Defining hypo-methylated regions of stem cell-specific promoters in human iPS cells derived from extra-embryonic amnions and lung fibroblasts

Abstract

Background: Human induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived. Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of "stemness" remain unknown in iPS cells derived from extra-embryonic and embryonic cells.

Methodology/principal findings: We examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells.

Conclusions/significance: We show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for "stemness" and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

Figure 1. The ratio of hyper-methylated sites in iPS/ES cells was significantly larger than that of the differentiated cells.

(A) Ratio of Low (methylation score≤0.3), Middle (0.3<score≤0.7), and High (0.7<score) methylated states in 24,949 CpG sites. (B) Clustering analysis. Heat map showing hyper-methylation in human iPS/ES cells compared with differentiated cells. The Heat map in hierarchical clustering analysis represented DNA methylation levels from completely methylated (red) to unmethylated (green). Epigenetic distances (Euclidean Distance) were calculated by NIA Array. (C) Comparisons of CpG sites between two groups show high similarities between AM-iPS and MRC-iPS cells or between human ES cells (HUESCs) and iPS cells (iPSCs). In contrast, 11.3% and 10.6% of CpG sites are differentially methylated in AM-iPS and MRC-iPS cells, respectively, compared to their parental cells (AM936EP and MRC5). It should be noted that 74.0% and 85.1% of the differentially methylated sites (DMSs) are hyper-methylated in AM-iPS and MRC-iPS cells, respectively, compared to their parental cells. (D) Comparison of the 24,949 CpG sites between iPS cells and their parental cells. (E) DMSs among human ES cells, AM- and MRC-iPS cells. The relative amount of inherited/aberrant DMSs is indicated in the pie chart. (F) Overlapped aberrant DMSs between MRC- and AM-iPS cells.

Figure 2. Pluripotent stem cells are significantly more hyper-methylated than differentiated cells.

(A) Principal component analysis (PCA) for DNA methylation states of 24,949 CpG sites with 16 human cell lines. The PC1 axis clearly distinguish iPS/ES cell group from differentiated cells, while human iPS cells are very close to human ES cells. (B) Stem cell-specific differently methylated regions (SS-DMRs) were defined by PC1. In the pluripotent stem cells, 60.5% of the SS-DMRs are located outside of CpG islands and 87.6% of the SS-DMRs are hyper-methylated. (C) DNA methylation levels at promoter regions in 12 representative genes determined by Illumina Infinium HumanMethylation27 assay and Bio-COBRA. Details of these genes are described in Table S6B. The promoter regions of these genes were defined as the SS-DMRs. The relative amount of methylated DNA ratio is indicated as the black area in the pie chart. The same methylation patterns in 12 regions were detected both by Infinium assay and COBRA. (D) Bisulfite sequencing analysis of the same regions that were analyzed by Infinium assay and COBRA assay in SOX15, SALL4, SP100 and GBP3. (Top) Schematic diagram of the genes. Arrows, open boxes and open circles represent transcription start site, first exon and position of CpG sites, respectively. (Bottom) Open and closed circles indicate unmethylated and methylated states, respectively. Red and blue arrowheads represent the position of CpG sites in Infinium assay and COBRA, respectively.

Figure 3. Expression and histone modification of the SS-DMRs related genes.

(A) Expression patterns of representative genes. RT-PCR analysis of 7 representative genes and methyltransferase genes. Methylation levels (Me) of each promoter are shown under each panel. H = High methylation (0.7<score); M = Middle methylation (0.3<score≤0.7); L = Low methylation (score≤0.3). (B) Comparable distribution of the SS-DMR and histone trimethylation (me3) of H3K4 and H3K27. Percentage of H3K4me3, H3K27me3, bivalent H3K4me3/K27me3 or non-modification on genes in SS-hyper-DMRs and in SS-hypo-DMRs. (C) Histone modification of 23 genes in Table 3.