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.

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.

T he eye-forming field in the diencephalon has been a classical paradigm in experimental biology, and the mechanism of how the eye primordium is specified has attracted the interest of embryologists over many decades. In addition, most retinal degeneration diseases in humans are caused by impairment in the neural retina, particularly in photoreceptor cells (1, 2). Therefore, in vitro generation of neural retinal precursors, if successful, would greatly contribute to medical and pharmaceutical researches for retinal diseases. To date, however, only infrequent expression of photoreceptor markers in ES cell-derived neural tissues has been reported (3, 4).The development of the neural retina involves multiple regulatory steps during embryogenesis (see Fig. 1A). A number of transcription factors have been isolated and implicated in the control of the step-wise differentiation of vertebrate retinal tissues (5). In contrast, relatively little has been known about the molecular nature of extracellular factors that mediate tissue interactions to form the retinal tissues in the exact location. To facilitate the study on retinal development, we have attempted to establish an in vitro differentiation system for neural retinal precursors from mouse ES cells.In our previous reports, it has been shown that retinal pigment epithelial cells can be induced at a moderate efficiency from primate ES cells by coculturing with PA6 cells [stromal cell-derived inducing activity (SDIA) method] (6, 7). However, as described below in this study, the frequency of the generation of neural retinal cells in the SDIA system has proven to be low. In addition to the SDIA method, we have recently established another in vitro culture system that induces efficient neural differentiation from ES cells. In this method, floating aggregates of ES cells are cultured under serum-free conditions in the optimized medium without retinoic acid (RA) or exogenous growth factors (serum-free floating culture of embryoid body-like aggregates, SFEB) (8). A characteristic feature of the SFEB culture is that it efficiently induces differentiation of rostral-most CNS tissues. Particularly, the addition of the Wnt antagonist Dkk1 (9) and the Nodal antagonist LeftyA (10) to the SFEB culture during the first 5 days facilitates both neural differentiation (up to 90%) and rostral specification of neural tissues (Ϸ35% are telencephalic tissues) (8).Here, we report efficient in vitro generation of neural retinal precursors from mouse ES cells by combining the SFEB culture and extracellular inductive signals. The ability of the ES cellderived progenitors to produce photoreceptors is also demonstrated. Materials and MethodsCell Culture. The methods of ES cell maintenance and the differentiation by the SDIA method have been described (11). For the SFEB method, 5 ϫ 10 4 dissociated ES cells (EB5 line) per milliliter were incubated in a bacterial-grade dish with ES differentiation medium (G-MEM, 5% KSR͞0.1 mM nonessential amino acids͞1 mM pyruvate͞0.1 mM 2-mercaptoethanol) (8). F...

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

During gastrulation of the amphibian embryo, specification of the three germ layers, endo-, ecto-, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-beta signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. Here, we report that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, we find that XFDL156 and its mammalian homologs interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53.

Although autologous nerve grafting is widely accepted as the gold standard treatment for segmental nerve defects, harvesting autologous nerves is highly invasive and leads to functional loss of the ablated part. In response, artificial nerve conduits made of artificial materials have been reported, but the efficacy of the nerve regeneration still needs improvement. The purpose of this study is to investigate the efficacy and mechanism of the Bio three-dimensional (3D) conduit composed of xenofree human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs). The 5-mm nerve gap of the sciatic nerve in immunodeficient rats was bridged with the Bio 3D conduit or silicone tube. Functional and histological recovery were assessed at 8 weeks after surgery. The regenerated nerve in the Bio 3D group was significantly superior to that in the silicone group based on morphology, kinematics, electrophysiology, and wet muscle weight. Gene expression analyses demonstrated neurotrophic and angiogenic factors. Macroscopic observation revealed neovascularization both inside and on the surface of the Bio 3D conduit. Upon their subcutaneous implantation, iMSCs could induce angiogenesis. The Bio 3D conduit fabricated from iMSCs are an effective strategy for nerve regeneration in animal model. This technology will be useful in future clinical situations. Peripheral nerve injury is often accompanied by trauma or tumor resection. The gold standard treatment when tensionless direct repair cannot be achieved is autologous nerve grafting 1,2. Unlike solid organ transplantation such as heart, liver, kidney, or lung, a grafted nerve does not function itself, but offers the best scaffold for axonal elongation 3. It does, however, have several potential disadvantages, including donor site morbidity, limited supply, risk of neuroma formation, mismatch of the caliber diameter, necessity of an extra surgical incision, and increased operative time 4. A nerve allograft resolves some of these disadvantages, but requires perioperative immunosuppression and a particular preservation method 5. Because peripheral nerve injury causes merely

Water is an important component in national development. Despite the efforts of the Kenyan government to increase water coverage throughout the country so that economic development of the nation remains unimpeded, recent statistics show that the rate of water supply improvement is unlikely to support the nation's long-term development goals. This paper examines literature, reports, studies and other relevant information regarding Kenya's water sector in order to expose the underlying causes of the challenges the sector management faces. It also analyses the interrelationships between the challenges and discusses opportunities for improvement. From the study, nine root causes of the challenges that are of technical, economic and social in nature were identified. As they evolve, they conjoin into other problem scenarios characterised by: unsustainability of water supply systems, low social acceptance of interventions, low investment in the sector and water-related conflicts. It is also observed that the sector management has opportunity for improving the current situation through adoption of elaborate monitoring strategies for water services and water resources, embracing sustainable technologies and involving target beneficiaries in water supply development. Great strides can be achieved by the sector through Adaptive Management (AM).

No abstract