Pluripotent stem cells are significantly more hyper-methylated than differentiated cells.

<p>(A) Unsupervised hierarchical clustering analysis based on DNA methylation. (B) Principal component analysis (PCA) of the DNA methylation states of 485,577 CpG sites from 10 human cell lines. The principle component 1 axis clearly distinguishes the iPS/ES cell group from differentiated cells, whereas human iPS cells are very close to human ES cells. (C) Heat map showing hyper-methylation in human iPS/ES cells compared with differentiated cells. The heat map of hierarchical clustering analysis represents DNA methylation levels from completely methylated (dark blue) to unmethylated (light blue). Epigenetic distances (Euclidean Distance) were calculated by NIA Array.</p

The ratio of CpG sites in iPSCs was significantly larger than that of the differentiated cells.

<p>(A) Comparisons of 485,577 CpG sites between two groups show high similarities between AF-IPS and FF-IPS cells. (B) In contrast, 12.34% and 12.47% of CpG sites are differentially methylated in AF-IPS and FF-IPS cells, respectively, compared with their parental cells (AF and FF). It should be noted that 89.46% and 86.06% of the differentially methylated sites (DMSs) are hypermethylated in AF-IPS and FF-IPS cells, respectively, compared with their parental cells.</p

Venn-like diagram showing stem cell-specific differentially methylated regions (SS-DMRs) that overlap CpG sites among ESCs, iPSCs and their parental cells.

<p>(A) A total of 28,777 ssDMRs were identified. (B) The number of sites with low, middle and high methylation status. (C) In the pluripotent stem cells, 92.72% of the SS-DMRs are hypermethylated. (D) A total of 86.36% of the hypermethylated SS-DMRs and 86.45% of the hypomethylated SS-DMRs are located outside of CpG islands.</p

Comparison of aberrant and inherited methylation in human iPSCs.

<p>The DMRs between ESCs and iPSCs are designated as ES-iPS-DMRs, and the DMRs between ESCs and parental cells are designated as ES-parental-DMRs. (A) A comparison of ES-iPS-DMRs from iPSC lines derived from each parental cell are shown (left). The number of regions inherited from parental cells in iPSCs (light blue) and the aberrant regions in iPSCs that differ from ESCs and parental cells (dark blue) in the ES-iPS-DMRs are shown as bars (right). (B) The proportion of the hyper- and hypo-methylated ES-iPS-DMRs in inherited and aberrant regions of each iPSC line. (C) The proportion of the ES-iPS-DMRs associated with CpG islands and non-CpG islands in inherited and aberrant regions of each iPSC line. (D) The number and distrubition of overlapping aberrant ES-iPS-DMRs in iPSCs. Chromosome ideogram showing the location of the individual DMRs specific for iPSCs from this study. Red dots indicate the location of the individual DMRs covering a particular gene; red circles denote those for intergene region. (E) Clustering analysis of aberrant ES-iPS-DMRs. The heat map shows the methylation pattern of aberrant DMRs in human iPS cells compared with ES cells. The heat map of hierarchical clustering analysis represents DNA methylation levels from completely methylated (red) to unmethylated (green). Epigenetic distances (Euclidean Distance) were calculated by NIA Array. The number of probes and the genes representing hyper and hypomethylation are shown (right). (F) Gene expression level of Ube3a and Fgr. Bar and line graphs showing the normalized expression level and the DNA methylationb-values for two interesting genes. Gene expression was normalized to the expression of Gapdh. The mean values were calculated from independent experiments performed in triplicate.</p

Pluripotent stem cells are significantly more hypermethylated than their corresponding parental cells.

<p>(A) Distribution of 485,577 CpG sites with their methylation scores in the parental cells, iPSCs and ESCs. (B) The average number of CpG sites with low (0–0.3), middle (0.3–0.7) and high (0.7–1.0) methylation. The iPSCs have more highly methylated sites than the parental cells.</p

Clustering analysis showing differential methylation in episomal iPS cells compared with virally induced iPS cells.

<p>(A) The heat map of hierarchical clustering analysis represents DNA methylation levels from completely methylated (red) to unmethylated (green). (B) 0.14% of CpG sites are differential in episomally derived AF-IPS cells compared with virally derived AF-IPS cells. It should be noted that 591 of the DMRs are hypomethylated.</p

A list of human cells analyzed for a methylation state in this study.

<p>Numbers in parenthesis with P indicate passage in culture on the cells used in the methylation analysis.</p><p>A list of human cells analyzed for a methylation state in this study.</p