Supplementary Figure 3: Methylation State of CpGs that Significantly Demethylate During Reprogramming of PBMCs to iPSCs

A) This boxplot depicts the methylation state of 3,896 CpG sites that significantly decline in methylation (p-value <0.05) and convert from mostly methylated to unmethylated during reprogramming of PBMCs to iPSCs (defined as CpGs with average an M-value > 1 (>67% methylation) in PBMCs and an average M-value < -1 (<33% methylation) in iPSCs). The methylation values of each CpG site are converted to a standardized Z-score. These sites globally display methylation levels that are, on average, ~1/3rd of a standard deviation higher in iPSCs derived from elderly vs. young individuals (center of boxplot - generalized Friedman rank sum test with replicated data, p-value < 2.2·10-16). B) DNA methylation profiles for selected iPSC lines were analyzed at early/intermediate and late passages. The methylation status of 3 CpG sites, whose methylation is not restored with passaging is plotted with black circle representing M-value for iPSCs at early passage and red triangle M-value for late passage in selected iPSC lines. TRAPPC3 (eR² = 0.38 p-value =0.0002; (lR²)= 0.53 p-value = 0.0002), AHRR (eR² = 0.24 p-value=0.0035; (lR²)= 0.24 p-value = 0.026), MYADML2 (eR² = 0.35 p-value=0.0003; (lR²)= 0.20 p-value = 0.04). Pearson correlation with two-tailed p-value. C) Quantitative RT-PCR for TET1, 2A, 2B, 3 and DNMT1, 3A, 3B. Correlation between gene expression and age is plotted. Relative mRNA expression was determined by the ΔΔ-Ct method with the average of young donors used as control. Gene expression was normalized using PPIA (peptidylprolyl isomerase A) as reference gene. TET1 (R² = 0.21 p-value=0.00015), TET2A (R² = 0.248 p-value=0.0005), TET2B (R² = 0.04 p-value=0.15), TET3 (R² = 0.46 p-value<0.0001). Pearson correlation with two-tailed p-value.