Journal of Assisted Reproduction and Genetics

, Volume 29, Issue 10, pp 1021–1028 | Cite as

Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells

  • Fredwell Hambiliki
  • Susanne Ström
  • Pu Zhang
  • Anneli Stavreus-Evers
Stem Cell Biology



NANOG and OCT4 are required for the maintenance of pluripotency in embryonic stem cells (ESCs). These proteins are also expressed in the inner cell mass (ICM) of the mouse pre-implantation embryo.


Immunohistochemistry was used to show the presence of NANOG and OCT4 protein, and in situ hybridization was used to localize NANOG mRNA in human embryos from two-cell to blastocyst stage, and in human ESCs (hESCs).


Nanog and Oct4 were co-localized in human embryos from morula and blastocyst stages. NANOG mRNA was detected in a group of cells in the morula, in cells of the ICM of blastocysts, and evenly in hESCs. All non-differentiated hESCs expressed NANOG and OCT4 protein. Pluripotent cells expressing NANOG and Oct4 were eccentrically localized, probably in polarized cells in a human compacted morula, which appears to be different from expression in murine embryos.


In this study, we demonstrate that whole mount in situ hybridization is amenable to localization of mRNAs in human development, as in other species.


Pluripotency Embryo Embryonic stem cells NANOG In situ hybridization 



The authors thank Outi Hovatta and Jennifer Nichols for stem cells, NANOG probe and valuable comments. The study was supported by grants from the Swedish Research Council (2005–7293), the Swedish Research Council, The Swedish Society of Medicine, Karolinska Institutet, Uppsala University, Goljes Foundation and Åke Wibergs Foundation


  1. 1.
    Bjuresten K, Hovatta O. Donation of embryos for stem cell research—how many couples consent? Hum Reprod. 2003;18:1353–5.PubMedCrossRefGoogle Scholar
  2. 2.
    Buehr M, Smith A. Genesis of embryonic stem cells. Philos Trans R Soc Lond B Biol Sci. 2003;358:1397–4402.PubMedCrossRefGoogle Scholar
  3. 3.
    Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of NANOG, a pluripotency sustaining factor in Embronic Stem cells. Cell. 2003;113:643–55.PubMedCrossRefGoogle Scholar
  4. 4.
    Chambers I, Smith A. Self renewal of teratocarcinoma and embryonic stem cells. Oncogene. 2004;23:7150–60.PubMedCrossRefGoogle Scholar
  5. 5.
    Du Puy L, Lopes SM, Haagsman HP, Roelen BA. Analysis of co-expression of OCT4, NANOG and SOX2 in pluripotent cells of the porcine embryo, in vivo and in vitro. Therienology. 2011;75:513–26.CrossRefGoogle Scholar
  6. 6.
    Galán A, Montaner D, Póo E, Valbuena D, Ruiz V, Aguillar C, Dopazo J, Simon C. Functional genomics of 5- to 8-cell stage human embryos to blastomere single-cell cDNA analysis. PLoS One. 2010;5:e13615.PubMedCrossRefGoogle Scholar
  7. 7.
    Göke J, Jung M, Behrens S, Chavez L, O’Keeffe S, Timmermann B, Lehrach H, Adjaye J, Vingron M. Combinatorial binding in human and mouse embryonic stem cells identifies conserved enhancers active in early embryonic development. PLoS Comput Biol. 2011;7:e1002304.PubMedCrossRefGoogle Scholar
  8. 8.
    Hart AH, Hartley L, Ibrahim M, Robb L. Identification, cloning and expression analysis of the pluripotency promoting NANOG genes in mouse and human. Dev Dyn. 2004;230:187–98.PubMedCrossRefGoogle Scholar
  9. 9.
    Hansis C, Grifo JA, Krey LC. Oct-4 expression in inner cell mass and trophectoderm of human blastocysts. Mol Hum Reprod. 2000;6:999–1004.PubMedCrossRefGoogle Scholar
  10. 10.
    Hansis C, Grifo JA, Tang Y, Krey LC. Assessment of beta-hCG and beta LH mRNA and ploidy in individual human blastomeres. RBM Online. 2002;5:156–61.PubMedGoogle Scholar
  11. 11.
    Hansis C, Edwards RG. Cell differentiation in the preimplantation human embryo. RBM Online. 2002;6:215–20.Google Scholar
  12. 12.
    Hatano SY, Tada M, Kimura H, Yamaguchi S, Kono T, Nakano T, Suemori H, Nakatsuji N, Tada T. Pluripotential competence of cells associated with NANOG activity. Mech Dev. 2005;122:67–79.PubMedCrossRefGoogle Scholar
  13. 13.
    Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 2003;18:1404–9.PubMedCrossRefGoogle Scholar
  14. 14.
    Inzunza J, Gertow K, Stromberg MA, Matilainen E, Blennow E, Skottman H, Wolbank S, Ahrlund-Richter L, Hovatta O. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells. 2005;23:544–9.PubMedCrossRefGoogle Scholar
  15. 15.
    Kimber SJ, Snedden SF, Bloor DJ, El-Bareg AM, Hawkhead JA, Metcalfe AD, Houghton FD, Leese HJ, Rutherford A, Lieberman BA, Brison DR. Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors. Reproduction. 2008;135:635–47.PubMedCrossRefGoogle Scholar
  16. 16.
    Kirchhof N, Carnwath JW, Lemme E, Anastassiadis K, Scholer H, Niemann H. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol Reprod. 2000;63:1698–705.PubMedCrossRefGoogle Scholar
  17. 17.
    Kuroda T, Tada M, Kubota H, Kimura H, Hatano SY, Suemori H, Nakatsuji N, Tada T. Octamer and Sox elements are required for transcriptional cis regulation of NANOG gene expression. Mol Cell Biol. 2005;25:2475–85.PubMedCrossRefGoogle Scholar
  18. 18.
    Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein NANOG is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003;113:631–6642.PubMedCrossRefGoogle Scholar
  19. 19.
    Mohr L, Trounson A. Cryopreservation of human embryos. Ann N Y Acad Sci. 1985;442:536–43.PubMedCrossRefGoogle Scholar
  20. 20.
    Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor OCT4. Cell. 1998;95:379–91.PubMedCrossRefGoogle Scholar
  21. 21.
    Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000;24:372–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990;60:461–72.PubMedCrossRefGoogle Scholar
  23. 23.
    Osterlund C, Wramsby H, Pousette A. Temporal expression of platelet-derived growth factor (PDGF)-A and its receptor in human preimplantation embryos. Mol Hum Reprod. 1996;2:507–12.PubMedCrossRefGoogle Scholar
  24. 24.
    Palmieri SL, Peter W, Hess H, Scholer HR. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol. 1994;166:259–67.PubMedCrossRefGoogle Scholar
  25. 25.
    Reubinoff BE, Pera MF, Vajta G, Trounson AO. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod. 2001;16:2187–94.PubMedCrossRefGoogle Scholar
  26. 26.
    Rosner MH, Vigano MA, Ozato K, Timmons PM, Poirier F, Rigby PW, Staudt LM. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature. 1990;345:686–92.PubMedCrossRefGoogle Scholar
  27. 27.
    Scholer HR, Dressler GR, Balling R, Rohdewohld H, Gruss P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 1990;9:2185–95.PubMedGoogle Scholar
  28. 28.
    Silva J, Nichols JM, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A. NANOG is the gateway to the pluripotent ground state. Cell. 2009;138:722–37.PubMedCrossRefGoogle Scholar
  29. 29.
    Smith AG. Embryo-derived stem cells: of mice and men. Ann Rev Cell Dev Biol. 2001;17:435–62.CrossRefGoogle Scholar
  30. 30.
    Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA. 1975;72:5099–102.PubMedCrossRefGoogle Scholar
  31. 31.
    Van Blerkom J, Davis P, Mathwig V, Alexander S. Domains of high polarized and low polarized mitochondria may occur in mouse and human oocytes and early embryos. Hum Reprod. 2002;17:393–406.PubMedCrossRefGoogle Scholar
  32. 32.
    Van Eijk MJ, van Rooijen MA, Modina S, Scesi L, Folkers G, van Tol HT, Bevers MM, Fisher SR, Lewin HA, Rakacolli D, et al. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol Reprod. 1999;60:1093–103.PubMedCrossRefGoogle Scholar
  33. 33.
    Yeom YI, Fuhrmann G, Ovitt CE, Brehm A, Ohbo K, Gross M, Hubner K, Scholer HR. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development. 1996;122:881–94.PubMedGoogle Scholar
  34. 34.
    Zhang P, Zucchelli M, Bruce S, Hambiliki F, Stavreus-Evers A, Levkov L, Skottman H, Kerkelä E, Kere J, Hovatta O. Transcriptome profiling of human pre-implantation development. PLoS One. 2009;4(11):e7844.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Fredwell Hambiliki
    • 1
    • 2
  • Susanne Ström
    • 1
  • Pu Zhang
    • 2
  • Anneli Stavreus-Evers
    • 2
  1. 1.Department of Clinical Science, Intervention and TechnologyKarolinska InstitutetStockholmSweden
  2. 2.Department of Women’s and Children’s HealthUppsala UniversityUppsalaSweden

Personalised recommendations