(A) Unsupervised hierarchical clustering of global gene expression data in human fibroblasts (hFibr), early-passage hiPSCs (e-hiPSC), late-passage hiPSCs (l-hiPSC), and hESCs. Expression values are presented as the log₂ ratio of the given gene divided by the average of the ESC lines (all subsequent expression heatmaps are presented in this manner). Individual cell lines used are indicated below the heatmap.
(B) e-hiPSC signature genes. As in (A) except for the 3947 genes significantly different between e-hiPSC and hESC based on two criteria, Student’s t test (p < 0.05) and at least a 1.5-fold change. Genes are ordered according to the decreasing average expression ratio between hESCs and e-hiPSCs. Expression data for passage 5 and 28 hESC (P5 and P28) for this set of genes are added to the right. (B′) e-hiPSC genes were divided into those expressed at higher levels in hESCs than e-hiPSCs and vice versa. Each of these two groups was further subclassified into two groups, either more highly expressed in hESCs than fibroblasts (red) or more highly expressed in fibroblasts than hESCs (blue). (B″) Boxplots of the absolute value of the log₂ fold change between hESC and the following: e-hiPSC, l-hiPSC, P5 or P28 hESC (*all p = 0). (B‴) Gene ontology (GO) analysis for e-hiPSC signature genes upon division into those for which hESC expression was greater than e-hiPSC and vice versa. Only significant GO-terms with an enrichment value >3 (p < 0.001) are presented.
(C) l-hiPSC signature genes. As in (B) except for the 860 genes significantly different between hESCs and late-passage-hiPSCs. (C′) Barplot similar to (B′) using l-hiPSC signature genes. (C″) Boxplot similar to (B″) for l-hiPSC signature genes.
(D) Common hiPSC signature genes were determined as the overlap between early- and late-passage signature genes from (B) and (C), respectively, as shown in the Venn diagram. The expression heatmap below is presented for the 318 genes in the overlap as in (B). (D′) Barplot similar to (B′) using common hiPSC signature genes. (D″) Boxplot similar to (B″) for common hiPSC signature genes.

iPSC signature genes defined as genes differentially expressed between hiPSC and hESC lines (Student’s t test [p < 0.05] and at least a 1.5-fold change) were obtained from Figure 1 of this study (Chin et al.) and from additional published reprogramming experiments. The matrix summarizes the overlap of hiPSC signature genes between the different experiments. The values on the diagonal designate the total number of genes identified as significantly different in expression between hESCs and the indicated hiPSCs. The intersection of the rows and columns give the number of genes that are in common between the two respective experiments and the corresponding significance (p value) as determine using Fisher’s exact test. For the Soldner et al. experiment (Soldner et al., 2009), data were analyzed before (2lox) and after (1lox) excision of the reprogramming factors. InYu et al. (2009), iPSCs were generated with episomal vectors and analyzed before (episomal) and after subcloning. Genomic integrations were not detected for any of these subclones. The Maherali iPSCs (Maherali et al., 2008) were reprogrammed with integrating lentiviruses.

(A) Unsupervised hierarchical clustering of global gene expression data from mouse (m) ESCs, miPSCs, and mouse embryonic fibroblasts (MEFs) obtained from the Maherali et al. data set (Maherali et al., 2007). The lines included are biological replicates of V6.5 and E14 mESC lines and of 1D4 and 2DA iPSC lines, respectively. All log₂ ratios are relative to averaged expression in mESCs.
(B) Comparison of iPSC signatures between human and mouse reprogramming experiments. Human early-passage iPSC signature genes as defined in Figure 1B of this study (Chin et al.) and mouse iPSC signature genes defined with the Student’s t test (p < 0.05) and an at least 1.5-fold change between miPSCs and mESCs were further annotated to only include genes that have homologous partners across the two species according to the HomoloGene database, resulting in 2834 genes for the early hiPSC signature and 1018 genes for the miPSC signature. The Venn diagram shows the overlap between these two groups of genes, and significance was determined using the Fisher’s exact test. The heatmap above displays the expression values for the 294 iPSC signature genes conserved between mouse and human reprogramming experiments across the different cell types and species, as log₂ ratio relative to the average ESC expression for each species.
(C) Similar to Figure 1B′, miPSC signature genes were divided into those expressed at higher levels in mESCs than miPSCs and vice versa. Each of these two groups was further subclassified into two groups, either more highly expressed in mESCs than fibroblasts (red color) or those more highly expressed in fibroblasts than hESCs (blue color).
(D) The heatmap depicts the log2 expression ratio between mESCs and miPSCs for miPSC signature genes ordered according to decreasing ratios. For these genes, the binding strength (−log10pXbar) of each reprogramming factor (Oct4 [O], Klf4 [K], Sox2 [S], or c-Myc [C]; data obtained from Sridharan et al., 2009) in miPSCs was subtracted from the binding strength in mESCs. Higher binding strength in mESCs is represented in yellow and in miPSCs in blue. The intensity of the colors increases as differential binding increases. The Pearson correlation of the differential binding strength relative to differential gene expression is given in the attached table.

(A) Tree-view representation of the hierarchical clustering of histone H3 K27 trimethylation in HSF1 (hESC1), HSF6 (hESC2), two human fibroblast lines (hFibr1 and -2), and late-passage hiPSCs (l-hiPSC1 and l-hiPSC18) across the promoter regions of all genes considered significantly differentially methylated between fibroblasts and hESCs (p = 0.05). Genes were classified as E (hESC-like, 950 genes), N (neutral, 19 genes), or F (fibroblast-like, 9 genes) based on the similarity of the methylation patterns in l-hiPSCs with hESCs or fibroblasts. For N and F class genes, the y axis is scaled 33 to make the methylation patterns visible. Each row represents the −5.5 kb to +2.5 kb region with respect to the transcription start site (TSS). The 8 kb promoter regions are divided into sixteen 500 bp regions displayed in pseudocolor based on the average log ratio of the IP to input probe signal intensity. Probes within a given 500 bp region are averaged. Dark gray coloring indicates missing values for enrichment due to the lack of probes. Expression levels for the represented genes are shown on the right for hESCs, e-hiPSCs, and l-hiPSCs relative to fibroblasts.
(B) Table showing the overlap between genes shown in (A) (which demonstrate differences in H3K27 methylation between hESCs and fibroblasts), grouped according to the methylation pattern in l-iPSCs, and the late and common hiPSC expression signature genes as defined in Figures 1C and 1D (these genes are differentially expressed between l-hiPSCs and hESCs). *p value = 0.0063.
(C) Histograms detailing the differences of H3K27me3 patterns between hESC and fibroblasts, measured as the Euclidean distance across the sixteen 500 bp regions of the promoters for the indicated gene sets. The black outlined bars denote the distribution of Euclidean distances for all genes on the array (17,000), while the red outlined bars show the distribution for the indicated subset of signature genes.
(D) As in (C) but for histone H3K4 trimethylation. Note: hESC signature genes must undergo a significantly larger degree of change in both H3K4me3 (**p value = 0.005) and H3K27me3 (***p < 10⁻⁷) than the global population of genes. None of the distributions for the other subsets of genes are significantly different from the global population.

(A) Hierarchical clustering of the expression data for the 105 microRNAs substantially expressed in either hESCs (H9 and HSF1), l-hiPSCs (lines 1, 2, and 18), or fibroblasts, given as log₂ ratios relative to expression in fibroblasts. Note: miRNA expression does not cluster the hESC lines distinctly from the hiPSC lines, whereas the fibroblasts, as expected, exhibit a different miRNA expression pattern.
(B) Table showing miRNAs that were differentially expressed between the two hESC lines (12 replicates total) and three l-hiPSC lines (15 replicates total). Expression values for the given cell lines are given as well as the p value derived from the Student’s t test. Asterisk indicates that miRNA was also found to be differentially expressed between hESC and hiPSC lines inWilson et al. (2009).

(A) Karyotype analysis of the only genomic abnormality detected in any of our hiPSC lines. A clonal duplication on chromosome 8 was found in 19 of 20 metaphase spreads in hiPSC line 1 at passage 44. Array CGH also identified this region (Z-score = 45) solely in the hiPSC1 line (see Table 1), and the array CGH data for this region in all hiPSC lines and fibroblasts are given. The duplicated region in hiPSC1 “steps” down from the midline, indicated by the red box.
(B) A cartoon schematic depicting all overlapping genomic abnormalities among hiPSC lines that were determined by array CGH. Alterations in l-hiPSC1 are indicated with green bars, in l-hiPSC2 with blue bars, and in l-hiPSC18 with red bars. These six regions were the only ones found to be shared between any two hiPSC lines. No genomic aberrations were found in all three l-hiPSC lines.