Anti cd15 mab coated magnetic microbeads

To initiate the differentiation of mouse iPSCs to ECs, medium was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 μM CHIR-99021 (Selleckchem). Starting on day 2, RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 2 μM CHIR-99021 (Selleckchem) was used. From day 4 to day 7, cells were exposed to RPMI-1640 EC medium containing 2% B-27 minus insulin plus 50 ng ml⁻¹ of mouse vascular endothelial growth factor (mVEGF, R&D Systems), 10 ng ml⁻¹ of mouse fibroblast growth factor basic (mFGFb, R&D Systems), 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). EC clusters were visible from day 7, and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell) plus supplements, 10% FCS heat-inactivated (HI) (Gibco), 1% penicillin–streptomycin, 25 ng ml⁻¹ of VEGF, 2 ng ml⁻¹ of FGFb, 10 μM Y-27632 (Sigma-Aldrich) and 1 μM SB 431542 (Sigma-Aldrich). The differentiation process was completed after 21 days, and undifferentiated cells were detached during the differentiation process. For purification, cells went through MACS purification using anti-CD15 mAb-coated magnetic microbeads (Miltenyi) for negative selection.

Differentiation into iCMs was performed as described in detail previously (3 (link)). Beating cells developed around days 11 to 14. Cells then underwent MACS purification using negative selection with anti-CD15 mAb-coated magnetic microbeads (Miltenyi). IF staining was performed using primary antibodies against alpha-sarcomeric actinin (EA-53, Abcam) and Troponin I (ab47003, Abcam) followed by the corresponding secondary antibodies conjugated with AF488 or AF555 (Invitrogen). The following primers were Gata4: (forward) 5′-CTG​TCA​TCT​CAC​TAT​GGG​CA-3′, (reverse) 5′-CCA​AGT​CCG​AGC​AGG​AAT​TT-3′; Myh6: (forward) 5′-ATC​ATT​CCC​AAC-​GAG​CGA​AAG-3′, (reverse) 5′-AAG​TCC​CCA​TAG​AGA​ATG​CGG-3′. All other primers were included in the Mouse ES/iPS Cell Pluripotency RT-PCR Kit (ASK-6001, Applied StemCell).

Mouse iPSCs were plated on gelatin in 6-well plates and maintained in iPSC media until they reached 60% confluency. Differentiation into iECs was performed as described in detail previously (3 (link)). Cells after differentiation underwent magnetic-activated cell sorting (MACS) purification using negative selection with anti-CD15 mAb-coated magnetic microbeads (Miltenyi). The iEC phenotype was confirmed by immunofluorescence (IF) for expression of Cd31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Biotechnology) with secondary antibodies conjugated with AF488 or AF555 (Invitrogen). For the tube formation assay, 2.5 × 10⁵ iECs were stained with 5 µM carboxyfluorescein succinimidyl ester (CFSE) and 0.1 μg/mL Hoechst (both Thermo Fisher) for 10 min at room temperature and plated on 10 mg/mL undiluted Matrigel (356231, Corning) in 24-well plates. After 48 h, tube formations were visualized by IF. PCR was performed using primers VE-cadherin (Cdh5): (forward) 5′-GGA​TGC​AGA​GGC​TCA​CAG​AG-3′, (reverse) 5′-CTG​GCG​GTT​CAC​GTT​GGA​CT-3′. All other primers were included in the Mouse ES/iPS Cell Pluripotency RT-PCR Kit (ASK-6001, Applied StemCell).