Differentiation of Embryonic Lineages

Differentiation to specific embryonic lineages was performed in CDM-PVA, which has the same composition as CDM-BSA, with polyvinyl alcohol (PVA, 1 mg/ml, Sigma) instead of BSA. For early mesoderm differentiation, cells were grown in CDM-PVA with FGF2 (20 ng/ml), LY294002 (10 μM, Sigma) and BMP4 (10 ng/ml, R&D) for 36 h then treated with CDM-PVA with FGF2 (20 ng/ml) and BMP4 (50 ng/ml) for 3.5 days to generate LM as previously described (Cheung et al., 2012 (link)). To generate epicardium, LM cells were differentiated in CDM-PVA with BMP4 (50 ng/ml), recombinant human WNT3A (25 ng/ml, R&D systems) and RA (4 μM, Sigma) at a seeding density of 2.5×10³/cm² for 10 days. To investigate the role of WNT pathway, LM cells were differentiated with IWP2 (2 μM, Tocris) along with BMP4 and RA. Subsequently, epicardial cells were re-suspended in CDM-PVA with PDGF-BB (10 ng/ml, Peprotech) and TGFβ1 (2 ng/ml, Peprotech), designated as PT for 12 days. Epicardial cells were differentiated with PT in the presence of Y27632 (2 μg/ml, Calbiochem) to study the role of RhoA/RhoK in EPI-SMC differentiation. To generate EPI-CFs, epicardial cells were differentiated in CDM-PVA with VEGF (50 ng/ml, Peprotech) and FGF2 (50 ng/ml) for 12 days. HESCs were grown in CDM-PVA with FGF2 (12 ng/ml) and SB431542 (10 μM, Tocris) for 7 days to generate neuroectoderm (Vallier et al., 2009 (link)).