Fibroblast growth factor 2 (fgf2)

The hESC (H1) and hiPSC (BC1, BC1-T, and JHUi181) lines were maintained on Matrigel-coated plates with E8 medium (Life Technologies) as described previously (23 (link)–25 (link)). For differentiation, single hPSCs were obtained for sequential EC-HC induction, as described previously (23 (link)–25 (link)) with minor modifications. Briefly, single hPSCs were plated at an optimized density at 6 × 10³ cells per well onto vitronectin (PeproTech)–coated 12-well plates in the STEMdiff APEL 2 Medium (STEMCELL Technologies) supplemented with 3 μM glycogen synthase kinase 3 inhibitor CHIR99021 [Applied Biological Materials (ABM)], activin A (2 ng/ml; PeproTech), BMP4 (10 ng/ml; PeproTech), or 10 μM rho kinase inhibitor Y-27632 (STEMCELL Technologies) on day 0. After 48 hours (day 2), the medium was changed to STEMdiff APEL Medium supplemented with vascular endothelial growth factor (VEGF; 40 ng/ml; PeproTech). On day 3, fibroblast growth factor 2 (FGF2; 40 ng/ml; 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 VEGF (40 ng/ml; PeproTech) and FGF2 (40 ng/ml; ABM) until day 6. Cultures were maintained at 37°C under normoxic (room air with 5% CO₂) or hypoxic (1% or 5% O₂) conditions as indicated. Where indicated, a MEK inhibitor (U0126; 3 μM; Selleck Chemicals) was included.