbic acid (Sigma-Aldrich/Merck, Darmstadt, Germany) in culture flasks coated with 0.1% gelatin (Sigma-Aldrich/Merck, Darmstadt, Germany). The culture medium was replaced every 3–4 days. The cells were subcultured upon reaching confluency. SCP-1 cells in passages between 10 and 20 and HUVECs in passages between 5 and 10 were used for the experiments.

Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Growth Basal Medium 2 (EBM-2, Peprotech, Hamburg, Germany) supplemented with 2% FCS, 1% Antibiotic/Antimycotic (A/A, PAA Laboratories, Toronto, ON, Canada), 0.5 ng/mL human VEGFA165, 10 ng/mL human FGF-b, 5 ng/mL human Epidermal growth factor (EGF), 20 ng/mL hRIGF-R3 (all Peprotech, Hamburg, Germany), 22.5 µg/mL Heparin (Leo Pharma, Bellerup, Denmark), 0.2 µg/mL Hydrocortisone (Pfizer, New York, NY, USA), and 1 µg/mL L-ascorbic acid (Sigma-Aldrich/Merck, Darmstadt, Germany). HUVECs were cultured on 0.1% Gelatin (Sigma-Aldrich/Merck, Darmstadt, Germany)-coated flasks and subcultured when confluent. The medium was changed every 2 to 3 days. For experiments, cells were used in passages 7 to 10.