We used CHARM to examine the methylome of the germ-line competent pluripotent stem cells, the tertiary reprogrammed B-NP-iPSC and NSC-NP-iPSC, and the drug-treated NP-iPSC (Fig. 4a). In pair-wise comparisons (Supplementary Table 1b), the NP-iPSC showed only a small number of DMRs relative to fESC (553), fewer than the numbers of DMRs distinguishing ntESC from fESC (679), indicating that selection using the Nanog-GFP reporter and derivation from young donor tissue yields more equivalently reprogrammed cells. Despite equivalent Nanog-GFP expression, B lymphocyte-derived Bl-iPSC harbored more DMRs (1485) relative to fESC than did the NP-iPSC. Cluster dendrogram analysis, employing the most variable DMRs that distinguish Bl-iPSC and NP-iPSC, showed NP-iPSC to be more similar to fESC than are Bl-iPSC, which represent a distinct cluster (Fig. 4c). These data suggest that neural progenitors are more completely reprogrammed to an ESC-like state than blood donor cells. Cluster dendrogram analysis failed to distinguish among NP-iPSC, ntESC, and fESC, but assessment of the overlap of DMRs with loci for highly expressed ESC-specific genes26 and core pluripotency transcription factor binding sites27 indicated differences among these three pluripotent cell types, and reveal that ntESC have the fewest DMRs affecting these critical loci (Supplementary Fig. 5b).
