Differentiation of human iPSCs to endothelial cells

Human induced pluripotent stem cells (SBAD-02-01) were kindly supplied Dr Zameel Cader, University of Oxford. Expansion and differentiation of hiPSCs was done on Matrigel (BD Biosciences) coated 6-well plates (Nunc) in mTeSR1 medium (STEMCELL Technologies). Differentiation of SBAD-02-01 was performed as described [15 (link)]. Briefly, cells were thawed from the frozen vial and were expanded until 70% confluent on a Matrigel coated 6 well plate. After expansion, cells were passaged by use of accutase solution (Sigma-Aldrich) and seeded at 7.5x10³ hiPSC/cm² on Matrigel coated 6-well plates. Cells were further expanded in mTeSR1 medium. After achieving an optimal cell density of 2.5 x10⁴–3.5 x10⁴ hiPSC/cm², the cells were shifted to unconditioned media (UC) for 6 days. UC medium consisted of 78.5% DMEM/F-12 + 20% KnockOutTM Serum Replacement (Life Technologies) + 1% MEM NEAA (Sigma-Aldrich) + 1mM L-glutamine (Sigma-Aldrich) + 0.1 mM β-mercaptoethanol (Sigma-Aldrich). The medium was changed completely every day. Cells were then cultured for 2 days in human Endothelial-SFM (Life Technologies), containing 0.5% B27^TM (50x) (Thermo Fisher Scientific) supplemented with 20 ng/mL hbFGF (PeproTech) and 10 μM all-trans Retinoic Acid (Sigma-Aldrich), called EC medium + hbFGF + RA.