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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.
The adenohypophysis, corresponding to the anterior and intermediate lobes of the pituitary gland, contains several types of endocrine cells, which secrete systemic hormones such as adenocorticotropic hormone (ACTH), GH, LH/FSH, TSH and prolactin1-3 in response to hypothalamus-derived releasing hormones. In contrast, the neurohypophysis (posterior pituitary) consists of the axons and secretory termini of hypothalamic vasopressin and oxytocin neurons. During early mammalian development, the adenohypophysis anlage originates as a placode in the non-neural head ectoderm rostral and adjacent to the anterior neural plate4,5 (Supplementary Fig. 1a) (hereafter, this part of non-neural ectoderm, expressing region-specific markers Pitx1 and Pitx2, is simply referred to as rostral head ectoderm; this includes the oral ectoderm as a large part). The thickened placodal epithelium invaginates and subsequently detaches from the oral ectoderm, becoming a hollowed epithelial vesicle, Rathke's pouch (Fig. 1a, b and Supplementary Fig. 1b, c). The development of Rathke's pouch depends on tissue interactions between the rostral head ectoderm and the rostral hypothalamus3-8. We sought to recapitulate this interactive microenvironment of the pituitary-forming morphogenetic field using three-dimensional ES cell culture.
Juxtaposing ectodermal layers formed in culture
Mouse ES cells can be induced to differentiate into rostral hypothalamic tissues when cultured as floating aggregates (typically 3,000 cells per aggregate) in a chemically defined medium lacking extrinsic growth factors (including no insulin; hereafter referred to as SFEBq...