Takafumi Wataya

A ROCK inhibitor permits survival of dissociated human embryonic stem cells

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 ∼1% to ∼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.

Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals

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.

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.

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.

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.

Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation

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.

High molecular weight neurofilament proteins are physiological substrates of adduction by the lipid peroxidation product hydroxynonenal

Protein adducts of the lipid peroxidation producttrans-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimers disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1–33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.

Cl--ATPase and Na+/K+-ATPase activities in Alzheimer's disease brains

Abstract The enzyme activities and the protein levels of Cl − -ATPase and Na + /K + -ATPase were examined in Alzheimers disease (AD) brains. Cl − -ATPase and Na + /K + -ATPase activities in AD brains ( n =13) were significantly lower than those in age-matched control brains ( n =12). In contrast, there was no significant difference in anion-insensitive Mg 2+ -ATPase activity between the two groups. Western blot analysis revealed that the protein levels of Cl − -ATPase, Na + /K + -ATPase and neuron specific Na + /K + -ATPase α 3 isoform were also significantly reduced in AD brains, while the amount of protein disulfide isomerase, one of the house keeping membrane proteins, was not different between the two groups. The data first demonstrated that Cl − -ATPase and Na + /K + -ATPase are selectively impaired in AD brains, which may reduce the gradients of Na + , K + and Cl − across the cell membranes to cause excitotoxic cellular response and the resulting neuronal death.

Reactive oxygen: Its sources and significance in Alzheimer disease

Over the past decade, oxidative stress has been established as the earliest cytological feature of Alzheimer disease and an attractive therapeutic target. The major challenges now are establishing the source of the reactive oxygen and what oxidative stress tells us about the etiology of Alzheimer disease. These are complex issues since a variety of enzymatic and non-enzymatic processes are involved in reactive oxygen formation and damage to macromolecules. In this review, we consider disease mechanisms that show the greatest promise for future research.

La función del estrés oxidativo en la patogénesis de la enfermedad de Alzheimer

La presencia de estres oxidativo es la caracteristica mas temprana de la Enfermedad de Alzheimer (EA), lo cual proporciona un atractivo objetivo para intervenciones terapeuticas. Entre los mayores retos que se presentan actualmente estan el establecimiento de la fuente de estres oxidativo y la determinacion de como este proceso puede influir en la etiologia de la Enfermedad de Alzheimer. Este es un tema complejo, pues varios procesos, enzimaticos y no-enzimaticos, estan implicados en la formacion de oxigeno reactivo y otras moleculas toxicas. En este articulo discutimos el progreso en el entendimiento de estos procesos