A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages


Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

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Figure 1: An ESC level of Oct4 marks pluripotency acquisition.
Figure 2: Low levels of Oct4 expression sustain self-renewal.
Figure 3: Oct4 expression at an ESC level is required for in vitro differentiation.
Figure 4: Oct4 expression at an ESC level is required for in vivo differentiation.
Figure 5: Oct4 binding is converse to Nanog and is linked to downregulation of naive pluripotency genes.
Figure 6: Oct4-low iPSCs self-renew in the absence of pluripotent culture requisites.
Figure 7: A defined Oct4 level is also required for downregulation of key naive pluripotency genes in ESCs.
Figure 8: A defined Oct4 level controls cell state transitions around pluripotency.

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  • 07 May 2013

    In the version of this Article originally published online, there were errors in Fig. 1k,l. This has been corrected in the HTML and PDF versions.


  1. 1

    Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem. Cell 4, 487–492 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Rosner, M. H. et al. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, 686–692 (1990).

    CAS  Article  Google Scholar 

  3. 3

    Scholer, H. R., Dressler, G. R., Balling, R., Rohdewohld, H. & Gruss, P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 9, 2185–2195 (1990).

    CAS  Article  Google Scholar 

  4. 4

    Okamoto, K. et al. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461–472 (1990).

    CAS  Article  Google Scholar 

  5. 5

    Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet 24, 372–376 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Theunissen, T. W. et al. Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions. Curr. Biol. 21, 65–71 (2011).

    CAS  Article  Google Scholar 

  9. 9

    Kim, J. B. et al. Direct reprogramming of human neural stem cells by OCT4. Nature 461, 649–643 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Yuan, X. et al. Combined chemical treatment enables Oct4-induced reprogramming from mouse embryonic fibroblasts. Stem Cells 29, 549–553 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Li, Y. et al. Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res. 21, 196–204 (2011).

    CAS  Article  Google Scholar 

  12. 12

    Kim, J. B. et al. Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411–419 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Ying, Q. L., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Wilkinson, D. G., Bhatt, S. & Herrmann, B. G. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659 (1990).

    CAS  Article  Google Scholar 

  16. 16

    Avilion, A. A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Monaghan, A. P., Kaestner, K. H., Grau, E. & Schutz, G. Postimplantation expression patterns indicate a role for the mouse forkhead/HNF- 3α,β and γ genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 119, 567–578 (1993).

    CAS  PubMed  Google Scholar 

  18. 18

    Kanai-Azuma, M. et al. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development 129, 2367–2379 (2002).

    CAS  PubMed  Google Scholar 

  19. 19

    Downs, K. M. Systematic localization of Oct-3/4 to the gastrulating mouse conceptus suggests manifold roles in mammalian development. Dev. Dyn. 237, 464–475 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem. Cell 3, 391–401 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K. & Niwa, H. Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135, 909–918 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Kobayashi, T. et al. The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells. Genes Dev. 23, 1870–1875 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Canham, M. A., Sharov, A. A., Ko, M. S. & Brickman, J. M. Functional heterogeneity of embryonic stem cells revealed through translational amplification of an early endodermal transcript. PLoS Biol. 8, e1000379 (2010).

    Article  Google Scholar 

  25. 25

    Festuccia, N. et al. Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells. Cell Stem. Cell 11, 477–490 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Hall, J. et al. Oct4 and LIF/Stat3 additively induce Kruppel factors to sustain embryonic stem cell self-renewal. Cell Stem. Cell 5, 597–609 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Niwa, H., Ogawa, K., Shimosato, D. & Adachi, K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460, 118–122 (2009).

    CAS  Article  Google Scholar 

  28. 28

    Van Oosten, A. L., Costa, Y., Smith, A. & Silva, J. C. JAK/STAT3 signalling is sufficient and dominant over antagonistic cues for the establishment of naive pluripotency. Nat. Commun. 3, 817 (2012).

    Article  Google Scholar 

  29. 29

    Niwa, H., Masui, S., Chambers, I., Smith, A. G. & Miyazaki, J. Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol. Cell Biol. 22, 1526–1536 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Vigano, M. A. & Staudt, L. M. Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1. Nucleic Acids Res. 24, 2112–2118 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Silva, J. et al. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008).

    Article  Google Scholar 

  32. 32

    Ying, Q. L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Thomson, M. et al. Pluripotency factors in embryonic stem cells regulate differentiation into germ layers. Cell 145, 875–889 (2011).

    CAS  Article  Google Scholar 

  34. 34

    Teo, A. K. et al. Pluripotency factors regulate definitive endoderm specification through eomesodermin. Genes Dev. 25, 238–250 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Du, P., Kibbe, W. A. & Lin, S. M. Lumi: a pipeline for processing Illumina microarray. Bioinformatics 24, 1547–1548 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Lin, S. M., Du, P., Huber, W. & Kibbe, W. A. Model-based variance-stabilizing transformation for Illumina microarray data. Nucleic Acids Res. 36, e11 (2008).

    Article  Google Scholar 

  37. 37

    Smyth, G. K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet Mol. Biol. 3, Article 3 (2004).

    Article  Google Scholar 

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We thank W. Mansfield and C-E. Dumeau for blastocyst injections and morula aggregations, R. Walker for flow cytometry, and M. McLeish and H. Skelton for histological processing of teratomas. We are grateful to H. Niwa for providing mice with different Oct4 genotypes and A. Smith and J. Betschinger for providing plasmids. We are also grateful to Y. Costa and P. Shliaha for technical assistance and H. Stuart for critical reading of the manuscript. The study was supported by Wellcome Trust Fellowship WT086692MA. J.C.R.S. is a Wellcome Trust Career Development Fellow. A.R. is a recipient of the Darwin Trust of Edinburgh Postgraduate Scholarship.

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A.R. performed and designed the experiments, analysed the data and wrote the manuscript. R.L.S. and G.L.B.C. performed experiments. T.W.T., L.F.C., A.R. and J.S. designed the study. J.N. analysed data. J.S. supervised the study, designed the experiments, analysed the data, and wrote and approved the manuscript.

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Correspondence to José C. R. Silva.

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Embryoid body outgrowths of PB-Oct4 iPSCs−/− contain beating heart cells 3 days after plating on gelatine-coated dishes. (AVI 4310 kb)

PB-Oct4 iPSCs−/− differentiate into beating heart cells.

Embryoid body outgrowths of PB-Oct4 iPSCs−/− contain beating heart cells 3 days after plating on gelatine-coated dishes. (AVI 4310 kb)

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Radzisheuskaya, A., Le Bin Chia, G., dos Santos, R. et al. A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nat Cell Biol 15, 579–590 (2013). https://doi.org/10.1038/ncb2742

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