Induced Pluripotent Stem Cell Differentiation into Endothelial Cells

Induction of endothelial differentiation was carried out using an established protocol (Wu et al., 2015 (link); Wanjare et al., 2017 (link)). Briefly, the iPSCs were supplemented with Wnt agonist CHIR 99021 (5 μM, Selleck), bone morphogenetic protein-4 (25 ng/mL, Peprotech), B27 supplement (Gibco), and N2 supplement (Gibco). After 3 days, the cells were dissociated with HyQtase (Fisher Scientific) and plated at a density of 3.3 × 10⁴ cells/cm² in StemPro media (Gibco), supplemented with forskolin (5 μM, LC Labs), vascular endothelial growth factor (VEGF, 50 ng/mL, Peprotech), and polyvinyl alcohol (2 mg/mL, Sigma). After 7 days, the cells were washed twice with PBS, and then cultured in endothelial growth media (EGM-2MV, Lonza) supplemented with additional VEGF (100 ng/ml) for 7 more days. For indicated in vivo experiments, the human iECs derived from induced pluripotent stem cells were further lentivirally transduced between day 11–14 of differentiation using a double fusion reporter construct consisting of the ubiquitin promoter driving firefly luciferase (luc) and green fluorescence protein (GFP) (Huang et al., 2010 (link); Nakayama et al., 2015 (link)).