Recipe for pituitary organoids

Abstract

Generation of a variety of organs and tissues from human pluripotent stem cells (hPSCs) has been attempted in vitro. We here present a simple and efficient method for induction of hypothalamic and pituitary tissues from hPSCs. On provision of exogenous agents important for early hypothalamus-pituitary organogenesis, including bone morphogenetic protein 4 and activators of sonic hedgehog, in three-dimensional culture, hPSCs spontaneously form spherical organoids with two distinct tissues, hypothalamus and adenohypophysis. The pituitary tissues derived from hPSCs not only secrete adenocorticotropic hormone, but also retain both positive and negative feedback mechanisms, recapitulating mature endocrine organs in vivo. Furthermore, the results of ectopic transplantation with mouse models of hypopituitarism suggest that these hypothalamus-pituitary organoids have potential as engraftment organs. In addition to their use in transplantation for patients with hypopituitarism they will allow establishment of disease models in vitro and enable research impossible in humans. Hypothalamus-pituitary organoids promise to be a powerful tool in regenerative medicine, drug discovery, and basic research into pituitary development.

Keywords: corticotroph; embryonic stem cells; induced pluripotent stem cells; organoid; pituitary; pluripotent stem cells.

Figure 1

The close relationship between hypothalamus and pituitary. (A) The hypothalamus-pituitary-adrenal axis. Created with BioRender (https://biorender.com). (B–D) Pituitary organogenesis in mice. The ventral diencephalon provides BMP4 and FGF 8/10/18. The oral ectoderm provides signals that activate sonic hedgehog (Shh) (B). Opposing dorsal→ventral FGF8/10/18 and ventral←dorsal BMP2 gradients are crucial for pituitary organogenesis (C). Ventral hypothalamus expresses Rax. Non-neural oral ectoderm expresses Paired like homeodomain 1 (Pitx1). LIM homeobox protein 3 (Lhx3) encodes an early pituitary marker.

Figure 2

Efficient pituitary differentiation from hPSCs. (A) Pituitary differentiation from hPSCs and critical points for succesful induction. Above, original conditions (7). Below, modified conditions (8). This article describes the modified method. (B) Human PSCs one day after passage. (C) Human PSCs three days after passage. Healthy hPSCs have well-defined colony edges and high cell density in the colony center. Suitable colony diameter for passage is 1mm–2mm. Scale bars: 100 µm (B, C).

Figure 3

Differentiation into early pituitary primordium in SFEBq culture. (A–K) Bright-field and EGFP fluorescence microscopy views of aggregates in SFEBq culture. We used the KhES-1_Rx::Venus (VA22-N37) cell line, carrying Venus knocked into the Rx locus (13). RX::Venus-expressing hypothalamic tissues are visible from day 9. On day 24, oral ectoderm-like tissue appears on the surface of the aggregate (H, magenta arrowheads). This feature can be clearly identified in bright-field. Scale bar: 500 μm.

Figure 4

Induction of differentiation of human iPSCs into corticotroph cells. From “Hypothalamic contribution to pituitary functions is recapitulated in vitro using 3D-cultured human iPS cells,” by T. Kasai and H. Suga, 2020, Cell Reports 30, 18-24. e15. https://doi.org/10.1016/j.celrep.2019.12.009. Creative Commons CC-BY license. (A–G) original condition. (H, I) modified condition. Scale bars: 50μm (A–F, H, I) and 20μm (G).

Figure 5

Kidney subcapsular transplantation of the hPSC-derived pituitary tissue into post-hypophysectomy mice. (A) Hypophysectomy of SCID mice. Created with BioRender (https://biorender.com). (B) Post-hypophysectomy mice are anaesthetized with isoflurane. Before the operation, mice are injected with 0.2mg dexamethasone and 2mg ampicillin intramuscularly. (C) Exposure of a kidney (yellow arrowheads). (D) The hPSC-derived pituitary tissue (yellow arrowheads) is transplanted under the renal capsule. (E) Post-transplantation mouse. Mice must be kept warm until they fully awaken from anesthesia.