The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells, which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor–mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs.
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We are grateful to B. Payer (Harvard University) for providing M. musculus × M. castaneus fibroblasts, D. Wesemann for advice on B-cell isolation and immunoglobulin rearrangements, A. Foudi for genomic DNA from B cells, H. Su for technical assistance and A. Riley for help with blastocyst injections. We thank past and present members of the Hochedlinger laboratory for suggestions. M.S. was supported by a postdoctoral fellowship from the Howard Hughes Medical Institute (HHMI), E.A. was supported by a Jane Coffin Childs postdoctoral fellowship, and support to K.H. was from the US National Institutes of Health (DP2OD003266 and R01HD058013).
The authors declare no competing financial interests.
Supplementary Figures 1–18 (PDF 9180 kb)
Differential H3K4me3 enrichment in B-iPSCs and ESCs (XLS 94 kb)
Coordinates of genomic regions analysed by pyrosequencing (XLS 38 kb)
Primer sequences used for SSLP and ChIP analyses (XLS 27 kb)
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Stadtfeld, M., Apostolou, E., Ferrari, F. et al. Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all–iPS cell mice from terminally differentiated B cells. Nat Genet 44, 398–405 (2012) doi:10.1038/ng.1110
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