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.

Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

We have searched for near-infrared extragalactic background light ( EBL) in the data from the Near-Infrared Spectrometer ( NIRS) on the Infrared Telescope in Space ( IRTS). After subtracting the contribution of faint stars and the zodiacal component based on modeling, a significant isotropic emission is obtained in the wavelength bands from 1.4 to 4.0 m. The spectrum is stellar-like but shows a spectral jump from the optical EBL. The emission obtained is isotropic over the observed sky, and the in-band flux amounts to $35 nW m À2 sr À1 , which is too bright to be explained by the integrated light from faint galaxies. Analyses of COBE DIRBE data, after removal of starlight, show essentially the same result within the uncertainty in the zodiacal light model, which implies that the isotropic emission observed by IRTS NIRS is of extragalactic origin. Significant fluctuations in sky brightness were also detected that cannot be explained by fluctuations due to faint stars, zodiacal components, and normal galaxies. The excess fluctuation amounts to $1/4 of the excess emission over the integrated light of galaxies and is consistent with fluctuations observed by COBE DIRBE. A two-point correlation analysis shows that IRTS NIRS data have an angular scale of fluctuations of a few degrees. The spectrum and brightness of the observed excess EBL emission could be explained by the redshifted UV radiation from the first generation of massive stars ( Population III stars), which caused the reionization of the universe. Recent Wilkinson Microwave Anisotropy Probe (WMAP) observations of the cosmic microwave background (CMB) polarization have indicated that reionization occurred at z $17 or earlier, while the spectral jump around 1 m in the observed excess EBL suggests that the Population III star formation terminated at z $ 9. The observed fluctuations, however, are considerably larger than the theoretical predictions for the Population III stars.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax ؉ /Six3 ؉ /Vax1 ؉ ) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax ؉ progenitors generate Otp ؉ /Brn2 ؉ neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax ؉ progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.chemically defined ͉ hypothalamus ͉ patterning ͉ vasopressin D ifferentiation culture of mouse ES (mES) cells is a versatile in vitro tool for understanding molecular and cellular controls in early mammalian neurogenesis (1-5). We previously established a mES cell culture system with reduced exogenous signals, namely, serum-free culture of embryoid body-like aggregates (SFEB culture) (4). In this method, ES cells are dissociated (to minimize possible effects of culture substrate matrix), reaggregated (over one day), and cultured as floating aggregates in serum-free medium containing knockout serum replacement (KSR) (6), but with no major exogenous inductive factors, such as Fgf, BMP, Wnt, or Nodal. SFEB-cultured mES cells spontaneously differentiate into neural progenitors that acquire a rostral forebrain fate and efficiently generate telencephalic progenitors positive for Bf1 (FoxG1; (7); see Fig. 1A). Efficient forebrain differentiation is also seen in SFEB-cultured human ES cells (8).In contrast to telencephalic development, relatively little has been known about regulatory signals for early diencephalic development, including the initial specification of the mammalian hypothalamic anlage (9, 10). In the present study, using a similar SFEB culture approach, we wished to steer ES cell differentiation into hypothalamic tissues, which arises from the rostral forebrain region adjacent to the embryonic telencephalon in vivo. However, we noticed at the beginning of this study that hypothalamic mark...