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.

Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.

Highlights d Hypothalamic-pituitary functional units can be generated from 3D-cultured human iPS cells d Maturation of induced pituitary through simultaneous culture with hypothalamic neurons d ACTH secretion ability of induced pituitary is comparable with adult mice d Induced pituitary cells respond to low-glucose stimulation through the CRH-ACTH pathway SUMMARYThe pituitary is a major hormone center that secretes systemic hormones responding to hypothalamusderived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs).Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a lifethreatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

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.