Norio Shimizu

Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes

Low concentrations (0.11-1.7 microg ml(-1)) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modified by amino groups, when added with nerve growth factor (NGF), promoted outgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and rat pheochromocytoma cell line PC12h cells in culture media. The quantity of active extracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 microg ml(-1) CNTs and NGF than that with NGF alone. CNTs increased the number of cells with neurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERK signaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after the addition of CNTs to the culture medium. These results demonstrate that CNTs may stimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs are biocompatible and are promising candidates for biological applications and devices.

Neurite outgrowths of neurons on patterned self-assembled monolayers

The effects of specific chemical functionalities on the neurite outgrowths of embryonic chick dorsal root ganglia (DRG) neurons and PC12h cells were investigated using a set of chemically functionalized surfaces prepared by self-assembled monolayers (SAMs) of alkanethiolates with R = NH2, COOH, and CH3 on patterned gold surfaces. The numbers of neurons with neurite outgrowths were compared in the course of a two-week cultivation period. Neurons with neurite outgrowths were observed predominantly on a patterned SAM of long-chain alkanethiolates with amino groups. After about two weeks, the neurons detached from the patterned SAM. However, the activity of beta-galactosidase immobilized via a patterned SAM did not decrease over a 13-d period, reflecting the long-term stability of the SAM. Therefore, the neurons became detached upon cell death. These results demonstrate that the patterned SAM of 11-amino-1-undecanethiolate is a scaffold suitable for making cell chips.

Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

When brain-derived neurotrophic factor (BDNF) is produced in the Escherichia coli periplasm, insoluble BDNF proteins with low biological activity and having mismatched disulfide linkages are formed. The coexpression of cysteine oxidoreductases (DsbA and DsbC) and membrane-bound enzymes (DsbB and DsbD), which play an important role in the formation of disulfide bonds in the periplasm, was investigated to improve the production of soluble and biologically active BDNF. The expression levels of Dsb proteins changed when the growth medium and the Dsb expression plasmids were changed, and the production rate of soluble BDNF was almost proportional to the expression level of DsbC protein with disulfide isomerase activity in the case of a low expression level of BDNF. The rate of soluble BDNF production with coexpression of DsbABCD was as high as 35%. These results show that coexpression of BDNF and Dsb proteins can effectively increase the production of soluble and biologically active BDNF.

Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia.

Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 μm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 μm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.

Neurite Outgrowths of Neurons Using Neurotrophin-Coated Nanoscale Magnetic Beads

Neurotrophin-coated nanoscale magnetic beads were used to regulate the differentiation and survival of neurons. The beads coated with nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) promoted neurite outgrowths of neurons in the same manner as soluble NGF or soluble BDNF, but beads coated with bovine serum albumin did not promote neurite outgrowths. When the volume of NGF-coated bead solution was increased, the number of neurons with neurite outgrowths increased. The addition of anti-NGF antibodies decreased the numbers of neurons with neurite outgrowths in proportion to the volume of anti-NGF antibodies added. NGF-coated beads appeared to bind to soma with neurite outgrowths as determined using fluorescence. In addition, hybrid beads coated with both NGF and BDNF promoted neurite outgrowths of PC12h cells, although the cells did not produce neurite outgrowths in response to BDNF. Neurons with neurite outgrowths could be concentrated within a particular area when NGF-coated beads were immobilized in a particular area of the culture plate surface using a magnet. The results demonstrate that neurotrophin-coated nanoscale magnetic beads allow us to cultivate neurons in a selected area of the culture plate surface by using a magnet. Thus, neurotrophin-coated nanoscale magnetic beads are applicable to micro-integrated systems and biosensors.

Promotion of mouse embryonic stem cell differentiation by Rho kinase inhibitor Y-27632

Rho kinase (ROCK) is one of the major downstream mediators of Rho. Rho plays crucial regulatory roles in the cellular proliferation and differentiation. Because a ROCK inhibitor, Y-27632, is known to inhibit the dissociation-induced cell death in human embryonic stem (ES) cells, we investigated the effects of this ROCK inhibitor on the differentiation of the mouse ES cells. The ROCK inhibitor promoted the differentiation of the ES cells into neurons, particularly motor and sensory neurons. The addition of both ROCK inhibitor and nerve growth factor (NGF) strongly stimulated the differentiation of the ES cells into neurons. Moreover, the ROCK inhibitor promoted the differentiation of the ES cells into muscle cells. The ES cells primarily differentiated into neurons rather than muscle cells. We found that the ROCK inhibitor may promote the neuronal differentiation of the ES cells by activating the extracellular signal-regulated kinase (ERK) signaling pathway. These results suggest that the ROCK inhibitor has a significant potential to regulate the differentiation of the ES cells.

Activation of the phospholipase C signaling pathway in nerve growth factor-treated neurons by carbon nanotubes

Low concentrations of carbon nanotubes (CNTs) promoted the number of nerve growth factor (NGF)-treated neurons with neurite outgrowth by activating extracellular signal-regulated kinase (ERK), even when MEK inhibitor was added to the neuron culture medium. We speculated that CNTs may activate ERK through the phospholipase C (PLC) signaling pathway independent of the Ras/Raf/MEK cascade involved in the ERK signaling pathway. CNTs enhanced phosphorylation of PLC-γ1 in NGF-treated neurons but failed to increase the number and length of neurites of NGF-treated neurons with neurite outgrowth when a PLC inhibitor, an inositol triphosphate receptor (IP3R) inhibitor, or an inhibitor of protein kinase C (PKC) in the PLC signaling pathway were added to the neuron culture medium. Furthermore, intracellular Ca(++) levels of cells treated with CNTs+NGF were higher than those of cells treated with NGF alone. Although the combination of CNTs and NGF increased the concentration of phosphorylated ERK (p-ERK) in MEK inhibitor-treated neurons, CNTs did not induce phosphorylation of ERK in PLC inhibitor-treated neurons. These data suggest that PKC in the PLC signaling pathway may activate ERK independent of the Ras/Raf/MEK cascade. In summary, we identified a role of PLC signaling in mediating neurite outgrowth of NGF-treated neurons in the presence of CNTs.

Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons

Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells.

Rho kinase inhibitor Y-27632 promotes neuronal differentiation in mouse embryonic stem cells via phosphatidylinositol 3-kinase.

Rho kinase (ROCK) regulates the functions of several target proteins via its kinase activity. Therefore, ROCK activity inhibition may provide new possibilities of controlling the in vitro neuronal differentiation of embryonic stem (ES) cells. When we investigated the effects of the ROCK inhibitor Y-27632 on ES cell differentiation, we found that this inhibitor promoted the differentiation of these cells into neurons. Furthermore, we found that ROCK inhibition may promote the neuronal differentiation of ES cells by activating extracellular signal-regulated kinase (ERK) involved in the ERK signaling pathway. In this study, we investigated the effects of specific inhibitors of several cellular signaling components on the promotion of neuronal differentiation in ES cells to clarify the roles of cellular signaling pathways in the ROCK inhibitor-mediated cell differentiation process. Our results suggest that ERK may be activated via the Ras/Raf/MEK, the PI3K/PKC, or the Cdc42/Rac signaling pathways in the ROCK inhibitor-mediated promotion of neuronal differentiation in ES cells.

Bioethanol production from steam-exploded rice husk by recombinant Escherichia coli KO11

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.