Pluripotent cells can be segregated based on the methylation/gene expression level of nine genes. (A) Heatmap and hierarchical clustering results of the cell lines used in this study using methylation patterns at CpG sites containing aberrant methylation in at least one hiPSC line. Similar aberrant epigenetic patterns were observed in hiPSCs derived from common somatic sources, and these lines accordingly tend to cluster together. (B) Graphical representation of an example of residual methylation and de novo methylation located on chromosome 15 (ISLR2 gene). Each circle corresponds to an individual CpG site and the level of methylation is represented in a colored pattern. In the example shown, NSChiPS2F retains the epigenetic pattern of its somatic progenitor (hNSC), showing residual methylation. HUVhiPS4F1 takes on an epigenetic pattern not observed in its somatic progenitor or any of the other pluripotent lines, showing a hiPSC line-specific de novo methylation. Methylation levels of the same CpG sites in hESC and hiPSC lines were included for comparison. (C and D) Types of methylation errors leading to epigenetic aberrations. Most aberrantly methylated CpG sites associated to genes showing de novo methylation (C) and residual methylation (D) in all hiPSC lines are characterized by overmethylation or partial methylation, respectively. (E) Heatmap and ordered dendrogram for the hiPSC and hESC described lines (11) based on the level of relative change observed at CpG sites associated to our nine signature genes. Note that hESC and hiPSC lines segregated in two different groups. (F) Hierarchical clustering of six hiPSC (ASThiPS4F2, 3, 4, and 5, HUVhiPS4F6, and FhiP4F2) and six hESC (H1, H9, HUES2, HUES6, HUES8, and HUES9) lines based on the gene expression level analyzed by real-time PCR of the nine common aberrantly methylated genes identified in hiPSC lines used in this study.

Identification and classification of the epigenetic changes occurring during cell reprogramming. (A) Hierarchical clustering of the indicated cell lines based on the methylation state of all characterized CpG sites. HUVEC, human umbilical vein endothelial cell; K-MMTA, keratinocyte cell line; MSC, mesenchymal stem cell; NSC, neural stem cell; PGP1F, HFF.XF, IMR90, fibroblasts lines. (B) Heatmap and ordered dendrogram for all hiPSC lines based on the level of relative change observed at each differentially methylated site compared with the values observed in each respective somatic cell of origin. Pearson’s correlation values were used to generate a single distance metric. (C) Reprogramming efficiency of somatic cell lines estimated after hiPSCs generation by retroviral infection of OCT4, SOX2, KLF4, and cMYC inversely correlates with the percentage of differential methylation achieved in hiPSC lines. Note that amount of epigenetic reorganization required appears to be a barrier to reprogramming. R², Pearson’s correlation value.

Reprogramming-associated epigenetic/transcriptional signatures segregate hiPSCs and hESCs after differentiation. (A) Percentage of aberrant CpG sites identified between hESC-derived lines and the corresponding hiPSC-derived lines classified in the following categories: aberrant methylation remains and is still aberrant compared with differentiated hESCs (A); aberrant methylation remains but is the same as the one found in differentiated hESCs (B); aberrant methylation is removed during differentiation reaching the level found in differentiated hESCs (C); and aberrant methylation changed to a new aberrant methylation state (D). (B) Genes with aberrantly methylated CpG sites and differential transcriptional abundance with at least a twofold cutoff were identified in the HUVhiPS4F1 cell line after comparison with H9 cells. Graph shows the relative fold change in the expression of genes still aberrantly methylated after differentiation between the differentiated HUVhiPS4F1 cell line and the differentiated hESC cell line. Note that differential expression was independent on whether Activin or BMP4-differentiated cells were analyzed. (C) Hierarchical clustering of hESC (H1, H9, HUES3, HUES6, and HUES9) and hiPSC (HUVhiPS4F1, HUVhiPS4F3, HUVhiPS4F6, ASThiPS4F4, and ASThiPS4F5) lines in their pluripotent and differentiated states based on the methylation level of the nine common aberrantly methylated genes identified in the hiPSC lines used in this study. (D) Hierarchical clustering of hESC (H9) and hiPSC (HUVhiPS4F1 and HUVhiPS4F3) lines in their pluripotent and differentiated states based on the gene expression level of the nine common aberrantly methylated genes identified in the hiPSC lines used in this study. Data were obtained from microarray analysis.