Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro-expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham-operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham-operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham-operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2Ј-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.

Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 lm and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 lm in diameter quickly by culturing them in plates with Ubottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of g-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs. V V C 2006 Wiley-Liss, Inc.

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta‐galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53‐62 and 141‐148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter‐domain region of the N‐terminal cAMP‐binding and C‐terminal DNA‐binding domains.

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Here we explicitly present the diffusion coefficients (D) and activation energies (E a ) of interstitial H in α-Fe over a temperature range of 100 to 1000 K. These values were predicted by applying path-integral molecular dynamics modeling based on first principles. The obtained D and E a values exhibit clear non-Arrhenius temperature dependence and a transition from quantum to classical behavior at around 500 K. Our results show that the quantum effects not only significantly lower the diffusion barrier but also change the diffusion pathway even at room temperature; thus, fast diffusion becomes possible.

To elucidate forces providing high mechanical strength during formation of carbon nanotube (CNT) yarns and sheets, internanotube static friction force was investigated using a transmission electron microscope with a nanomanipulation system. Results show that the static friction force depends strongly on the CNT surface state. That force between two as-grown CNTs grown by chemical vapor deposition is much larger than that for highly crystalline CNTs. The as-grown CNT surfaces generally have amorphous carbon and defects. For CNT yarns and sheets, the frictional force attributable to surface roughness, rather than the van der Waals force, affects interactions among CNTs. #