Figure 4

Change in pluripotent (A), mesoderm (B), ectoderm (C), and endoderm (D) gene expression during spontaneous differentiation of ESD3 embryonic stem cells due to changes in substrate stiffness. For pluripotent markers (Rex1, Oct4, and Sox2) there was either down regulation or no upregulation of gene expression after 5 days of differentiation. FGF8 (B, red line) was the only mesoderm marker that demonstrated any level of upregulation. The primitive ectoderm marker FGF5 (C, red line) was dramatically upregulated when compared to undifferentiated cells, however, this increase was notable for all substrate stiffnesses. The endoderm genes (D) CXCR4 and AFP demonstrated a strong upregulation in relation to the alginate substrates, while SOX17 and FOXA2 did not.