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.

Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Subcutaneous transplantation of human embryonic stem cells-derived pituitary organoids.

Introduction: The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied. Methods: hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment. Results: During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 - 0.05 and 0.001 - 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration. Conclusions: Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.

Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells.

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

Generation and purification of ACTH-secreting hPSC-derived pituitary cells for effective transplantation.

Pituitary organoids are promising graft sources for transplantation in treatment of hypopituitarism. Building on development of self-organizing culture to generate pituitary-hypothalamic organoids (PHOs) using human pluripotent stem cells (hPSCs), we established techniques to generate PHOs using feeder-free hPSCs and to purify pituitary cells. The PHOs were uniformly and reliably generated through preconditioning of undifferentiated hPSCs and modulation of Wnt and TGF-ß signaling after differentiation. Cell sorting using EpCAM, a pituitary cell-surface marker, successfully purified pituitary cells, reducing off-target cell numbers. EpCAM-expressing purified pituitary cells reaggregated to form three-dimensional pituitary spheres (3D-pituitaries). These exhibited high adrenocorticotropic hormone (ACTH) secretory capacity and responded to both positive and negative regulators. When transplanted into hypopituitary mice, the 3D-pituitaries engrafted, improved ACTH levels, and responded to in vivo stimuli. This method of generating purified pituitary tissue opens new avenues of research for pituitary regenerative medicine.

Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

Functional Lactotrophs in Induced Adenohypophysis Differentiated From Human iPS Cells.

Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.

Generation of four induced pluripotent stem cell lines (FHUi003-A, FHUi003-B, FHUi004-A and FHUi004-B) from two affected individuals of a familial neurohypophyseal diabetes insipidus family.

Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms.

Tanycyte-Like Cells Derived From Mouse Embryonic Stem Culture Show Hypothalamic Neural Stem/Progenitor Cell Functions.

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.

Improved methods for the differentiation of hypothalamic vasopressin neurons using mouse induced pluripotent stem cells.

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.

Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue.

The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20(+)/Prox1(+) granule neurons and Zbtb20(+)/KA1(+) pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.