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Background: Neural stem cells (NSCs) represent an optimal tool for studies and therapy of neurodegenerative diseases. We recently established a v-myc immortalized human NSC (IhNSC) line, which retains stem properties comparable to parental cells. Oxygen concentration is one of the most crucial environmental conditions for cell proliferation and differentiation both in vitro and in vivo. In the central nervous system, physiological concentrations of oxygen range from 0.55 to 8% oxygen. In particular, in the in the subventricular zone niche area, it's estimated to be 2.5 to 3%., Methodology/principal Findings: We investigated in vitro the effects of 1, 2.5, 5, and 20% oxygen concentrations on IhNSCs both during proliferation and differentiation. The highest proliferation rate, evaluated through neurosphere formation assay, was obtained at 2.5 and 5% oxygen, while 1% oxygen was most noxious for cell survival. The differentiation assays showed that the percentages of beta-tubIII+ or MAP2+ neuronal cells and of GalC+ oligodendrocytes were significantly higher at 2.5% compared with 1, 5, or 20% oxygen at 17 days in vitro. Mild hypoxia (2.5 to 5% oxygen) promoted differentiation into neuro-oligodendroglial progenitors as revealed by the higher percentage of MAP2+/Ki67+ and GalC+/Ki67+ residual proliferating progenitors, and enhanced the yield of GABAergic and slightly of glutamatergic neurons compared to 1% and 20% oxygen where a significant percentage of GFAP+/nestin+ cells were still present at 17 days of differentiation., Conclusions/significance: These findings raise the possibility that reduced oxygen levels occurring in neuronal disorders like cerebral ischemia transiently lead to NSC remaining in a state of quiescence. Conversely, mild hypoxia favors NSC proliferation and neuronal and oligodendroglial differentiation, thus providing an important advance and a useful tool for NSC-mediated therapy of ischemic stroke and neurodegenerative diseases like Parkinson's disease, multiple sclerosis, and Alzheimer's disease.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Recent studies have raised appealing possibilities of replacing damaged or lost neural cells by transplanting in vitro-expanded neural precursor cells (NPCs) and/or their progeny. Magnetic resonance (MR) tracking of superparamagnetic iron oxide (SPIO)-labeled cells is a noninvasive technique to track transplanted cells in longitudinal studies on living animals. Murine NPCs and human mesenchymal or hematopoietic stem cells can be efficiently labeled by SPIOs. However, the validation of SPIO-based protocols to label human neural precursor cells (hNPCs) has not been extensively addressed. Here, we report the development and validation of optimized protocols using two SPIOs (Sinerem and Endorem) to label human hNPCs that display bona fide stem cell features in vitro. A careful titration of both SPIOs was required to set the conditions resulting in efficient cell labeling without impairment of cell survival, proliferation, self-renewal, and multipotency. In vivo magnetic resonance imaging (MRI) combined with histology and confocal microscopy indicated that low numbers (5 x 10(3) to 1 x 10(4)) of viable SPIO-labeled hNPCs could be efficiently detected in the short term after transplantation in the adult murine brain and could be tracked for at least 1 month in longitudinal studies. By using this approach, we also clarified the impact of donor cell death to the MR signal. This study describes a simple protocol to label NPCs of human origin using SPIOs at optimized low dosages and demonstrates the feasibility of noninvasive imaging of labeled cells after transplantation in the brain; it also evidentiates potential limitations of the technique that have to be considered, particularly in the perspective of neural cell-based clinical applications.

Neural stem cells (NSCs) have been identified in the mature central nervous system (CNS), and they reside in specific areas. Cultures of NSCs can be successfully established in vitro by exploiting the NeuroSphere assay. This methodology relies on the continuous exposure of neural cells to mitogens such as epidermal growth factor and fibroblast growth factor-2. Under these conditions, only NSCs and highly undifferentiated progenitors proliferate, whereas committed precursors and terminally differentiated cells are eliminated from the culture. The proper application of this method to the cells allows the establishment of long-term expanding stable NSC lines, starting from different neural tissues as the adult rodent CNS and human brain tumor specimens.

The discovery of stem cell populations in the adult central nervous system (CNS) that continually produce neurons and glial cells, and the hypothesis that they could contribute to neural plasticity/repair, has opened new and exciting areas of research in basic cell biology and regenerative medicine. The success of these studies relies on understanding the functional features and the normal fate of neural stem cells (NSCs) in vivo as well on the development of in vitro culture conditions enabling isolation, extensive propagation, and rigorous characterization of the "putative" NSCs. The neurosphere assay (NSA) has emerged as a valuable tool for isolating embryonic and adult CNS stem cells and for studying their biology. However, because this assay may select and expand a heterogeneous stem/progenitor cell population, rigorous clonal and serial subcloning analyses are required to detect and document stem cell activity and to unequivocally identify bona fide stem cells. We illustrate and discuss methods for the isolation, propagation, cryopreservation, and functional characterization of NSCs, focusing on the essential issue of their clonogenic capacity.

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

Background: HHV-6 is the etiologic agent of exanthem subitum, a pediatric illness that may be associated with clinical and laboratory signs of central nervous system involvement. The absence of suitable experimental models has so far hampered the elucidation of the mechanisms of HHV-6-mediated neural cell damage. Recently, the growing knowledge in neurobiology has permitted the establishment of long-term cultures of human neural stem cells (hNSC) that, by virtue of their self-renewal capacity and multipotentiality, provide a valuable tool for the study of neurodegenerative disorders., Objectives and Study Design: We studied the effects of HHV-6 infection in differentiated cultures of hNSC derived from the telencephalic and diencephalic regions of a 13.5 week post conception (pcw) fetal brain. The prototypic HHV-6 strain GS (subgroup A) was used., Results: hNSC were differentiated ex vivo to obtain mixed cultures encompassing astrocytes, neurons and oligodendrocytes. These differentiated hNSC cultures were found to be susceptible to productive HHV-6A infection, resulting in the formation of syncytia associated with phenotypic alterations., Conclusion: These results demonstrate that hNSC may provide a physiologically relevant model to investigate the pathogenic role of HHV-6 in central nervous system disorders.

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.

Aquaporins (AQP) are water channel proteins that play important roles in the regulation of water homeostasis in physiological and pathological conditions. AQP4 and AQP9, the main aquaporin subtypes in the brain, are expressed in the adult forebrain subventricular zone (SVZ), where neural stem cells (NSCs) reside, but little is known about their expression and role in the NSC population, either in vivo or in vitro. Also, no reports are available on the presence of these proteins in human NSCs. We performed a detailed molecular and phenotypical characterization of different AQPs, and particularly AQP4 and AQP9, in murine and human NSC cultures at predetermined stages of differentiation. We demonstrated that AQP4 and AQP9 are expressed in adult murine SVZ-derived NSCs (ANSCs) and that their levels of expression and cellular localization are differentially regulated upon ANSC differentiation into neurons and glia. AQP4 (but not AQP9) is expressed in human NSCs and their progeny. The presence of AQP4 and AQP9 in different subsets of ANSC-derived glial cells and in different cellular compartments suggests different roles of the two proteins in these cells, indicating that ANSC-derived astrocytes might maintain in vitro the heterogeneity that characterize the astrocyte-like cell populations in the SVZ in vivo. The development of therapeutic strategies based on modulation of AQP function relies on a better knowledge of the functional role of these channels in brain cells. We provide a reliable and standardized in vitro experimental model to perform functional studies as well as toxicological and pharmacological screenings., (2005 Wiley-Liss, Inc.)

Widespread demyelination and axonal loss are the pathological hallmarks of multiple sclerosis. The multifocal nature of this chronic inflammatory disease of the central nervous system complicates cellular therapy and puts emphasis on both the donor cell origin and the route of cell transplantation. We established syngenic adult neural stem cell cultures and injected them into an animal model of multiple sclerosis--experimental autoimmune encephalomyelitis (EAE) in the mouse--either intravenously or intracerebroventricularly. In both cases, significant numbers of donor cells entered into demyelinating areas of the central nervous system and differentiated into mature brain cells. Within these areas, oligodendrocyte progenitors markedly increased, with many of them being of donor origin and actively remyelinating axons. Furthermore, a significant reduction of astrogliosis and a marked decrease in the extent of demyelination and axonal loss were observed in transplanted animals. The functional impairment caused by EAE was almost abolished in transplanted mice, both clinically and neurophysiologically. Thus, adult neural precursor cells promote multifocal remyelination and functional recovery after intravenous or intrathecal injection in a chronic model of multiple sclerosis.

The appropriate control of proliferation of neural precursors has fundamental implications for the development of the central nervous system and for cell homeostasis/replacement within specific brain regions throughout adulthood. The role of genetic determinants in this process is largely unknown. We report the expression of the homeobox transcription factor Emx2 within the periventricular region of the adult telencephalon. This neurogenetic area displays a large number of multipotent stem cells. Adult neural stem cells isolated from this region do express Emx2 and down-regulate it significantly upon differentiation into neurons and glia. Abolishing or, increasing Emx2 expression in adult neural stem cells greatly enhances or reduces their rate of proliferation, respectively. We determined that altering the expression of Emx2 affects neither the cell cycle length of adult neural stem cells nor their ability to generate neurons and glia. Rather, when Emx2 expression is abolished, the frequency of symmetric divisions that generate two stem cells increases, whereas it decreases when Emx2 expression is enhanced.

The lateral walls of the forebrain lateral ventricles are the richest source of stem cells in the adult mammalian brain. These stem cells give rise to new olfactory neurons that are renewed throughout life. The neurons originate in the subventricular zone (SVZ), migrate within the rostral extension (RE) of the SVZ along the rostral migratory stream (RMS) within tube-like structures formed of glial cells, to eventually reach the olfactory bulb (OB). We demonstrate that, contrary to the current view, multipotential (neuronal-astroglial-oligodendroglial) precursors with stem cell features can be isolated not only from the SVZ but also from the entire RE, including the distal portion within the OB. Specifically, these stem cells do not derive from the migratory neuroblasts coming from the SVZ. Interestingly, stem cells isolated from the proximal RE generate significantly more oligodendrocytes, and those from the distal RE proliferate significantly more slowly than stem cells derived from the SVZ and other RE regions. These findings demonstrate that stem cells are not confined to the forebrain periventricular region and indicate that stem cells endowed with different functional characteristics occur at different levels of the SVZ-RE pathway.

Stem cells play an essential role during the processes of embryonic tissue formation and development and in the maintenance of tissue integrity and renewal throughout adulthood. The differentiation potential of stem cells in adult tissues has been thought to be limited to cell lineages present in the organ from which they derive, but there is evidence that somatic stem cells may display a broader differentiation repertoire. This has been documented for bone marrow stem cells (which can give rise to muscle, hepatic and brain cells) and for muscle precursors, which can turn into blood cells. The adult central nervous system (CNS) has long been considered incapable of cell renewal and structural remodeling. Recent findings indicate that, even in postnatal and adult mammals, neurogenesis does occur in different brain regions and that these regions actually contain adult stem cells. These cells can be expanded both in vivo and ex vivo by exposure to different combinations of growth factors and subsequently give rise to a differentiated progeny comprising the major cell types of the CNS. Almost paradoxically, adult neural stem cells display a multipotency much broader than expected, since they can differentiate into non-CNS mesodermal-derivatives, such as blood cells and skeletal muscle cells. We review the recent findings documenting this unforeseen plasticity and unexpected developmental potential of somatic stem cells in general and of neural stem cells in particular. To better introduce these concepts, some basic notions on the functional properties of adult neural stem cells will also be discussed, particularly focusing on the emerging role of the microenvironment in determining and maintaining their peculiar characteristics.