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Results for “Neural stem cell”

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PubMed1999

Intraspinal delivery of neurotrophin-3 using neural stem cells genetically modified by recombinant retrovirus.

Neural stem cells have been shown to participate in the repair of experimental CNS disorders. To examine their potential in spinal cord repair, we used retroviral vectors to genetically modify a clone of neural stem cells, C17, to overproduce neurotrophin-3 (NT-3). The cells were infected with a retrovirus construct containing the NT-3.IRES.lacZ/neo sequence and cloned by limiting dilution and selection for lacZ expression. We studied the characteristics of the modified neural stem cells in vitro and after transplantation into the intact spinal cord of immunosuppressed adult rats. Our results show that: (i) most of the genetically modified cells express both NT-3 and lacZ genes with a high coexpression ratio in vitro and after transplantation; and (ii) large numbers of the xenografted cells survive in the spinal cord of adult rats for at least 2 months, differentiate into neuronal and glial phenotypes, and migrate for long distances. We conclude that genetically modified neural stem cells, acting as a source of neurotrophic factors, have the potential to participate in spinal cord repair.

Animals↗
PubMed1999

Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors.

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.

Animals↗
PubMedDate not supplied

Passaging protocols for mammalian neural stem cells in suspension bioreactors.

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.

Animals↗
PubMed2006

[Isolation of neural stem cells from the spinal cords of human fetus.].

Neural stem cells are a potential therapeutic source for cellular transplantation therapy in neurological diseases. The present paper was aimed to investigate whether neural stem cells could be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C without any additional treatments for 2, 6 and 12 h before use. The spinal cords were anatomized out and divided into cervical cords, thoracic cords and lumbar/sacral cords. Then the spinal cord segments were used for cell culture separately. Neural stem cells were isolated from the segments and cultured in bFGF, EGF and N2 supplement containing free-serum DMEM/F12 (1:1) medium. In order to examine the differentiation potential, the stem cells were induced to differentiate with 5% fetal bovine serum on poly-l-lysine substrate. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Indirect fluorescent immunocytochemistry was used to examine the expressions of neural stem cell marker (nestin), neuron marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. One-way analysis of variance and Kruskal-Wallis test were used for the statistical comparison. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. Both the cells on the surface and inside the neurospheres showed nestin immunoreactivity. Therefore, nearly all the cells that composed the neurospheres were nestin-positive undifferentiated cells. When the spheres were induced to differentiate, they could yield GFAP-positive astrocytes and MAP2-positive neurons including ChAT-positive cholinergic neurons. Primary neurospheres could be dissociated mechanically, expand in subcultures and maintain the differentiation potential. In clonal cultures, single cells from a single primary sphere could give rise to new neurospheres, which had the same differentiation potential as the primary spheres. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation time of the fetus was prolonged to 12 h, the number of primary neurospheres decreased sharply. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for experimental and potential therapeutic purposes.

Cell Differentiation↗
PubMed2004

Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors.

Pluripotent neural stem cells (NSCs) have been used as replacement cells in a variety of neurological disease models. Among the many different NSCs that have been used to date, most robust results have been obtained with the immortalized neural stem cell line (C17.2) isolated from postnatal cerebellum. However, it is unclear if other NSCs isolated from different brain regions are similar in their potency as replacement therapies. To assess the properties of NSC-like C17.2 cells, we compared the properties of these cells with those reported for other NSC populations identified by a variety of different investigators using biological assays, microarray analysis, RT-PCR, and immunocytochemistry. We show that C17.2 cells differ significantly from other NSCs and cerebellar granule cell precursors, from which they were derived. In particular, they secrete additional growth factors and cytokines, express markers that distinguish them from other progenitor populations, and do not maintain karyotypic stability. Our results provide a caution on extrapolating results from C17.2 to other nonimmortalized stem cell populations and provide an explanation for some of the dramatic effects that are seen with C17.2 transplants but not with other cells. We suggest that, while C17.2 cells can illustrate many fundamental aspects of neural biology and are useful in their own right, their unique properties cannot be generalized.

Animals↗
PubMed2005

Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal.

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.

Animals↗
PubMed2003

PARP inhibition improves the effectiveness of neural stem cell transplantation in experimental brain trauma.

Neural stem cell transplantation is a promising new treatment of ischemic or traumatic brain injury. We have now investigated the involvement of the peroxynitrite - poly(ADP-ribose) polymerase (ONOO- - PARP) activation cascade in brain trauma and neural stem cell transplantation. The forelimb motor cortex of adult male rats was exposed to cold lesion (-60 degrees C) and motor function was monitored. Neural stem cells isolated from E14 rat embryos were labeled with brome deoxyuridine (BrDU) and injected into the injured cortex 6 days after the lesion. After another 6 days, the survival and differentiation of the grafted cells were investigated with immunohistochemistry. Increased production of ONOO- revealed by tyrosine nitration was seen in the lesion 2 days after transplantation. Animals treated with the ONOO- decomposition catalyst FP15 or the PARP inhibitor PJ34 had a significantly improved motor score, when compared to vehicle-treated controls. The neurological score further improved following stem cell grafting in the PJ34 treated, but not in the control animals. Six days after transplantation, differentiated BrDU positive cells were found in the cortical penumbra. The majority of these differentiated cells expressed an astrocyte marker and some of the cells expressed oligodendrocyte or neuronal markers. The number of surviving transplanted cells was significantly higher in the PJ34 treated group. Inhibition of the ONOO- - PARP activation cascade significantly improves the effectiveness of neural stem cell transplantation and promotes rapid functional recovery.

Animals↗
PubMed2001

Upregulated expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in differentiated neural stem cells.

Adult rat hippocampus-derived neural stem cells are incorporated into neural tissues, and differentiate to neuronal and glial cells. However, the cell surface protein molecules are, to date, undefined. RT-PCR, immunoblotting and immunocytochemistry showed the increased expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in the neural stem cells after the differentiation induced by retinoic acid. Our data indicate that N-syndecan may be involved in the differentiation of neural stem cells.

Animals↗
PubMed2004

Identification of c-Kit receptor as a regulator of adult neural stem cells in the mammalian eye: interactions with Notch signaling.

Neural stem cells are present in specific regions of the adult central nervous system (CNS). Recent evidence suggests that the ciliary epithelium (CE), a CNS derivative, in the adult mammalian eye, harbors a quiescent population of neural stem cells. Here, we report the identification of c-Kit signaling as one of the regulators of adult CE neural stem cells in vitro. c-Kit receptors are expressed in proliferating adult CE neural stem cells and colocalized with neural progenitor markers. Perturbation of c-Kit signaling influences the self-renewal and differentiation of CE neural stem cells, thus demonstrating the role of c-Kit signaling in the maintenance of these cells. In addition, we observed an influence of c-Kit-mediated signaling on the expression of Notch1, another critical regulator of neural stem cells. Our observations suggest that, given the importance of preservation of a stem cell pool for generating different cell types at different times, multiple signaling pathways act in concert for the maintenance of neural stem cells.

Animals↗
PubMed2003

Postnatal astrocytes promote neural induction from adult human bone marrow-derived stem cells.

Neural stem cells (NSCs) have generated considerable interest because of their potential as a source of defined cells for drug screening or cell-based therapies for neurodegenerative diseases. Ethical and practical considerations limit the availability of human fetal-derived neural tissue and highlight the need to consider alternative sources of human NSCs. Because of their ready availability, their ability to be easily expanded, and reports of neural potential, bone marrow-derived populations have become the focus of intense study with regard to their potential clinical utility. However, recent identification of spontaneous cell fusion and limited neuronal differentiation has tempered initial optimism. In this study, we demonstrate the monoclonal neural and mesodermal potential of adult human bone marrow mesenchymal cells. Critically, we show that sequential treatment with the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) followed by postnatal hippocampal astrocyte conditioned medium significantly promotes the generation of neurofilament(+)/beta-tubulin(+) cells from bone marrow precursors. The ability to generate almost limitless numbers of neural precursors from a readily accessible autologous adult human source provides a platform for further studies and potentially has important therapeutic implications.

Actins↗
PubMed2006

Neural stem cells in mammalian development.

Neural stem cells (NSCs) are primary progenitors that give rise to neurons and glia in the embryonic, neonatal and adult brain. In recent years, we have learned three important things about these cells. First, NSCs correspond to cells previously thought to be committed glial cells. Second, embryonic and adult NSCs are lineally related: they transform from neuroepithelial cells into radial glia, then into cells with astroglial characteristics. Third, NSCs divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. These advances challenge our traditional perceptions of glia and stem cells, and provide the foundation for understanding the molecular basis of mammalian NSC behavior.

Animals↗
PubMed1999

Human neural stem cells: isolation, expansion and transplantation.

Neural stem cells, with the capacity to self renew and produce the major cell types of the brain, exist in the developing and adult rodent central nervous system (CNS). Their exact function and distribution is currently being assessed, but they represent an interesting cell population, which may be used to study factors important for the differentiation of neurons, astrocytes and oligodendrocytes. Recent evidence suggests that neural stem cells may also exist in both the developing and adult human CNS. These cells can be grown in vitro for long periods of time while retaining the potential to differentiate into nervous tissue. Significantly, many neurons can be produced from a limited number of starting cells, raising the possibility of cell replacement therapy for a wide range of neurological disorders. This review summarises this fascinating and growing field of neurobiology, with a particular focus on human tissues.

Animals↗
PubMed2004

Low-level tyrosine hydroxylase (TH) expression allows for the generation of stable TH+ cell lines of human neural stem cells.

Genetic engineering of neurotransmitter metabolic routes is important for the development of neurotransmitter-producing cells for the ex vivo gene therapy of many CNS diseases. Human neural stem cells (hNSCs) are excellent candidates to serve this role, but, for the case of Parkinson's disease, the cells do not normally express the rate-limiting dopamine (DA) synthesis enzyme tyrosine hydroxylase (TH), and are not equipped with the detoxifying mechanisms needed to prevent the neurotoxicity associated with the DA phenotype. In this study we have examined the capacity of hNSCs for ectopic expression of human TH. High-level TH expression (from viral promoters) leads to growth arrest and hNSC death (associated with an increase in p53 expression and nuclear fragmentation), which can be counteracted by treatment with a pan-caspase inhibitor. As a consequence, stable TH-expressing hNSC sublines could not be derived using viral promoters. In contrast, moderate TH expression (from a human housekeeping promoter, polyubiquitin gene), allows for stable TH+ subclone derivation, seemingly originating from low-expressing cells. Our results are thus compatible with the view that stable TH-expressing hNSC lines can be generated if TH expression levels are kept at a moderate level, and that the goal normally set of aiming at high-level TH expression may need to be reconsidered. These results may be relevant for the generation of TH/DA-producing human neural cells for in vitro and neurotransplantation research in Parkinson's disease.

Cell Division↗
PubMed2005

Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation.

BACKGROUND: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. RESULTS: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. CONCLUSION: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

Animals↗
PubMed2000

Asymmetric division of Drosophila neural stem cells: a basis for neural diversity.

Recent studies of Drosophila neural precursor cells have unveiled the essential roles played by asymmetric cell divisions in the determination of cell fates during neural development. Our understanding now extends to the molecular nature of the cell polarity that underlies asymmetric divisions. This polarity is conserved among neural stem cells, epithelial cells and fertilized eggs.

Animals↗
PubMed2001

Neural stem cells.

In recent papers on neural stem cells, two areas of progress stand out. First, there has been a substantial growth in the published examples of 'transdifferentiation'. Second, there has been progress, albeit more modest, in studies of repair by neural stem cells following transplantation into animal models of disease.

Animals↗
PubMed2006

Experimental study on plasticity of proliferated neural stem cells in adult rats after cerebral infarction.

OBJECTIVE: To investigate whether there is endogenous neural stem cell proliferation and whether these proliferated neural stem cells represent neural plasticity in the adult rats after cerebral infarction. METHODS: Cerebral infarction models of rats were established and the dynamic expression of bromodeoxyuridine (BrdU), BrdU/polysialylated neural cell adhesion molecule (PSA-NCAM) were determined by immunohistochemistry and immunofluorescence staining. BrdU was used to mark dividing neural stem cells. PSA-NCAM was used to mark the plasticity of neural stem cells. RESULTS: Compared with controls, the number of BrdU-positive cells in the subventricular zone (SVZ) and hippocampus increased significantly at 1st day after cerebral infarction (P < 0.05), reached maximum at 7th day, decreased markedly at 14th day, but it was still elevated compared with that of the controls (P < 0.05). The number of BrdU-labeled with PSA-NCAM-positive cells increased significantly at 7th day (P < 0.05), reached maximum at 14th day, markedly decreased at 28th day, but it was still elevated compared with that of the controls (P < 0.05). It was equal to 60% of the number of BrdU-positive cells in the same period. CONCLUSION: Cerebral infarction may stimulate the proliferation of endogenous neural stem cells in situ and most proliferated neural stem cells represent neural plasticity.

Animals↗
PubMed2002

Human neural stem cells: electrophysiological properties of voltage-gated ion channels.

We have characterized the profile of membrane currents in an immortalized human neural stem cell line, HB1.F3 cells, using whole-cell patch clamp technique. Human neural stem cell line generated from primary cell cultures of embryonic human telencephalon using a replication-incompetent retroviral vector containing v-myc expresses nestin, a cell type-specific marker for neural stem cells. The human neural stem cells expressed both outward and inward K(+) currents with no evidence for Na(+) currents. The density of the outward, delayed rectifying type K(+) current was 1.8 +/- 0.015 nA/pF, and that of the inwardly rectifying K(+) current was 0.37 +/- 0.012 nA/pF (at 30 mM of [K(+)](o)). In order to induce neuronal differentiation of the neural stem cells, a full-length coding region of NeuroD, a neurogenic transcription factor, was transfected into HB1.F3 cells. Introduction of NeuroD induced expression of Na(+) currents with the current density of 0.042 +/- 0.011 nA/pF. The presence of two types of K(+) currents and expression of Na(+) currents induced by NeuroD appear to reflect the characteristic physiological features of human neural stem cells.

Basic Helix-Loop-Helix Proteins↗