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Although human induced pluripotent stem cell (hiPSC) derivatives are considered promising cellular resources for regenerative medicine, their tumorigenicity potentially limits their clinical application in hiPSC technologies. We previously demonstrated that oncogenic hiPSC‐derived neural stem/progenitor cells (hiPSC‐NS/PCs) produced tumor‐like tissues that were distinct from teratomas. To gain insight into the mechanisms underlying the regulation of tumorigenicity in hiPSC‐NS/PCs, we performed an integrated analysis using the Infinium HumanMethylation450 BeadChip array and the HumanHT‐12 v4.0 Expression BeadChip array to compare the comprehensive DNA methylation and gene expression profiles of tumorigenic hiPSC‐NS/PCs (253G1‐NS/PCs) and non‐tumorigenic cells (201B7‐NS/PCs). Although the DNA methylation profiles of 253G1‐hiPSCs and 201B7‐hiPSCs were similar regardless of passage number, the methylation status of the global DNA methylation profiles of 253G1‐NS/PCs and 201B7‐NS/PCs differed; the genomic regions surrounding the transcriptional start site of the CAT and PSMD5 genes were hypermethylated in 253G1‐NS/PCs but not in 201B7‐NS/PCs. Interestingly, the aberrant DNA methylation profile was more pronounced in 253G1‐NS/PCs that had been passaged more than 15 times. In addition, we identified aberrations in DNA methylation at the RBP1 gene locus; the DNA methylation frequency in RBP1 changed as 253G1‐NS/PCs were sequentially passaged. These results indicate that different NS/PC clones have different DNA methylomes and that DNA methylation patterns are unstable as cells are passaged. Therefore, DNA methylation profiles should be included in the criteria used to evaluate the tumorigenicity of hiPSC‐NS/PCs in the clinical setting. S tem  C ells   2017;35:1316–1327

Whole‐Genome DNA Methylation Analyses Revealed Epigenetic Instability in Tumorigenic Human iPS Cell‐Derived Neural Stem/Progenitor Cells