We postulated that the differing methylation signatures of B-iPSC, F-iPSC, and ntESC reflect disparate reprogramming, and confirmed this by two independent computational analyses. First, we overlapped DMRs that distinguish B-iPSC, F-iPSC, and ntESC from fESC with genes specifically expressed in undifferentiated murine fESC26. By this analysis, ntESC showed the fewest DMRs at loci corresponding to the most highly expressed fESC-specific genes, and B-iPSC showed fewer DMRs at these loci than F-iPSC (Supplementary Fig. 5a). Second, we overlapped DMRs with the DNA binding locations for seven transcription factors that compose a core protein network of pluripotency27, and found the fewest DMRs at core transcription factor binding sites in ntESC, and less overlap in B-iPSC than in F-iPSC (Supplementary Table 5). These analyses indicate that F-iPSC harbor more residual methylation than B-iPSC at loci directly linked to the gene expression and pluripotency networks of fESC, whereas ntESC show the least differential methylation and appear closest to fESC at these critical loci.
