Fig 1: MEG induces nearly homogeneous definitive endoderm differentiation in combination with the AWS condition. (A) Schematic overviews of definitive endoderm differentiation conditions used in this study. (B) Summary of FACS results showing the percentages of SOX17+FOXA2+ population acquired after 7 days of definitive endoderm differentiation of H1 and H9 hESCs under various plating densities and differentiation conditions. Results from six independent experiments (n = 6) are shown as mean ± SD; 3–5 data points were collected and analyzed for each condition. (C) FACS analysis of SOX17 and FOXA2 in H1 hESCs treated with definitive endoderm differentiation conditions including AWS, MEG+AWS, and MEG->AWS for 7 days. Cells were plated at 1.0 × 105 cells/cm2. (D) Immunofluorescent staining of SOX17 (red) and FOXA2 (green) in H9 hESCs induced to undergo definitive endoderm differentiation under the MEG->AWS condition for 1, 3, and 7 days. Nuclei were stained with DAPI (blue). Scale bar: 100 µm. (E) Western blotting of SOX17, FOXA2, CER1, and AFP in H9 hESCs induced to undergo definitive endoderm differentiation under AWS, MEG+AWS, and MEG->AWS conditions for 7 days. Cells cultured in mTeSR1 medium were used as a control (mTeSR1). Cells were plated at 0.25 × 105 cells/cm2 for the AWS condition, 1.0 × 105 cells/cm2 for the MEG+AWS condition, and 1.5 × 105 cells/cm2 for the MEG->AWS condition. a-TUBULIN was used as a loading control. (F) Quantitative-PCR analysis of endoderm markers in H9 hESCs under AWS and MEG->AWS conditions for 5 days. All values were normalized to the mRNA level (=1) in cells cultured in mTeSR1 medium. Each bar represents the mean ± SD (error bars). n = 2 independent experiments. ACTB (ß-actin) was used as a loading control.