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.

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 a Three-Dimensional Retinal Tissue from Self-Organizing Human ESC Culture.

A three-dimensional (3D) tissue generated in vitro is a promising source to study developmental biology and regenerative medicine. In the last decade, Yoshiki Sasai's group have developed a 3D stem cell culture technique known as SFEBq and demonstrated that embryonic stem cells (ESCs) have an ability to self-organize stratified neural tissue including 3D-retina. Furthermore, we have reported that ESC-derived retinal tissue can form an optic cup and a ciliary margin, which are unique structures in the developing retina. In this review, we focus on self-organizing culture technique to generate 3D-retina from human ESCs.

Novel phototoxicity assay using human embryonic stem cell-derived retinal pigment epithelial cells.

Some chemicals are harmful in to light-exposed tissues such as skin and eyes. The 3T3 Neutral Red Uptake Phototoxicity Test has been validated and adopted by the Organization of Economic and Community Development (OECD) as a method of evaluating chemical phototoxicity using mouse 3T3 fibroblasts. However, the high rate of false positive results associated with this test eventually led to increased laboratory animal usage. Although the eye is vulnerable to light damage because of constant exposure to environmental radiation, few approaches are available to predict ocular phototoxicity in humans. Here, we propose a tier one test that identifies the potential ocular phototoxicity of chemical substances. Using a three-dimensional culture technique, human embryonic stem cells (hESCs) were differentiated to retinal pigment epithelial cell (RPE) precursors. The precursors after prolonged treatment with FBS formed a uniform hexagonal lattice of cells with well-developed tight junctions and time-dependent elevation of melanin content and RPE maturation marker levels. Hierarchical clustering of gene transcripts revealed that hESC-derived RPEs were very similar to tissue-derived adult RPEs. Interestingly, there were a high percentage of chemicals eliciting a positive response in 3T3 cells and negative in hESC-derived RPEs under the experimental conditions used in the phototoxicity test. The response to treatment of hESC-derived RPEs with these negative chemicals became positive at a higher dose of UVA irradiation; however, the biological responses to these chemicals differed between the two cells. Taken together, we conclude that hESC-derived RPEs are novel tool for future toxicological and mechanistic studies of ocular phototoxicity in humans.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration.

Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.

Optimized Culture System to Induce Neurite Outgrowth From Retinal Ganglion Cells in Three-Dimensional Retinal Aggregates Differentiated From Mouse and Human Embryonic Stem Cells.

PURPOSE: To establish a practical research tool for studying the pathogenesis of retinal ganglion cell (RGC) diseases, we optimized culture procedures to induce neurite outgrowth from three-dimensional self-organizing optic vesicles (3D-retinas) differentiated in vitro from mouse and human embryonic stem cells (ESCs). MATERIALS AND METHODS: The developing 3D-retinas isolated at various time points were placed on Matrigel-coated plates and cultured in media on the basis of the 3D-retinal culture or the retinal organotypic culture protocol. The number, length, and morphology of the neurites in each culture condition were compared. RESULTS: First, we confirmed that Venus-positive cells were double-labeled with a RGC marker, Brn3a, in the 3D-retina differentiated from Fstl4::Venus mouse ESCs, indicating specific RGC-subtype differentiation. Second, Venus-positive neurites grown from these RGC subsets were positive for beta-III tubulin and SMI312 by immunohistochemistry. Enhanced neurite outgrowth was observed in the B27-supplemented Neurobasal-A medium on Matrigel-coated plates from the optic vesicles isolated after 14 days of differentiation from mouse ESCs. For the differentiated RGCs from human ESCs, we obtained neurite extension of >4 mm by modifying Matrigel coating and the culture medium from the mouse RGC culture. CONCLUSION: We successfully optimized the culture conditions to enhance lengthy and high-frequency neurite outgrowth in mouse and human models. The procedure would be useful for not only developmental studies of RGCs, including maintenance and projection, but also clinical, pathological, and pharmacological studies of human RGC diseases.

Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.

In the developing neural retina (NR), multipotent stem cells within the ciliary margin (CM) contribute to de novo retinal tissue growth. We recently reported the ability of human embryonic stem cells (hESCs) to self-organize stratified NR using a three-dimensional culture technique. Here we report the emergence of CM-like stem cell niches within human retinal tissue. First, we developed a culture method for selective NR differentiation by timed BMP4 treatment. We then found that inhibiting GSK3 and FGFR induced the transition from NR tissue to retinal pigment epithelium (RPE), and that removing this inhibition facilitated the reversion of this RPE-like tissue back to the NR fate. This step-wise induction-reversal method generated tissue aggregates with RPE at the margin of central-peripherally polarized NR. We demonstrate that the NR-RPE boundary tissue further self-organizes a niche for CM stem cells that functions to expand the NR peripherally by de novo progenitor generation.

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.

Self-formation of optic cups and storable stratified neural retina from human ESCs.

In this report, we demonstrate that an optic cup structure can form by self-organization in human ESC culture. The human ESC-derived optic cup is much larger than the mouse ESC-derived one, presumably reflecting the species differences. The neural retina in human ESC culture is thick and spontaneously curves in an apically convex manner, which is not seen in mouse ESC culture. In addition, human ESC-derived neural retina grows into multilayered tissue containing both rods and cones, whereas cone differentiation is rare in mouse ESC culture. The accumulation of photoreceptors in human ESC culture can be greatly accelerated by Notch inhibition. In addition, we show that an optimized vitrification method enables en bloc cryopreservation of stratified neural retina of human origin. This storage method at an intermediate step during the time-consuming differentiation process provides a versatile solution for quality control in large-scale preparation of clinical-grade retinal tissues.

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.

Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.

Human embryonic stem cells (hESCs), unlike mouse ones (mESCs), are vulnerable to apoptosis upon dissociation. Here, we show that the apoptosis, which is of a nonanoikis type, is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr, a unique Rho-GEF family factor containing a functional Rac-GAP domain, is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation, but not in Abr-depleted hESCs or mESCs. Furthermore, artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus, the Abr-dependent "Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs.