Vascular endothelial growth factor (vegf)

Single-cell suspensions of hPSCs were obtained by treating the hPSC cultures at 70%−80% confluency with TrypLE (Thermo Fisher Scientific). Single cells were plated at an optimized density at 6×10³ cells/well onto vitronectin (Peprotech)-coated 12-well plates in STEMdiff APEL Medium (STEMCELL Technologies) supplemented with 3 μM GSK3β inhibitor, CHIR99021 (abm Inc), 2 ng/ml ActivinA (Peprotech), 10 ng/ml BMP4 (Peprotech) and 10 μM Rho kinase inhibitor, Y-27632 (STEMCELL Technologies) on day 0. After 48 hr (day 2), the medium was changed to STEMdiff APEL Medium supplemented with 10 ng/ml VEGF (Peprotech). On day 3, additional 10 ng/ml FGF2 (abm) was added to the cultures without aspirating the old medium. From day 4, the medium was changed to STEMdiff APEL 2 Medium supplemented with 10 ng/ml VEGF and 10 ng/ml FGF2 until day 6. From day 6, the medium was changed to STEMdiff APEL 2 Medium supplemented with 10 ng/ml VEGF, 10 ng/ml FGF2, 50 ng/ml SCF (Peprotech), 50 ng/ml Flt-3L (Peprotech), 50 ng/ml TPO (Peprotech), 10 ng/ml IL-3 (Peprotech) and 10 ng/ml IL-6 (Peprotech). Differentiation was conducted in a 1%−5% hypoxic condition from day 0 to day 6. The TrypLE was used to dissociate and collect cells for analysis.

H1 hESCs were obtained from WiCell Research Institute (Madison, WI). H1 hESC line, the iETS1 H1 hESC line and the tdTomato H1 hESC lines were maintained on matrigel in mTeSR1 medium. Cells were passaged every 3–4 days using 0.5 mM EDTA in PBS. The hESC lines were differentiated on ColIV coating plate (Uenishi et al., 2014 (link)). Singular iETS1 hESCs were plated 10,000 cells/cm² to 6 well plates coated with ColIV (Sigma). After 1 day, media was changed to IF9S media with 50 ng/ml FGF (Peprotech), 50 ng/ml BMP4 (Peprotech), 15 ng/ml Activin A (Peprotech) and 2 mM LiCl (Sigma). On day 2, media was changed to IF9S media with 50 ng/ml FGF and 50 ng/ml VEGF (Peprotech). On day 4, media was changed to IF9S media with 50 ng/ml FGF, 50 ng/ml VEGF, 50 ng/ml TPO (Peprotech), 50 ng/ml SCF (Peprotech), 50 ng/ml IL-6 (Peprotech) and 10 ng/ml IL-3 (Peprotech). On day 6, IF9S media with cytokines the same as day 4 media was added to the culture.

For VEGF and NaHS treatment +/− Compound C (10μM) (Fig. 5D, Fig. S6A), HUVEC were pretreated for 1hr with 50ng/ml VEGF (Peprotech) or 100μM NaHS. Cells were then depleted in Krebs-Ringer Bicarbonate Buffer (KRB; NaH₂PO₄/Na₂HPO₄ 10mM, NaCl 136mM, KCl 4.7mM, MgSO4 1.25mM, CaCl2 1.25mM, pH7.4), without glucose and serum for 30min and then incubated for 6min in a solution containing 0.5μCi ³H-2DG. On ice, cells were then washed in cold PBS 3 times, lysed, and sample counted in a liquid scintillation counter. Samples were normalized to protein content as measured from the same cells by BCA. For remaining glucose uptake experiments (Fig. S5B, S6E, I), EC were treated with NaHS (100μM), KCN (10μM), Antimycin A (2.5μM), Oligomycin (2μM), or FCCP (1.5μM) for 45, 75 or 195min in EGM-2. When indicated, inhibitors were added 30min before the addition of the tracer. Fifteen min before the termination of the experiment, 0.4μCi of ³H-2DG was added to each well (1mL final volume, 12-well format). At the end of the incubation the plate was rapidly transferred on ice, media removed and washed 4 times with PBS + BSA 0.1%. Finally, cell lysis was performed with NaOH 0.2% + SDS 0.5%. Lysate (500uL) was mixed with scintillation fluid (5mL) and sample radioactivity measured in a scintillation counter (Beckman).