The LARGE Principle in AFiPSCs Figure 2. Pluripotency and in vitro and in vivo differentiation of AFiPSCs. (A) Microarray-derived gene expression levels of pluripotency- associated genes. Significantly up-regulated gene expression in AFiPSCs compared to AFCs is indicated by asterisks (*) FDR-adjusted P-Value ,0.05, **) FDR-adjusted P-Value ,0.01, ***) FDR-adjusted P-Value ,0.001). The heatmap is colored by LOG2 average expression signals according to the color key on the bottom. (B) qRT-PCR for most commonly used pluripotency genes in AFiPSCs and ESC line H1. Bars and error bars represent LOG2 ratios (AFiPSCs or H1 relative to AFCs, respectively) and standard deviation. (C, D) AFiPSCs have the capacity to differentiate into derivatives representative of the three embryonic germ layers. (C) Embryoid body (EB)-based differentiation of AFiPSCs into various lineages in vitro as confirmed by immunofluorescent stainings of distinct germ layer marker proteins (lower panels); SOX17, alpha-fetoprotein (AFP); smooth muscle actin (SMA); nestin (NES) and class III beta-tubulin (TUJ1) (scale bars = 200 mm). (D) Histological structures of ectodermal, mesodermal and endodermal lineages observed in teratomas. doi:10.1371/journal.pone.0013703.g002 Global gene expression analyses of AFCs, AFiPSCs and ESCs Transcriptomes were profiled employing the Illumina Bead- Studio platform to investigate the relatedness between primary AFCs, AFiPSCs and the ESC lines H1 and H9. Hierarchical clustering (Pearson’s correlation) as well as linear correlation coefficient analysis based on expression signals of detected genes revealed similar, though not identical, transcriptomes of AFiPSCs and ESCs (average linear R2<0.94). In contrast, the AFiPSC transcriptomes are distinct from those of primary AFCs at passage 6 (average linear R2<0.67) and passage 17 (average linear R2<0.50) (Figure 4A, B). The same analysis was repeated for separate replicate samples of HFF1-derived FiPSCs generated in our laboratory and ESC lines H1 and H9 [31]. This resulted in an average linear correlation coefficient of R2<0.87, reflecting heterogeneity of iPSCs of different somatic origins. A Venn diagram was generated to highlight overlapping and distinct gene expression patterns in AFCs versus AFiPSCs and ESCs. We identified gene signatures representative of cellular housekeeping functions (6934 genes, e.g. GAPDH, ACTB, PGK1, LDHA), self- renewal and pluripotency (1299 genes, e.g. POU5F1, SOX2, NANOG, LIN28), a donor cell memory (350 genes, e.g. KRT7, RGS7), ESC-specificity (257 genes, e.g. PRDM14, GSC, WNT3A), donor cell (AFCs)-specificity (665 genes, e.g. OXTR, HHAT, RGS5, PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13703