Clone 89106

Cells were dissociated with Accutase for 20min and resuspended in FACS buffer containing 1% BSA, 2mM EDTA, 30ug/mL DNAse I and Normocin in PBS. Cells were washed and incubated in FACS buffer with antibody for 30 minutes on ice at 4 degrees in the dark. Cells were washed and resuspended in FACS buffer and strained through 40uM caps to eliminate cell clumps. Data collection was done on a BD-LSRII and FACSDiva v8. Gating (Fig. S2) and subsequent analysis was done using FlowJo software v10.5.3. FACS antibodies used include KDR-PE – R&D, FAB357P, Clone 89106 at 1:60, CD235A-APC – BD Biosciences at 1:100, 551336, Clone GA-R2, CD41-Apc/Cy7 – Biolegend, 303715, Clone HIP8, CD43-PerCP/Cy5.5 -BD Biosciences, 563521, Clone 1G10, CD45-FITC – BD Biosciences, 560976, Clone HI3O, CX3CR1-PE – Biolegend, 341604, 2A9–1, and Cd11b-APC/Cy7 – Biolegend, 301351, ICRF44 all at 5ul/100ul test.

iPSC-ECs were harvested with TrypLE and stained with either isotype control or antigen-specific antibodies. Cell-surface antigen expression was analyzed using fluorescently conjugated antigen-specific antibodies against VEGFR2/KDR (R&D Systems, clone 89106), CD31 (BD Biosciences, clone WM59), CD105 (eBiosciences, clone SN6) and CD144 (eBiosciences, clone 16B1). The cells were also tested for contaminating iPSCs using an antibody against TRA-1-81 to confirm a negative result (Stemgent, San Diego, CA). Internal antigen expression was analyzed using methanol fixed cells permeabilized with PBS + 0.1% saponin/BSA. Unconjugated polyclonal rabbit anti-human von Willebrand Factor (Dako, Carpinteria, CA) was detected with an Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR). Cells were analyzed on an Accuri flow cytometer (BD Biosciences) using propidium iodide, when appropriate, to exclude dead cells. iPSC-ECs exhibited a purity of ≥ 90% as measured by high coexpression of CD31 with CD105 or CD144 by flow cytometry.