To identify transplanted hiPSC-ECs, a primary antibody specifically against human CD31 (hCD31, mouse anti-human CD31-Biotin) was used and visualized by mouse anti-Biotin-VioBright 515 (both from Miltenyi Biotec, Germany). Fluorescence images were taken with an Olympus IX71 fluorescence microscope.
Neovascularization in ischemic limb was determined as described (Su et al., 2018 ). Cryosections were stained for CD31 expression (rabbit anti-CD31, Abcam, United States), which targets both human and mouse ECs), to evaluate total vessel density, and smooth muscle actin (SMA) expression (Cy3-conjugated mouse anti-SMA antibodies, Sigma-Aldrich), which targets SMCs, to evaluate arteriole density. Vascular structures that were positive for CD31 expression and for both CD31 and SMA expression were counted for all animals in each group.
Neovascularization in ischemic limb was determined as described (Su et al., 2018 ). Cryosections were stained for CD31 expression (rabbit anti-CD31, Abcam, United States), which targets both human and mouse ECs), to evaluate total vessel density, and smooth muscle actin (SMA) expression (Cy3-conjugated mouse anti-SMA antibodies, Sigma-Aldrich), which targets SMCs, to evaluate arteriole density. Vascular structures that were positive for CD31 expression and for both CD31 and SMA expression were counted for all animals in each group.