Collagen 4

96-well plates were coated overnight at 4°C with vitronectin (1 μg/cm²; R&D Systems), fibronectin (10 μg/cm²; R&D Systems), Matrigel (5 μg/cm²; BD Biosicences) or collagen IV (10 μg/cm²; R&D Systems) and then blocked for 1 h with 0.5% bovine serum albumin. Cells were seeded at a density of 4 × 10⁴ cells/well and incubated at 37°C for 1.5 h. Non-adherent cells were removed by washing with PBS, and adherent cells were fixed with cold methanol and stained with 0.1% crystal violet for 25 min at room temperature. After removing the crystal violet solution, the stained cells were washed with water and 10% acetic acid was added to dissolve the crystal violet. Absorbances were measured at 590 nm using a microplate spectrophotometer.

Four different clones of human iPS Cells were differentiated using StemPro‐34 SFM serum free media (Thermo Fisher Scientific) supplemented with BMP4 (Thermo Fisher Scientific), Activin A (R&D), fibroblast growth factor (FGF) (Miltenyi Biotec, Germany), and vascular endothelial growth factor (VEGF) (Thermo Fisher Scientific) for 5 days. The differentiated cells were seeded on collagen IV (R&D), while CD144 positive cells were magnetically sorted on day 6 using MicroBeads Kit (Miltenyi BIotec) and cultured in EGM‐2 media (LONZA) (iPS‐ECs). FSTL3 was overexpressed or knocked down by transfection or lentiviral gene transfer in iPS‐ECs and the cells were harvested 2–3 days later for further analysis or used for in vivo angiogenesis and hind limb ischemia assays. Equal cell numbers were used between control and treated conditions in all experiments in this study.

We coated 96-well plates overnight at 4 °C with recombinant Npnt (5 μg/ml, R&D Systems), collagen I (10 μg/ml, Nippi), collagen IV (10 μg/ml, Nippi), laminin I (10 μg/ml, Nippi), and fibronectin (10 μg/ml, Nippi), then they were washed with PBS and blocked with 3% bovine serum albumin for 1 hour at 37 °C. M3H1 cells were then plated at a concentration of 2 × 10⁴ cells/well and cultured for 48 hours. Sox2+ cells were determined by immunohistochemistry using an anti-Sox2 antibody. Total cells were counted using DAPI, then the ratio of Sox2+ cells was determined under a fluorescence microscope.