Showing papers on "Neurosphere published in 2002"

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro.

Abstract: Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem-like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage-specific proteins. The high frequencies of clone-forming cells (about 0.1-10 x 10(-3)) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT-PCR analyses of populations of normal brain- and tumor-derived sister clones revealed transcripts for nestin, neuron-specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor-derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch-signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma-derived clones contain cells immunopositive separately for GFAP or neuronal beta-III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors.

Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells.

Abstract: Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-Jκ, a key molecule in Notch signaling, by using RBP-Jκ−/− embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-Jκ−/− or Notch1−/− mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1+/− adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.

Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons

Abstract: Neural stem cells are present both in the developing nervous system and in the adult nervous system of all mammals, including humans. Little is known, however, about the extent to which stem cells in adults can give rise to new neurons. We used immunocytochemistry, electron microscopy, fluorescence microscopy (FM imaging) and electrophysiology to demonstrate that progeny of adult rat neural stem cells, when co-cultured with primary neurons and astrocytes from neonatal hippocampus, develop into electrically active neurons and integrate into neuronal networks with functional synaptic transmission. We also found that functional neurogenesis from adult stem cells is possible in co-culture with astrocytes from neonatal and adult hippocampus. These studies show that neural stem cells derived from adult tissues, like those derived from embryonic tissues, retain the potential to differentiate into functional neurons with essential properties of mature CNS neurons.

Adult Rodent Neurogenic Regions: The Ventricular Subependyma Contains Neural Stem Cells, But the Dentate Gyrus Contains Restricted Progenitors

Abstract: Neurogenesis persists in two adult brain regions: the ventricular subependyma and the subgranular cell layer in the hippocampal dentate gyrus (DG). Previous work in many laboratories has shown explicitly that multipotential, self-renewing stem cells in the subependyma are the source of newly generated migrating neurons that traverse the rostral migratory stream and incorporate into the olfactory bulb as interneurons. These stem cells have been specifically isolated from the subependyma, and their properties of self-renewal and multipotentiality have been demonstrated in vitro. In contrast, it is a widely held assumption that the “hippocampal” stem cells that can be isolated in vitro from adult hippocampus reside in the neurogenic subgranular layer and represent the source of new granule cell neurons, but this has never been tested directly. Primary cell isolates derived from the precise microdissection of adult rodent neurogenic regions were compared using two very different commonly used culture methods: a clonal colony-forming (neurosphere) assay and a monolayer culture system. Importantly, both of these culture methods generated the same conclusion: stem cells can be isolated from hippocampus-adjacent regions of subependyma, but the adult DG proper does not contain a population of resident neural stem cells. Indeed, although the lateral ventricle and other ventricular subependymal regions directly adjacent to the hippocampus contain neural stem cells that exhibit long-term self-renewal and multipotentiality, separate neuronal and glial progenitors with limited self-renewal capacity are present in the adult DG, suggesting that neuron-specific progenitors and not multipotential stem cells are the source of newly generated DG neurons throughout adulthood.

Transplantation of in vitro‐expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats

Abstract: Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetus-derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.

A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells

Abstract: The unique property of stem cells to self-renew suggests specific mechanisms that regulate their cell-cycle progression. In the present study, we identify a novel protein, nucleostemin, found in the nucleoli of CNS stem cells, embryonic stem cells, and several cancer cell lines and preferentially expressed by other stem cell-enriched populations. It contains an N-terminal basic domain and two GTP-binding motifs. When stem cells differentiate, nucleostemin expression decreases rapidly prior to cell-cycle exit both in vitro and in vivo. Depletion or overexpression of nucleostemin reduces cell proliferation in CNS stem cells and transformed cells. Mutation analysis indicates that excessive nucleostemin, particularly mutants that lack the GTP-regulatory domain, prevents cells from entering mitosis and causes apoptosis in a p53-dependent manner. The N-terminal basic domain specifies nucleolar localization, the p53 interaction, and is required for the cell death caused by overexpression. This work describes a novel nucleolar mechanism that controls the cell-cycle progression in CNS stem cells and cancer cells.

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

Abstract: The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 × 106 neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.

Multipotent Neural Stem Cells Reside into the Rostral Extension and Olfactory Bulb of Adult Rodents

Abstract: 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.

Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres.

Abstract: Neural stem cells (NSCs) in vitro are able to generate clonal structures, "neurospheres," that exhibit intra-clonal neural cell-lineage diversity; i.e., they contain, in addition to NSCs, neuronal and glial progenitors in different states of differentiation. The present study focuses on a subset of neurospheres derived from fresh clinical specimens of human brain by using an in vitro system that relies on particular growth factors, serum, and anchorage withdrawal. Thirty individual and exemplary cDNA libraries from these neurosphere clones were clustered and rearranged within a panel after characterization of differentially expressed transcripts. The molecular phenotypes that were obtained indicate that clonogenic NSCs in our in vitro system are heterogeneous, with subsets reflecting distinct neural developmental commitments. This approach is useful for the sorting and expansion of NSCs and facilitates the discovery of genes involved in cell proliferation, communication, fate control, and differentiation.

RNA-binding protein Musashi family: Roles for CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption and antisense ablation

Abstract: Homologues of the Musashi family of RNA-binding proteins are evolutionarily conserved across species. In mammals, two members of this family, Musashi1 (Msi1) and Musashi2 (Msi2), are strongly coexpressed in neural precursor cells, including CNS stem cells. To address the in vivo roles of msi in neural development, we generated mice with a targeted disruption of the gene encoding Msi1. Homozygous newborn mice frequently developed obstructive hydrocephalus with aberrant proliferation of ependymal cells in a restricted area surrounding the Sylvius aqueduct. These observations indicate a vital role for msi1 in the normal development of this subpopulation of ependymal cells, which has been speculated to be a source of postnatal CNS stem cells. On the other hand, histological examination and an in vitro neurosphere assay showed that neither the embryonic CNS development nor the self-renewal activity of CNS stem cells in embryonic forebrains appeared to be affected by the disruption of msi1, but the diversity of the cell types produced by the stem cells was moderately reduced by the msi1 deficiency. Therefore, we performed antisense ablation experiments to target both msi1 and msi2 in embryonic neural precursor cells. Administration of the antisense peptide-nucleotides, which were designed to specifically down-regulate msi2 expression, to msi1−/− CNS stem cell cultures drastically suppressed the formation of neurospheres in a dose-dependent manner. Antisense-treated msi1−/− CNS stem cells showed a reduced proliferative activity. These data suggest that msi1 and msi2 are cooperatively involved in the proliferation and maintenance of CNS stem cell populations.

N-CoR controls differentiation of neural stem cells into astrocytes.

Abstract: Understanding the gene programmes that regulate maintenance and differentiation of neural stem cells is a central question in stem cell biology. Virtually all neural stem cells maintain an undifferentiated state and the capacity to self-renew in response to fibroblast growth factor-2 (FGF2)1,2,3,4,5. Here we report that a repressor of transcription, the nuclear receptor co-repressor (N-CoR), is a principal regulator in neural stem cells, as FGF2-treated embryonic cortical progenitors from N-CoR gene-disrupted mice display impaired self-renewal and spontaneous differentiation into astroglia-like cells. Stimulation of wild-type neural stem cells with ciliary neurotrophic factor (CNTF), a differentiation-inducing cytokine3, results in phosphatidylinositol-3-OH kinase/Akt1 kinase-dependent phosphorylation of N-CoR, and causes a temporally correlated redistribution of N-CoR to the cytoplasm. We find that this is a critical strategy for cytokine-induced astroglia differentiation and lineage-characteristic gene expression. Recruitment of protein phosphatase-1 to a specific binding site on N-CoR exerts a reciprocal effect on the cellular localization of N-CoR. We propose that repression by N-CoR, modulated by opposing enzymatic activities, is a critical mechanism in neural stem cells that underlies the inhibition of glial differentiation.

Human cord blood-derived cells attain neuronal and glial features in vitro

Abstract: Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells.

Engraftment of sorted/expanded human central nervous system stem cells from fetal brain

Abstract: Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133+, CD34–, CD45–, and CD24–/lo (CD133+ cells) Fluorescence-activated cell sorted (FACS) CD133+ cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages CD133–, CD34–, and CD45– sorted cells (∼95% of total fetal brain tissue) fail to initiate neurospheres Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (β-tubulin III and glial fibrillary acidic protein) are not species specific To trace the progeny of human cells after transplantation, CD133+-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter After transduction, GFP+ cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP GFP+ cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model © 2002 Wiley-Liss, Inc

In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord

Abstract: Stem cells isolated from the fourth ventricle and spinal cord form neurospheres in vitro in response to basic fibroblast growth factor (FGF2)+heparin (H) or epidermal growth factor (EGF)+FGF2 together. To determine whether these growth factor conditions are sufficient to induce stem cells within the fourth ventricle and spinal cord to proliferate and expand their progeny in vivo, we infused EGF and FGF2, alone or together, with or without H, into the fourth ventricle for 6 days via osmotic minipumps. Animals were injected with bromodeoxyuridine (BrdU) on days 4, 5 and 6 of infusion in order to label cells proliferating in response to the growth factors. Infusions of EGF+FGF2+H into the fourth ventricle resulted in the largest proliferative effect, a 10.8-fold increase in the number of BrdU+ cells around the fourth ventricle, and a 33.5-fold increase in the number of BrdU+ cells around the central canal of the spinal cord, as compared to vehicle infused controls. The majority of the cells were nestin+ after 6 days of infusion. Seven weeks post-infusion, 22 and 30% of the number of BrdU+ cells induced to proliferate after 6 days of EGF+FGF2+H infusions were still detected around the fourth ventricle and central canal of the spinal cord, respectively. Analysis of the fates of the remaining cells showed that a small percentage of BrdU+ cells around the fourth ventricle and in the white matter of the spinal cord differentiated into astrocytes and oligodendrocytes. BrdU+ neurons were not found in the brainstem or in the grey matter of the cervical spinal cord 7 weeks post-infusion. These results show that endogenous stem cells and progenitors around the fourth ventricle and central canal of the spinal cord proliferate in response to exogenously applied growth factors, but unlike in the lateral ventricle where they generate some new neurons, they only produce new astrocytes and oligodendrocytes at 7 weeks post-infusion.

Disruption of neurogenesis in the subventricular zone of adult mice, and in human cortical neuronal precursor cells in culture, by amyloid beta-peptide: implications for the pathogenesis of Alzheimer's disease.

Abstract: The adult mammalian brain contains populations of stem cells that can proliferate and then differentiate into neurons or glia. The highest concentration of such neural progenitor cells (NPC) is located in the subventricular zone (SVZ) and these cells can produce new olfactory bulb and cerebral cortical neurons. NPC may provide a cellular reservoir for replacement of cells lost during normal cell turnover and after brain injury. However, neurogenesis does not compensate for neuronal loss in age-related neurodegenerative disorders such as Alzheimer’s disease (AD), suggesting the possibility that impaired neurogenesis contributes to the pathogenesis of such disorders. We now report that amyloid β-peptide (Aβ), a self-aggregating neurotoxic protein thought to cause AD, can impair neurogenesis in the SVZ/cerebral cortex of adult mice and in human cortical NPC in culture. The proliferation and migration of NPC in the SVZ of amyloid precursor protein (APP) mutant mice, and in mice receiving an intraventricular infusion of Aβ, were greatly decreased compared to control mice. Studies of NPC neurosphere cultures derived from human embryonic cerebral cortex showed that Aβ can suppress NPC proliferation and differentiation, and can induce apoptosis. The adverse effects of Aβ on neurogenesis were associated with a disruption of calcium regulation in the NPC. Our data show that Aβ can impair cortical neurogenesis, and suggest that this adverse effect of Aβ contributes to the depletion of neurons and the resulting olfactory and cognitive deficits in AD.

Neural stem cell lineages are regionally specified, but not committed, within distinct compartments of the developing brain

Abstract: Regional patterning in the developing mammalian brain is partially regulated by restricted gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progeny. Whether or not neural stem cells, which are considered at the top of the neural lineage hierarchy, are regionally specified remains unknown. Here we show that the cardinal properties of neural stem cells (self-renewal and multipotentiality) are conserved among embryonic cortex, ganglionic eminence and midbrain/hindbrain, but that these different stem cells express separate molecular markers of regional identity in vitro, even after passaging. Neural stem cell progeny derived from ganglionic eminence but not from other regions are specified to respond to local environmental cues to migrate ventrolaterally, when initially deposited on the germinal layer of ganglionic eminence in organotypic slice cultures. Cues exclusively from the ventral forebrain in a 5 day co-culture paradigm could induce both early onset and late onset marker gene expression of regional identity in neural stem cell colonies derived from both the dorsal and ventral forebrain as well as from the midbrain/hindbrain. Thus, neural stem cells and their progeny are regionally specified in the developing brain, but this regional identity can be altered by local inductive cues.

Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat

Abstract: Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell-based therapy to replace lost neurons in neural injury or neurodegenerative disorders.

In vitro differentiation of size-sieved stem cells into electrically active neural cells

Abstract: Size-sieved stem (SS) cells isolated from human bone marrow and propagated in vitro are a population of cells with consistent marker typing, and can form bone, fat, and cartilage. In this experiment, we demonstrated that SS cells could be induced to differentiate into neural cells under experimental cell culture conditions. Five hours after exposure to antioxidant agents (beta-mercaptoethanol +/- retinoic acid) in serum-free conditions, SS cells expressed the protein for nestin, neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), and neuron-specific tubulin-1 (TuJ-1), and the mRNA for NSE and Tau. Immunofluorescence showed that almost all the cells (>98%) expressed NeuN and TuJ-1. After 5 days of beta-mercaptoethanol treatment, the SS cells expressed neurofilament high protein but not mitogen-activated protein-2, glial filament acidic protein, and galactocerebroside. For such long-term-treated cells, voltage-sensitive ionic current could be detected by electrophysiological recording, and the intracellular calcium ion, Ca(2+), concentration can be elevated by high potassium (K(+)) buffer and glutamate. These findings suggest that SS cells may be an alternative source of undifferentiated cells for cell therapy and gene therapy in neural dysfunction.

Somatic epidermal stem cells can produce multiple cell lineages during development.

Abstract: It has been demonstrated that several types of somatic stem cells have the remarkable capacity to differentiate into other types of tissues. We demonstrate here that stem cells from the skin, the largest organ of the body, have the capacity to form multiple cell lineages during development. Using our recently developed sorting technique, we isolated viable homogenous populations of somatic epidermal stem and transient amplifying cells from the skin of 3-day old transgenic mice, who carried the enhanced green fluorescent protein transgene, and injected stem, TA, or unsorted basal epidermal cells into 3.5-day C57BL/6 blastocysts. Only the stem-injected blastocysts produced mice with GFP+ cells in their tissues. We found GFP+ cells in ectodermal, mesenchymal, and neural-crest-derived tissues in E13.5 embryos, 13-day-old neonates, and 60-day-old adult mice, proving that epidermal stem cells survived the blastocyst injection and multiplied during development. Furthermore, the injected stem cells altered their epidermal phenotype and expressed the appropriate proteins for the tissues into which they developed, demonstrating that somatic epidermal stem cells have the ability to produce cells of different lineages during development. These data suggest that somatic epidermal stem cells may show a generalized plasticity expected only of embryonic stem cells and that environmental (extrinsic) factors may influence the lineage pathway for somatic stem cells. Thus, the skin could be a source of easily accessible stem cells that are able to be reprogrammed to form multiple cell lineages.

Large-scale sources of neural stem cells

Abstract: Large-scale sources of neural stem cells are crucial for both basic research and novel approaches toward treating neurological disorders. Three sources that produce neural cells closely resembling their normal counterparts are now available: oncogene immortalized stem cells, neurospheres, and embryonic stem cell (ES)-derived neural cells. Cells including multiple subtypes of CNS and PNS neurons, as well as oligodendrocytes, Schwann cells, and astrocytes, are modeled by these large-scale sources. Although most cell lines were originally from rodents, their human counterparts are being discovered and characterized.