Figure 2: Distinct mesendodermal identities are manifested within 24 h of differentiation.

(a) Representative density plots for NCAM/EpCAM and (b) analysis of NCAM⁺ of eight independent experiments on day 3. *P<0.05, evaluated by one-way analysis of variance (ANOVA) with post hoc Bonferroni analysis. (c) Flow cytometric analysis for cKIT⁺/CXCR4⁺ of three independent experiments on day 3 (n=3 independent experiments). (d,e) Representative density plots showing MIXL1-GFP on day 1 in 2D monolayer (upper panel), 3D suspension (lower panel) and respective quantification in n=5 independent experiments. *P<0.001, evaluated by one-way ANOVA with post hoc Bonferroni analysis. (f,g) Representative density plots showing T-brachyury on day 1 from suspension-based differentiation and respective quantification (n=5 independent experiments). *P<0.05, evaluated by one-way ANOVA with post hoc Bonferroni analysis. (h) Principal component analysis of microarray data. Each dot represents an independent sample collected after 24 h of differentiation and undifferentiated hESCs. (i) Venn diagram of >2-fold regulated genes in the four conditions after 24 h compared with undifferentiated cells. (j) Top-ranked gene ontology terms without pre-selection⁵⁸ associated with >5-fold regulated gens in the four conditions. (k) Spatial allocation of the each cornerstone condition to the mouse epiblast (E7.0) based on zipcode mapping of whole-transcriptome data along the primitive streak. Red=high correlation; green=low correlation. (l) Heatmap of differentially regulated genes (P<0.001) associated with pluripotency, primitive streak, early endoderm, early mesoderm and neural plate border. (m,n) Correlation of MIXL1/T-brachyury and NANOG/CDX2 in CHIR-treated conditions. (o,p) Flow cytometric analysis of CDX2 and NANOG on day 1. n=5 and 3 of independent experiments, respectively. *P<0.01 compared with 7.5 μM conditions evaluated by one-way ANOVA with post hoc Bonferroni analysis. All bars shown in this figure represent mean±s.e.m. See also Supplementary Figs 2 and 3. NS, not significant.