Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

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.

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases.

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We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH ؉ ) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH ؉ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH ؉ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (Ϸ5 weeks). In addition, 8% of the colonies contain large patches of Pax6 ؉ -pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.

The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

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...