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. 2023 Aug 4;24(15):12423. doi: 10.3390/ijms241512423

Figure 4.

Figure 4

RNA expression profiling of BM- and TCM-exposed EBs. (A) Principal component analysis (PCA) of top 500 most highly expressed genes of D4 and D8 EBs grown in BM and TCM. The greatest variance is introduced by day of differentiation, as expected. However, principal component 2 (PC2) distinguishes BM from TCM effects and shows a greater divergence of samples at D4 compared to D8. (B) 3-dimensional PCA plot of D4 EBs based on germ layer lineage marker expression. Mesoderm markers separate TCM- from BM-grown EBs along PC2 which accounts for 34% of variance, whereas endoderm markers separate these samples only along PC3 with 1% variance. Ectoderm markers do not separate the samples. (C) Heatmaps of gene expression levels of selected germ layer lineage markers in D4 EBs cultured in TCM vs. BM. The greatest divergence is seen for mesoderm markers that are collectively and uniformly down-regulated in TCM conditions, as indicated by the brightest colour intensity differences compared to markers of the endoderm and ectoderm lineages.