Lineage-Specific Differentiation of hESCs

Lineage-specific differentiation of hESCs was conducted as previously described [19 (link)]. Briefly, hESCs were passaged to 12-well plates at a ratio of 1:12. Differentiation starts on day 2 after passaging for endoderm differentiation and starts on day 1 after passaging for all the other lineages. For trophoblast differentiation, cells were differentiated in E7 medium (E8 medium without FGF2) with 20 ng/ml BMP4 for 6 days with medium change every 2 days and then harvested on day 6 for RT-qPCR analysis of CGA, CGB, GCM1, GATA2 and TROP2. For mesoderm differentiation, cells were differentiated in E8 medium supplemented with 20 ng/ml BMP4 for 2 days and harvested on D2 for RT-qPCR analysis of TBXT and MIXL1 expression. For endoderm differentiation, cells were treated with 5 uM CHIR in E8 for 24 h and then changed to E8 medium supplemented with activin A (10 ng/ml) for 3 days. Expression of SOX17 and FOXA2 was analyzed by RT-qPCR. For ectoderm differentiation, cells were treated with E6 medium (E8 without FGF2 and TGFβ) with 10 μM SB431542 (Selleck) for 1 day and subsequently treated with 10 μM SB431542 and 100 nM LDN193189 (Selleck) in E6 medium for 4 days. Expression levels of PAX3 and PAX6 were examined by RT-qPCR.