A novel three-dimensional culture system for isolation and clonal propagation of neural stem cells using a thermo-reversible gelation polymer

In the present study, we examined the possible utility of a three-dimensional culture system using a thermo-reversible gelation polymer to isolate and expand neural stem cells (NSCs). The polymer is a synthetic biologically inert polymer and gelates at temperatures higher than the gel-sol transition point ( approximately 20 degrees C). When fetal mouse brain cells were inoculated into the gel, spherical colonies were formed ( approximately 1% in primary culture and approximately 9% in passage cultures). The spheroid-forming cells were positive for expression of the NSC markers nestin and Musashi. Under conditions facilitating spontaneous neural differentiation, the spheroid-forming cells expressed genes characteristic to astrocytes, oligodendrocytes, and neurons. The cells could be successively propagated at least to 80 poly-D-lysines over a period of 20 weeks in the gel culture with a growth rate higher than that observed in suspension culture. The spheroids formed by fetal mouse brain cells in the gel were shown to be of clonal origin. These results indicate that the spheroid culture system is a convenient and powerful tool for isolation and clonal expansion of NSCs in vitro.

Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis

Adult neural stem cells (aNSCs) derived from the subventricular zone of the brain show therapeutic effects in EAE, an animal model of the chronic inflammatory neurodegenerative disease MS; however, the beneficial effects are modest. One critical weakness of aNSC therapy may be an insufficient antiinflammatory effect. Here, we demonstrate that i.v. or i.c.v. injection of aNSCs engineered to secrete IL-10 (IL-10–aNSCs), a potent immunoregulatory cytokine, induced more profound functional and pathological recovery from ongoing EAE than that with control aNSCs. IL-10–aNSCs exhibited enhanced antiinflammatory effects in the periphery and inflammatory foci in the CNS compared with control aNSCs, more effectively reducing myelin damage, a hallmark of MS. When compared with mice treated with control aNSCs, those treated with IL-10–aNSCs demonstrated differentiation of transplanted cells into greater numbers of oligodendrocytes and neurons but fewer astrocytes, thus enhancing exogenous remyelination and neuron/axonal growth. Finally, IL-10–aNSCs converted a hostile environment to one supportive of neurons/oligodendrocytes, thereby promoting endogenous remyelination. Thus, aNSCs engineered to express IL-10 show enhanced ability to induce immune suppression, remyelination, and neuronal repair and may represent a novel approach that can substantially improve the efficacy of neural stem cell–based therapy in EAE/MS.

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.

Pro-neural transcription factors as cancer markers

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours.

RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers.

CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

Reproductive isolation and genetic differentiation of ferox trout from sympatric brown trout in Loch Aweand Loch Laggan, Scotland

Molecular marker studies reported here, involving allozymes, mitochondrial DNA and microsatellites, demonstrate that ferox brown trout Salmo trutta in Lochs Awe and Laggan, Scotland, are reproductively isolated and genetically distinct from co-occurring brown trout. Ferox were shown to spawn primarily, and possibly solely, in a single large river in each lake system making them particularly vulnerable to environmental changes. Although a low level of introgression seems to have occurred with sympatric brown trout, possibly as a result of human-induced habitat alterations and stocking, ferox trout in these two lakes meet the requirements for classification as a distinct biological, phylogenetic and morphological species. It is proposed that the scientific name Salmo ferox Jardine, 1835, as already applied to Lough Melvin (Ireland) ferox, should be extended to Awe and Laggan ferox.

A neural network based tool for semi-automatic code transformation

A neural network based tool has been developed to assist in the process of code transformation. The tool offers advice on appropriate transformations within a knowledge-driven, semi-automatic parallelisation environment. We have identified the essential characteristics of codes relevant to loop transformations. A Kohonen network is used to discover structure in the characterised codes thus revealing new knowledge that may be brought to bear on the mapping between codes and transformations or transformation sequences. A transform selector based on this process has been developed and successfully applied to the parallelisation of sequential codes.

An investigation into the role of Bcl-2 in neuroendocrine differentiation

INTRODUCTION: In addition to its role in apoptosis suppression, Bcl-2 has been reported to be co-expressed with neuroendocrine markers in several tissues, leading to speculation that this oncoprotein may promote neuroendocrine differentiation. AIM: This study investigated whether Bcl-2 modulated neuroendocrine biopeptide expression. METHODS: Levels of chromogranin A, neurone specific enolase, protein gene peptide 9.5, pancreatic polypeptide, and the chromogranin-derived peptides, intervening peptide and vasostatin-1 were examined by immunocytochemistry in rat phaeochromocytoma (PC12) cell lines genetically engineered to over-express Bcl-2 and their mock-transfected controls. Intensity of fluorescence was graded using a semi-quantitative scale from (-) indicating negative expression to (+++) indicating intense positivity. RESULTS: Mann-Whitney U analysis indicated that no significant differences in expression existed between control and Bcl2 over-expressing cell lines for any of the six peptides examined. CONCLUSIONS: The results of this study do not support the hypothesis that Bcl-2 promotes the acquisition of a neuroendocrine phenotype.