Kanako Saito

The hypothalamus is comprised of heterogenous cell populations and includes highly complex neural circuits that regulate the autonomic nerve system. Its dysfunction therefore results in severe endocrine disorders. Although recent experiments have been conducted for in vitro organogenesis of hypothalamic neurons from embryonic stem (ES) or induced pluripotent stem (iPS) cells, whether these stem cell-derived hypothalamic neurons can be useful for regenerative medicine remains unclear. We therefore performed orthotopic transplantation of mouse ES cell (mESC)-derived hypothalamic neurons into adult mouse brains. We generated electrophysiologically functional hypothalamic neurons from mESCs and transplanted them into the supraoptic nucleus of mice. Grafts extended their axons along hypothalamic nerve bundles in host brain, and some of them even projected into the posterior pituitary (PPit), which consists of distal axons of the magnocellular neurons located in hypothalamic supraoptic and paraventricular nuclei. The axonal projections to the PPit were not observed when the mESC-derived hypothalamic neurons were ectopically transplanted into the substantia nigra reticular part. These findings suggest that our stem cell-based orthotopic transplantation approach might contribute to the establishment of regenerative medicine for hypothalamic and pituitary disorders.

Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary cells, as well as their ectodermal precursors. EpCAM was not expressed on hypothalamic lineages; thus, anterior pituitary cells were successfully enriched by magnetic separation of EpCAM+ cells from iPSC-derived HP organoids. The enriched pituitary population contained functional corticotrophs and their progenitors; the former responded normally to a corticotropin-releasing hormone stimulus. Our findings would extend the applicability of organoid culture as a novel source of human anterior pituitary cells, including stem/progenitor cells and their endocrine descendants.

Morphogenesis and organ development should be understood based on a thorough description of cellular dynamics. Recent studies have explored the dynamic behaviors of mammalian neural progenitor cells (NPCs) using slice cultures in which three-dimensional systems conserve in vivo-like environments to a considerable degree. However, live observation of NPCs existing truly in vivo, as has long been performed for zebrafish NPCs, has yet to be established in mammals. Here, we performed intravital two-photon microscopic observation of NPCs in the developing cerebral cortex of H2B-EGFP or Fucci transgenic mice in utero. Fetuses in the uterine sac were immobilized using several devices and were observed through a window made in the uterine wall and the amniotic membrane while monitoring blood circulation. Clear visibility was obtained to the level of 300 μm from the scalp surface of the fetus, which enabled us to quantitatively assess NPC behaviors, such as division and interkinetic nuclear migration, within a neuroepithelial structure called the ventricular zone at embryonic day (E) 13 and E14. In fetuses undergoing healthy monitoring in utero for 60 min, the frequency of mitoses observed at the apical surface was similar to those observed in slice cultures and in freshly fixed in vivo specimens. Although the rate and duration of successful in utero observations are still limited (33% for ≥10 min and 14% for 60 min), further improvements based on this study will facilitate future understanding of how organogenetic cellular behaviors occur or are pathologically influenced by the systemic maternal condition and/or maternal-fetal relationships.