Figure 8. Epiblast stem cells undergo definitive endoderm differentiation in the presence or absence of BMP.

(A) Epiblast stem cells (EpiSCs) of the EpiSC9 line (Najm et al., 2011) were cultured in the presence of 12 ng/ml FGF2 and 20 ng/ml ACTIVIN A (F/A) on fibronectin. EpiSCs were then plated overnight onto Laminin-coated micropatterns (−24 hr) in N2B27 medium with F/A. Various conditions were used for further differentiation - F/A, BMP4, WNT3A (+BMP) or F/A, WNT3A (-BMP). Colonies were analyzed after 72 hr differentiation. (B) MIPs of immunostained 72 hr colonies. Scale bars, 100 μm. (C) Quantification of immunostaining. Voxel fluorescence intensity was measured from colony center (0) to edge (500). Data represents average voxel intensity across multiple colonies (n = 10/condition) and is shown relative to maximum voxel intensity for each marker across both conditions. (D) Graphs showing the expression level of a number of genes from the published microarray dataset of Hayashi et al. from E5.75 in vivo epiblast, EpiLCs and EpiSCs (Hayashi et al., 2011). Data shown is from amplified RNA samples and represents the mean ± S.D for two independent replicates.