We investigated in vitro the properties of selected populations of cancer stem-like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice. The angiogenic potential of tumorospheres and glioblastoma-derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance-associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells' positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies.

The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.

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