Zd6474

To initiate differentiation, ESCs were dissociated with TrypLE express (ThermoFisher Scientific) and cultured in a 3:1 mixture of IMDM (ThermoFisher Scientific Cat. 12440053) and Ham’s F12 (ThermoFisher Scientific Cat. 11765054) medium supplemented with 0.5x N-2 supplement, 0.5x B27 supplement, 1x penicillin-streptomycin, 2mM Glutamine, 0.5 mM ascorbic acid (MilliporeSigma Cat. A4544), 450 μM mono-thioglycerol, 0.05% BSA at the density of 0.1 million cells per mL in a 10-cm Petri dish (Becton Dickenson) for inducing embryoid bodies.^{24 (link),25 (link)} After 48 h, the embryoid bodies (EBs) were dissociated with TrypLE express. For mesoderm induction, the dissociated EBs were re-aggregated in the ABV regimen, defined as the StemPro-34 SFM medium (ThermoFisher Scientific Cat. 10639011) supplemented with 2mM Glutamine, 0.5 mM ascorbic acid, 450 μM mono-thioglycerol, 200 μg/mL human transferrin (MilliporeSigma Cat. T8158), 6 ng/mL human bFGF (R&D systems Cat. 233FB), 1 ng/mL human BMP4 (R&D systems Cat. 314BP), 8 ng/mL human Activin A (R&D systems Cat. 338AC), 5 ng/mL mouse VEGF (R&D systems Cat. 494MV). For mesoderm induction in the AB regimen, the culture medium without 5 ng/mL VEGF was used. FLK1 inhibitor, 1.2 μM ZD6474 (SelleckChem Cat. S1046) was treated from day 2 to day 3.75.