Matrigel coated plate

The generation of hiPSCs from a healthy control donor was performed as previously described [40 ]. Peripheral blood mononuclear cells from healthy control donor were collected for iPSC induction. Cells were transduced with the integration-free CytoTune-iPS Sendai Reprogramming Kit (Life Technologies, Carlsbad, CA, USA), which utilizes Sendai virus particles of the four factors (OCT4, SOX2, c-MYC, and KLF4). Transduced cells were plated on vitronectin-coated culture dishes and fed iPSC medium, which was replaced by StemPro 34 SFM (Life Technologies) from days 3 to 7. On day 7, the medium was replaced by feeder-free mTeSR1medium (STEMCELL Technologies, Vancouver, BC, Canada) until small colonies were formed. The growth of small colonies was maintained for another 3–4 weeks, and cell colonies were manually picked and mechanically dissociated for the first four passages. The hiPSCs were maintained on Matrigel-coated plates (BD Bioscience, Franklin Lakes, NJ, USA) with mTeSR1 medium (STEMCELL Technologies), and passaged every 4–5 days using 1 mg/mL dispase (Life Technologies). All experimental protocols including human stem cell use were approved by the Ethics Committee at the First Affiliated Hospital of Sun Yat-sen University.

Serum-starved EPCs were seeded onto Matrigel-coated plates (BD Biosciences, USA) in EBM medium and incubated at 37°C for 24 h, and tubular structures of EPCs in the Matrigel were analyzed by phase-contrast microscopy. To quantify the length of newly formed tubes, 6 random phase-contrast photomicrographs per well were taken and the length of each tube was measured using Quantity One Image software.

A schematic representation of differentiation protocol is presented in Figure 1A. In brief, hESC and hiPSC were seeded at 1.7 × 10⁴ cells per square centimeter 30 on BD Matrigel‐coated plates and kept in mTeSR1 medium for two days. Rho kinase inhibitor, Y27632 (Chemdea, NJ, USA) [10 µM) was added to the mTeSR1 medium for the first 24 hours. Eight differentiation induction media listed in Figure 1B were introduced to the cells according to the respective groups on day 3 and maintained until day 9. The cells were then replated at 1.7 × 10⁴ cells per square centimeter onto 0.05 mg/ml collagen‐IV–coated plates 22 on day 9 and supplemented with CnT‐Prime medium (CELLnTEC, Switzerland) and 10% serum for the next 11 days. Three technical and three biological repeats were set up for each differentiation group.