Fig. 1

Schematic diagram of the protocols to differentiate mESCs to cardiovascular lineage. A Monolayer differentiation of mESCs to cardiomyocyte lineage. The mESCs were cultured in mESC growth medium (MGM) for three days (day − 3 to 0). On day 0, the medium was replaced with RPMI/B27-insulin as the serum-free differentiation medium (SFDM). Cells were treated with ascorbic acid (AA) from day 0, which continued until the end of the differentiation protocol (day 14). From days 3–5, cells were treated Wnt inhibitor (Wi), and from days 6–9, they were treated with retinoic acid inhibitor (RAi). B Three-dimensional differentiation of mESCs by EB method. EBs were generated by the hanging drop technique. On day 0, mESCs were dissociated, counted, and pipetted as drops of media on the inner side of a petri dish lid. The lid was inverted, and the petri dish was closed to form hanging drops. On day 2, spherical EBs was formed. On day 3, EBs were flushed with SFDM and cultured in suspension in an uncoated petri dish. On day 5, EBs were transferred to a 24-well plate containing 0.1% gelatin-coated coverslips. From day 5 to 30, EBs were cultured and differentiated in a monolayer. From day 6–9, cells were treated with RAi.