Ms macs separation column

iECs were isolated and expanded as described previously.^[66, ^67] In brief, CD31‐expressing cells were isolated via magnetic‐activated cell sorting (MACS; Miltenyi Biotec Bergisch Gladbach), following the manufacturer's protocol, on day 8 of differentiation. After rinsing the cells with 1x phosphate‐buffered saline (PBS; Thermo Fisher Scientific), cells were trypsinized with TrypLE Express and resuspended in MACS buffer (0.5 Ethylenediaminetetraacetic acid [MilliporeSigma] and 0.5% Bovine Serum Albumin (BSA; [MilliporeSigma] in PBS). After resuspension, cells were incubated with 10 µL of PE‐conjugated anti‐human CD31 (BD Biosciences) for 10 min at 4 °C. To remove the unbound primary antibody, cells were washed twice with MACS buffer. After washing, cells were resuspended in 80 µL MACS buffer, and 20 µL of anti‐PE microbeads (Miltenyi Biotec Bergisch Gladbach) were added to the cell suspension. Cells were incubated for an additional 15 min at 4 °C, followed by a washing step with MACS buffer before separation using the MS MACS separation column (Miltenyi Biotec Bergisch Gladbach). Finally, CD31⁺ cells were seeded on collagen type I‐coated plates and maintained in ECGM supplemented with 50 ng mL⁻¹ VEGF and 10 × 10⁻⁶ m SB‐431542.

CD31 expressing cells were isolated on day 8 of differentiation via magnetically activated cell sorting (MACS; Miltenyi Biotec Bergisch Gladbach, Germany) following the manufacturer’s protocol. After washing with 1× phosphate-buffered saline (PBS, ThermoFisher Scientific), cells were harvested with TrypLE Express and resuspended in MACS buffer (0.5 EDTA and 0.5% BSA in PBS). Cells were then incubated with 10 μl of PE-conjugated anti-human CD31 (BD Biosciences, San Jose, CA) for 10 min at 4 °C. After incubation, the unbound primary antibody was removed by washing with MACS buffer twice. Next, 20 μl of anti-PE microbeads (Miltenyi Biotec Bergisch Gladbach, Germany) were added to 80 μl of cells suspended in MACS buffer and incubated for an additional 15 min at 4 °C. Cells were washed with MACS buffer and separated using the MS MACS separation column (Miltenyi Biotec). Following separation, CD31 and VECAD enrichment were confirmed using flow cytometry as previously^{71 (link)} and detailed below in a separate section. Finally, CD31+ cells were seeded on type I collagen-coated plates and maintained in EC differentiation media. For all experiments, media was switched to ECGM without growth factor supplementation for 24 h unless otherwise noted. All experiments used iECs between passages 1 and 3.