Abstract
It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.
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Acknowledgements
We thank our colleague Dr Bing Zhu for his critical comments on the manuscript. We thank Prof Rudolf Jaenisch (the Whitehead Institute of MIT) for generously supplying the lentivirus vectors. We are also grateful to our laboratory colleagues for their assistance with experiments and preparation of the manuscript. This work was supported by the Ministry of Science and Technology (2011CB812700, 2010CB944900, and 2011CB964800 to SG, 2011AA100303 and 2011BAD19B01 to JT, 2011ZX09102-010-04 to TC) and the National Natural Science Foundation of China (31000656 to GC).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Figure S1
The pluripotency evaluation of iPCCs used in high-throughput sequencing and reads coverage plots at the transcription start site ± 2KB regions. (PDF 308 kb)
Supplementary information, Figure S2
The validation of some cell-line specific transcripts in high-throughput sequencing iPSCs using quantitative PCR. (PDF 371 kb)
Supplementary information, Figure S3
The DNA methylation, gene expression of Zrsr1, Murr1, and Dlk1-Dio3 gene cluster. (PDF 440 kb)
Supplementary information, Figure S4
DNA methylation state of Zrsr1 in Vc-iPSCs. (PDF 81 kb)
Supplementary information, Table S1
Summary of the iPSC lines used (PDF 97 kb)
Supplementary information, Table S2
The evaluation of high-throughput sequencing datasets (XLS 60 kb)
Supplementary information, Table S3
Summary of cell line-specific deviations in HT iPSCs (PDF 11 kb)
Supplementary information, Table S4
The top ten candidate loci between “4N-ON” and “4N-OFF” iPSCs (XLS 50 kb)
Supplementary information, Table S5
The reads distribution of MeDIP-Seq at the well-known DMRs (XLS 56 kb)
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Chang, G., Gao, S., Hou, X. et al. High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells. Cell Res 24, 293–306 (2014). https://doi.org/10.1038/cr.2013.173
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DOI: https://doi.org/10.1038/cr.2013.173
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