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Embryonic stem cell potency fluctuates with endogenous retrovirus activity


Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

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Figure 1: The MuERV-L retrovirus and a reporter driven by its LTR marks the 2C state.
Figure 2: ES cells enter the 2C state regularly, but remain in the state transiently owing to cell intrinsic and extrinsic factors.
Figure 3: The 2C state is associated with an active epigenetic signature and is antagonized by repressive chromatin-modifying enzymes.
Figure 4: Activation of the 2C state is associated with expanded potency in chimaeric embryos towards extraembryonic lineages.
Figure 5: Model of the role of the MuERV-L-LTR-linked 2C gene network in regulating embryonic potency.

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Gene Expression Omnibus

Data deposits

Microarray and RNA-seq files have been submitted to the NCBI Gene Expression Omnibus database under accession GSE33923.


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We would like to thank members of the Pfaff laboratory for discussion; S. Andrews, D. Chambers, Y. Dayn, T.-C. Sung, J. Fitzpatrick, M. Joens, Y. Sigal, D. Gibbs and L. Ouyang for technical assistance, Y. Shinkai and D. Gilbert for the G9a mutant ES cells, and T. Heidmann for MuERV-L-Gag antibodies. This research was supported by the National Institute of Neurological Disorders and Stroke (R37NS037116) and the Marshall Heritage Foundation. T.S.M. and W.D.G. were supported by the California Institute for Regenerative Medicine and S.L.P. is an investigator of the Howard Hughes Medical Institute and Benjamin H. Lewis Chair in Neurobiology.

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Authors and Affiliations



T.S.M. designed and performed all experiments with assistance from W.D.G., S.D., D.B. and K.L. under the supervision of S.L.P. D.T. generated Kap1 mutant ES cells and H.M.R. and D.T. provided mRNA-seq data from these cells. A.F. and O.S. generated and provided iPS cell lines and lentivirus constructs. T.S.M., W.D.G. and S.L.P. wrote the manuscript.

Corresponding author

Correspondence to Samuel L. Pfaff.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-11 and legends for Supplementary Movies 1-3. (PDF 4173 kb)

Supplementary Tables

This zipped file contains Supplementary Tables 1-7. (ZIP 1714 kb)

Supplementary Movie 1

The 2C::tomato reporter is restricted to the zygote and 2C/4C stage - see Supplementary Information fie for full legend. (MOV 543 kb)

Supplementary Movie 2

2C::tomato is transiently expressed in ES cultures - see Supplementary Information fie for full legend. (MOV 2199 kb)

Supplementary Movie 3

Entrance into the 2C::tomato (+) state is more rapid in Kdm1a mutant ES cells - see Supplementary Information fie for full legend. (MOV 2201 kb)

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Macfarlan, T., Gifford, W., Driscoll, S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

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