Mesoderm in vitro induction was performed as previously reported (26 (link)). 1.5 × 105 ESCs were seeded per well in ultra-low attachment surface six-well plate (Corning) in modified serum-free differentiation (SFD) medium for 2 days and small EBs were formed. The SFD medium is consisted of IMDM (Gibco, 12440053) and Ham-F12 (Cellgro, 10-080-CVR), supplemented with B27 (Gibco, 12587-010) and N2 supplements (Gibco, 17502-048), Glutamine (Gibco, 25030-081), 10% bovine serum albumin (Invitrogen, P2489), Ascorbic acid (Sigma-Aldrich, A4544), and MTG (Sigma-Aldrich, M6145). To direct mesoderm differentiation, EBs were dissociated into single cells by trypsin and 1.5 × 105 cells were seeded per well in ultra-low attachment surface six-well plate (Corning) in SFD medium containing 5ng/ml Activin A (R&D, 338-AC-010), 5ng/ml VEGF (R&D, 293-VE-010), 0.5 ng/ml BMP4 (Stemgent, 03-0007) for 48 h. To trace the middle stage of cell differentiation, 24 and 48 h samples were collected for analysis.
Sun X., Ren Z., Cun Y., Zhao C., Huang X., Zhou J., Hu R., Su X., Ji L., Li P., Mak K.L., Gao F., Yang Y., Xu H., Ding J., Cao N., Li S., Zhang W., Lan P., Sun H., Wang J, & Yuan P. (2020). Hippo-YAP signaling controls lineage differentiation of mouse embryonic stem cells through modulating the formation of super-enhancers. Nucleic Acids Research, 48(13), 7182-7196.
+ Open protocol