Rpmi and b 27 supplement minus insulin

iPSC-ECs were generated using a 2D monolayer differentiation protocol based on a modification of a previous protocol by Gu’s group (Gu, 2018 (link)). Briefly, iPSCs (over passage 25) were grown to 80% confluence and placed in differentiation medium (RPMI and B-27 supplement minus insulin, Life Technologies) toward the mesodermal lineage with 6 mM CHIR-99021 (Selleck Chemicals) for 2 days, followed by 3 mM CHIR-99021 for 2 days. The medium was then changed to a differentiation medium composed of RPMI-B27 without insulin and endothelial medium EGM2 (Lonza Group) supplemented with 20 ng/mL bone morphogenetic protein-4 (PeproTech), 50 ng/mL vascular endothelial growth factor (PeproTech), and 20 ng/mL basic fibroblast growth factor (BD Biosciences). Cells were subjected to medium change every 2 days. On day 12, iPSC-ECs were sorted for CD144+ using antibody-coated beads and a magnetic-activated cell sorter (Miltenyi Biotec) and expanded on 0.2% gelatin coated plates and maintained in EGM-2 BulletKit (Lonza Group) with 10 μM SB431542 (Selleck Chemicals), a TGF-β inhibitor at 37°C, 21% O₂, and 5% CO₂ in a humidified incubator with medium changes every 48 h. Cells were passaged once they reached 80%–90% confluence. iPSC-ECs used for experiments were between passages 3 and 10.