Directed differentiation of human pluripotent stem cells into the mesoderm (SSEA-5−KNA+) and ECFC (NRP-1+CD31+) lineages

To start differentiation, the hiPSC culture medium was switched to Stemline II medium (Sigma-Aldrich). After 2 days (day 0 of mesodermal differentiation), Activin-A (10 ng/ml; R&D Systems), FGF-2 (10 ng/ml; Stemgent), VEGF₁₆₅ (10 ng/ml; R&D Systems), and BMP4 (20 ng/ml; R&D Systems) were added to the medium for 24 hours to promote mesodermal differentiation. The next day, medium replaced with Stemline II medium containing FGF-2, VEGF₁₆₅, and BMP4. At day 3 of mesodermal differentiation, the medium was replaced with fresh Stemline II differentiation medium, and ECFC mesoderm cell sorting was performed at day 4 of mesodermal differentiation. KDR⁺NCAM⁺APLNR⁺, KDR⁺NCAM⁺APLNR⁻, and KDR⁺NCAM⁻APLNR⁻ mesoderm fractions ± SSEA-5 depletion were sorted using flow cytometry at day 4. Sorted cells (100,000 cells/75 cm²) from these fractions were each cultured in Matrigel-coated tissue culture flasks with fresh Stemline II medium supplemented with BMP4, VEGF₁₆₅, and FGF-2 (10 ng/ml) for additional 8 days (4 plus 8 days, total of 12 days) with fresh medium change every other day to examine for the emergence of NRP-1⁺CD31⁺ ECFCs as previously described (17).