Fig 2: 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.

Fig 3: MEG enhances definitive endoderm differentiation of hiPSCs and of variations of definitive endoderm induction conditions. (A) FACS analysis of SOX17 and FOXA2 in H9 hESCs cultured in mTeSR1 medium. Cells were stained with SOX17-APC and FOXA2-Alexa488 antibodies (SOX17-APC/FOXA2-Alexa488) or non-specific IgG isotype control antibodies conjugated with the same fluorophores (isotype control). Isotype IgG, when used as a gating control, gave rise to a high percentage of false-positive cells in the undifferentiated mTeSR1 culture (~50% APC+/Alexa488+ and ~98% Alexa488+ in the SOX17-APC/FOXA2-Alexa-488 stained sample). n > 10 independent experiments. (B) FACS analysis of SOX17 and FOXA2 in MMW2 hiPSCs cells induced to undergo definitive endoderm differentiation under AWS, MEG+AWS, and MEG->AWS conditions for 7 days. Cells were plated at 1.0 × 105 cells/cm2 for AWS and MEG+AWS conditions, and at 0.5 × 105 cells/cm2 for MEG->AWS condition. Cells cultured in mTeSR1 medium were used as a gating control. n = 2 independent experiments. (C) FACS analysis of SOX17 and FOXA2 in H9 hESCs induced to undergo definitive endoderm differentiation for 5 days in AW-B27, MEG+AW-B27, and MEG->AW-B27 conditions. B-27 supplement was used in these conditions instead of serum. Cells were plated at 1.0 × 105 cells/cm2. n > 3 independent experiments. (D) FACS analysis of SOX17 and FOXA2 in H9 hESCs induced to undergo definitive endoderm differentiation for 5 days in AW-ITS, MEG+AW-ITS, and MEG->AW-ITS conditions. ITS (insulin, transferrin, and selenium) supplement was used in these conditions instead of serum. Cells were plated at 2.0 × 105 cells/cm2. n = 2 independent experiments.

Fig 4: MEG functions at the onset of mesendoderm differentiation but shows no effect on Nodal and Wnt signaling. (A) FACS analysis of T and EOMES in H9 hESCs differentiated in MEG->A-BVF, A-BVF, and delayed MEG+A-BVF (with MEG added on day 1 instead of day 0) conditions. (B) FACS analysis of SOX17 and FOXA2 in H1 hESCs differentiated for 7 days in AWS condition and MEG+AWS condition with MEG added on day 0 or day 1. Cells were plated at 1.5 × 105 cells/cm2. (C) Western blotting of Phospho-SMAD2 (P-SMAD2) in H1 hESCs pre-treated with DMSO and MEG in mTeSR1 medium overnight, followed by 0.5 hr treatment of Activin A (10 ng/ml) in RPMI-1640 medium. SMAD2/3 was used as a loading control. n = 3 independent experiments. (D) Western blotting of Phospho-SMAD2 (P-SMAD2) in H1 hESCs untreated or pre-treated with DMSO and MEG (10 µM) in mTeSR1 medium overnight, followed by treatment of Activin A (10 ng/ml) in RPMI-1640 medium with DMSO and MEG for various lengths of time. SMAD2/3 was used as a loading control. (E) Western blotting of Phospho-ß-CATENIN (P-ß-CATENIN) in H1 hESCs untreated (Control) or treated with MEG at 10 µM and 25 µM in mTeSR1 medium overnight. ß-CATENIN was used as a loading control. n = 2 independent experiments.

Fig 5: H3K4me1 Deposition during Pancreatic Differentiation(A) Tornado plot and metapeak of the ATAC-seq stage-specific group show the profile of H3K4me1 and H3K27ac histone modifications in WT and FOXA2 KO cells. The maximum value (TPM) of the y axis is indicated in the plot. The WT signal is marked in black and the FOXA2 KO signal is in red in the tornado plots.(B) Primed PP1-specific ATAC-seq group was defined by excluding peaks that contained the H3K27ac signal at the DE or the FG stage (n = 1,136). See also Table S7. Metapeak analysis shows FOXA2, H3K4me1, ATAC-seq, and the H3K27ac signal of the primed PP1-specific group. WT is marked in black, and FOXA2 KO is in red. The maximum value (TPM) of the y axis is annotated in the plot.(C) Beeswarm plot to show the expression of 686 genes associated with the primed ATAC-seq group. Z scores of normalized counts are plotted, with each cell type averaged over replicates. Five WT samples, PP1 samples, and PP2 samples are grouped by themselves for comparison. See Figure S6C for the beeswarm plot of all cell types. The graph shows p values for significant increases in gene expression levels when comparing PP1 versus DE and PP2 versus DE using one-sided unpaired Wilcoxon rank-sum tests. There are also significant increases when comparing PP1 versus FG (p = 4.639 × 10-18), PP2 versus FG (p = 1.603 × 10-27), and PP2 versus PP1 (p = 6.329 × 10-6).See also Figure S6.