Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Self-formation of functional adenohypophysis in three-dimensional culture


The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke’s pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Adjacent formation of head ectoderm and hypothalamic epithelia in ES cell culture.
Figure 2: Spontaneous generation of Rathke’s pouch-like vesicles in ES cell culture.
Figure 3: Differentiation of ES-cell-derived pituitary progenitors into hormone-producing cell lineages.
Figure 4: CRH-induced ACTH release from ES-cell-derived pituitary tissues.
Figure 5: Increased systemic ACTH and glucocorticoid levels by grafting ES-cell-derived pituitary tissues.


  1. Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C. & Dattani, M. T. Genetic regulation of pituitary gland development in human and mouse. Endocr. Rev. 30, 790–829 (2009)

    Article  CAS  Google Scholar 

  2. Romero, C. J., Nesi-França, S. & Radovick, S. The molecular basis of hypopituitarism. Trends Endocrinol. Metab. 20, 506–516 (2009)

    Article  CAS  Google Scholar 

  3. Rizzoti, K. & Lovell-Badge, R. Early development of the pituitary gland: induction and shaping of Rathke’s pouch. Rev. Endocr. Metab. Disord. 6, 161–172 (2005)

    Article  Google Scholar 

  4. Takuma, N. et al. Formation of Rathke’s pouch requires dual induction from the diencephalon. Development 125, 4835–4840 (1998)

    CAS  PubMed  Google Scholar 

  5. Suh, H., Gage, P. J., Drouin, J. & Camper, S. A. Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification. Development 129, 329–337 (2002)

    CAS  PubMed  Google Scholar 

  6. Zhu, X., Gleiberman, A. S. & Rosenfeld, M. G. Molecular physiology of pituitary development: signaling and transcriptional networks. Physiol. Rev. 87, 933–963 (2007)

    Article  CAS  Google Scholar 

  7. Rizzoti, K. & Lovell-Badge, R. Early development of the pituitary gland: induction and shaping of Rathke’s pouch. Rev. Endocr. Metab. Disord. 6, 161–172 (2005)

    Article  Google Scholar 

  8. Ericson, J., Norlin, S., Jessell, T. M. & Edlund, T. Integrated FGF and BMP signaling controls the progression of progenitor cell differentiation and the emergence of pattern in the embryonic anterior pituitary. Development 125, 1005–1015 (1998)

    CAS  PubMed  Google Scholar 

  9. Wataya, T. et al. Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation. Proc. Natl Acad. Sci. USA 105, 11796–11801 (2008)

    Article  CAS  ADS  Google Scholar 

  10. Watanabe, K. et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nature Neurosci. 8, 288–296 (2005)

    Article  CAS  Google Scholar 

  11. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008)

    Article  CAS  Google Scholar 

  12. Basch, M. L. & Bronner-Fraser, M. Neural crest inducing signals. Adv. Exp. Med. Biol. 589, 24–31 (2006)

    Article  CAS  Google Scholar 

  13. Eiraku, E. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011)

    Article  CAS  ADS  Google Scholar 

  14. Jones, C. M., Lyons, K. M. & Hogan, B. L. Involvement of bone morphogenetic protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development 111, 531–542 (1991)

    CAS  PubMed  Google Scholar 

  15. Danjo, T. et al. Subregional specification of ES cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals. J. Neurosci. 31, 1919–1933 (2011)

    Article  CAS  Google Scholar 

  16. Treier, M. et al. Hedgehog signaling is required for pituitary gland development. Development 128, 377–386 (2001)

    CAS  PubMed  Google Scholar 

  17. Ellsworth, B. S., Butts, D. L. & Camper, S. A. Mechanisms underlying pituitary hypoplasia and failed cell specification in Lhx3-deficient mice. Dev. Biol. 313, 118–129 (2008)

    Article  CAS  Google Scholar 

  18. Sheng, H. Z. et al. Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 272, 1004–1007 (1996)

    Article  CAS  ADS  Google Scholar 

  19. Kikuchi, M. et al. Changes in E- and N-cadherin expression in developing rat adenohypophysis. Anat. Rec. 290, 486–490 (2007)

    Article  CAS  Google Scholar 

  20. Thor, S., Ericson, J., Brännström, T. & Edlund, T. The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat. Neuron 7, 881–889 (1991)

    Article  CAS  Google Scholar 

  21. Bharti, K., Gasper, M., Bertuzzi, S. & Arnheiter, H. Lack of the ventral anterior homeodomain transcription factor VAX1 leads to induction of a second pituitary. Development 138, 873–878 (2011)

    Article  CAS  Google Scholar 

  22. Mason, I. Initiation to end point: the multiple roles of fibroblast growth factors in neural development. Nature Rev. Neurosci. 8, 583–596 (2007)

    Article  CAS  Google Scholar 

  23. Davis, S. W., Mortensen, A. H. & Camper, S. A. Birthdating studies reshape models for pituitary gland cell specification. Dev. Biol. 352, 215–227 (2011)

    Article  CAS  Google Scholar 

  24. Lamolet, B. et al. A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104, 849–859 (2001)

    Article  CAS  Google Scholar 

  25. Zhu, X. et al. Sustained Notch signaling in progenitors is required for sequential emergence of distinct cell lineages during organogenesis. Genes Dev. 20, 2739–2753 (2006)

    Article  CAS  Google Scholar 

  26. Kita, A. et al. Hes1 and Hes5 control the progenitor pool, intermediate lobe specification, and posterior lobe formation in the pituitary development. Mol. Endocrinol. 21, 1458–1466 (2007)

    Article  CAS  Google Scholar 

  27. Zhao, Y. et al. Reduced expression of the LIM-homeobox gene Lhx3 impairs growth and differentiation of Rathke’s pouch and increases cell apoptosis during mouse pituitary development. Mech. Dev. 123, 605–613 (2006)

    Article  CAS  Google Scholar 

  28. Davis, S. W. et al. Molecular mechanisms of pituitary organogenesis: in search of novel regulatory genes. Mol. Cell. Endocrinol. 323, 4–19 (2010)

    Article  CAS  Google Scholar 

  29. Hemming, F. J., Bégeot, M., Dubois, M. P. & Dubois, P. M. Fetal rat somatotropes in vitro: effects of insulin, cortisol, and growth hormone-releasing factor on their differentiation: a light and electron microscopic study. Endocrinology 114, 2107–2113 (1984)

    Article  CAS  Google Scholar 

  30. Ogasawara, K. et al. Hormonal regulation of prolactin cell development in the fetal pituitary gland of the mouse. Endocrinology 150, 1061–1068 (2009)

    Article  CAS  Google Scholar 

  31. Gleiberman, A. S., Fedtsova, N. G. & Rosenfeld, M. G. Tissue interactions in the induction of anterior pituitary: role of the ventral diencephalon, mesenchyme, and notochord. Dev. Biol. 213, 340–353 (1999)

    Article  CAS  Google Scholar 

  32. Ingle, D. J. The effects of administering large amounts of cortin on the adrenal cortices of normal and hypophysectomized rats. Am. J. Physiol. 124, 369–371 (1938)

    Article  CAS  Google Scholar 

  33. Falconi, G. & Rossi, G. L. Transauricular hypophysectomy in rats and mice. Endocrinology 74, 301–303 (1964)

    Article  CAS  Google Scholar 

  34. Melmed, S. The Pituitary 3rd edn, 61 (Academic, 2011)

    Google Scholar 

Download references


We are grateful to H. Enomoto, R. Ladher and M. Eiraku for invaluable comments, to K. Misaki for electron microscopy analysis, and to members of the Y.S. laboratory for discussion. This work was supported by grants-in-aid from Ministry of Education, Culture, Sports, Science and Technology (Y.S., Y.O.), the Knowledge Cluster Initiative at Kobe, and the Leading Project for Realization of Regenerative Medicine (Y.S.).

Author information

Authors and Affiliations



H.S. and Y.S. designed the project and wrote the manuscript. H.S., T.K., M.O. and M.M. performed the experiments with the technical help and advice of T.N., N.T., M.S., K.M., H.M., S.Y. and T.W., and Y.O. provided critical advice on the research strategy and design.

Corresponding author

Correspondence to Yoshiki Sasai.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with legends and legends for Supplementary Movies 1-2. (PDF 13724 kb)

Supplementary Movie 1

This movie shows the formation of Lim3+ vesicles in SFEBq-cultured ESC Aggregates - see Supplementary Information file for full legend. (MOV 15058 kb)

Supplementary Movie 2

This movie shows improved locomotor activity in hypophysectomized mice receiving SAG+DAPT-treated aggregates - see Supplementary Information file for full legend. (MOV 13716 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Suga, H., Kadoshima, T., Minaguchi, M. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing