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Self-formation of functional adenohypophysis in three-dimensional culture

Abstract

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.

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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.

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Acknowledgements

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.).

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Contributions

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.

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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)

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Suga, H., Kadoshima, T., Minaguchi, M. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011). https://doi.org/10.1038/nature10637

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