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The possible use of cell therapies for neurological lesions and disorders is regarded as a very promising strategy. However, many issues related to cell type, tissue donor, expected biological action etc., are still open. In this study human mesenchymal stem cells derived from different fetal and adult tissues were examined in order to explore growth and neurotrophic factor synthesis and biological action, also considering the individual variability of the donors. Cells were derived from different human tissues and characterized according to the guidelines of the International Society for Cellular Therapy. Growth and neurotrophic factor synthesis was evaluated by real time PCR, biological assays and ELISA. It was found that human mesenchymal stem cells produce vascular endothelial-, nerve-growth factor (VEGF, NGF), brain-derived-, ciliary- and glial-derived neurotrophic factors (BDNF, CDGF, GDNF), which are neuroprotective molecules, but the source and the donor influence the synthesis rate. Accordingly, it is suggested that the source and the individual variability are key issues to be considered in the perspective of the clinical use of mesenchymal stem cells in neurological disorders.

J. Neurochem. (2010) 115, 897–909. Abstract Polychlorinated dibenzo-dioxins, furans and dioxin-like polychlorinated biphenyls are ubiquitous in foodstuffs of animal origin and accumulate in the fatty tissues of animals and humans. The most toxic congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a lipophilic endocrine-disrupting molecule that accumulates in adipose tissue, placenta and milk. polychlorinated biphenyls and TCDD are known to interfere with thyroid hormone metabolism and signaling in the developing brain. As thyroid hormone is critical in the myelination process during development, we investigated the effect of a single dose of TCDD prenatal exposure (gestational day 18) on the myelination process. A semi-quantitative analysis of oligodendrocyte markers at different stages of maturation was performed in the offspring’s medulla oblongata, cerebellum, diencephalon and telenchephalon at different postnatal days (2/3, 14, 30 and 135). The most significant alterations observed were: (i) cerebellum and medulla oblongata: altered expression of oligodendroglial lineage and platelet-derived growth factor alpha receptor, myelin basic protein (MBP) mRNAs (P2/3, P135) and MBP protein (P135); (ii) diencephalon: increase in platelet- derived growth factor alpha receptor mRNA level (P2/3); (iii) telenchephalon: decrease in MBP mRNA expression. The oligodendroglial generation capability of adult neural stem/precursor cells obtained ex vivo from TCDD and vehicle-treated dams was then explored. TCDD impairs neurosphere proliferation and retards CNPase-positive cell generation from adult neurospheres.

M. Paradisi, M. Fernandez, G. Del Vecchio, G. Lizzo, G. Marucci, M. Giulioni, E. Pozzati, T. Antonelli, G. Lanzoni, G. P. Bagnara, L. Giardino and L. Calza (2010) Neuropathology and Applied Neurobiology36, 535–550 Ex vivo study of dentate gyrus neurogenesis in human pharmacoresistant temporal lobe epilepsy Aims: Neurogenesis in adult humans occurs in at least two areas of the brain, the subventricular zone of the telencephalon and the subgranular layer of the dentate gyrus in the hippocampal formation. We studied dentate gyrus subgranular layer neurogenesis in patients subjected to tailored antero-mesial temporal resection including amygdalohippocampectomy due to pharmacoresistant temporal lobe epilepsy (TLE) using the in vitro neurosphere assay. Methods: Sixteen patients were enrolled in the study; mesial temporal sclerosis (MTS) was present in eight patients. Neurogenesis was investigated by ex vivo neurosphere expansion in the presence of mitogens (epidermal growth factor + basic fibroblast growth factor) and spontaneous differentiation after mitogen withdrawal. Growth factor synthesis was investigated by qRT-PCR in neurospheres. Results: We demonstrate that in vitro proliferation of cells derived from dentate gyrus of TLE patients is dependent on disease duration. Moreover, the presence of MTS impairs proliferation. As long as in vitro proliferation occurs, neurogenesis is maintained, and cells expressing a mature neurone phenotype (TuJ1, MAP2, GAD) are spontaneously formed after mitogen withdrawal. Finally, formed neurospheres express mRNAs encoding for growth (vascular endothelial growth factor) as well as neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor). Conclusion: We demonstrated that residual neurogenesis in the subgranular layer of the dentate gyrus in TLE is dependent on diseases duration and absent in MTS.

AIM: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer’s disease and for testing new molecules. METHODS: Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software. RESULTS: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt. CONCLUSION: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.

While the study of in vitro regulation of neural stem cell lineage from both embryonic and adult neurospheres is greatly advanced, much less is known about factors acting in situ for neural stem cell lineage in adult brain. We reported that neurotrophin low affinity receptor p75 NTR is present in the subventricular zone (SVZ) in adult male rats. We then characterized co-distribution of markers associated with precursor cells (nestin and PSA-NCAM) with growth factor receptors (p75 NTR , trkA, EGFr) and proliferation-associated antigens (Ki67 and BrDU-uptake) in adult male rat by immunocytochemistry and confocal laser scan microscopy. Distribution of p75 NTR -immunoreactivity (IR) was investigated using different mono- and polyclonal antisera. p75NTR− is not co-distributed with glial fibrillary acid protein. It was found to be co-distributed with a small number of nestin-IR cells, whereas no coexistence with PSA-NCAM-IR was observed. Conversely, p75 NTR -IR was present in numerous dividing cells (Ki-67-positive) and co-distributed with EGFr. In order to verify the possible association between p75 NTR and cell death, we investigated co-distribution of p75 NTR -IR with nuclear condensation images as visualized by Hoechst 33258 staining. While few images indicating nuclear condensation were observed in the SVZ, no coexistence with p75 NTR was found. TrkA- and trkB-IR was not found in the SVZ. We also investigated p75 NTR immunostaining on post-natal day 1 and day 16, because of the dramatic reduction of proliferating cells in SVZ over this time-interval. p75 NTR -IR was not increased in the early post-natal phase. Thus, p75 NTR seems to be associated with cell cycle regulation in SVZ in adult rat brain.