Showing papers on "Neurosphere published in 1995"

In vitro differentiation of embryonic stem cells into glial cells and functional neurons.

Abstract: Mouse embryonic stem cells were induced to differentiate in culture with retinoic acid Putative precursors of neurons and glial cells (nestin-positive cells) were clearly identified as early as three days after the onset of differentiation At day 6, neuron-like cells could be clearly identified, either as isolated cells or as cellular networks Some of these cells were positive for astrocyte- or oligodendrocyte-specific antigens (GFAP or O4 antigens, respectively) Other cells were positive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin) Some neuronal-like cells were also positive for acetylcholinesterase activity or glutamic acid decarboxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons Electrophysiological analyses performed in voltage clamp conditions showed that cell membranes contained voltage-dependent channels Overshooting action potentials could be triggered by current injection Taken together, these data provide evidence that embryonic stem cells can differentiate first into neuron-glia progenitors, and later into glial cells and functional neurons, in vitro This technique provides an unique system to study early steps of neuronal differentiation in vitro

In vitro growth and proliferation of genetically modified multipotent neural stem cells and their progeny

Abstract: A method for producing genetically modified neural cells comprises culturing cells derived from embryonic, juvenile, or adult mammalian neural tissue with one or more growth factors that induce multipotent neural stem cells to proliferate and produce multipotent neural stem cell progeny which include more daughter multipotent neural stem cells and undifferentiated progeny that are capable of differentiating into neurons, astrocytes, and oligodendrocytes. The proliferating neural cells can be transfected with exogenous DNA to produce genetically modified neural stem cell progeny. The genetic modification can be for the production of biologically useful proteins such as growth factor products, growth factor receptors, neurotransmitters, neurotransmitter receptors, neuropeptides and neurotransmitter synthesizing genes. The multipotent neural stem cell progeny can be continuously passaged and proliferation reinitiated in the presence of growth factors to result in an unlimited supply of neural cells for transplantation and other purposes. Culture conditions can be provided that induce the genetically modified multipotent neural stem cell progeny to differentiate into neurons, astrocytes, and oligodendrocytes in vitro.

In vitro growth and proliferation of multipotent neural stem cells and their progeny

Abstract: A method for the in vitro proliferation and differentiation of neural stem cells and stem cell progeny comprising the steps of (a) isolating the cells from a mammal, (b) exposing the cells to a culture medium containing a growth factor, (c) inducing the cells to proliferate, and (d) inducing the cells to differentiate is provided.

Human neuroblastoma I-type cells are malignant neural crest stem cells.

Abstract: Human neuroblastoma I-type cells isolated from cell lines in vitro are morphologically intermediate between neuroblastic (N) cells, with properties of embryonic sympathoblasts, and substrate-adherent (S) cells having properties of embryonic Schwann/glial/melanocytic cells of the neural crest. I cells have biochemical features of both N and S cells. We propose that the I-type cell represents a malignant neural crest stem cell. The strongest evidence in support of this hypothesis is that: (a) I cells can generate progeny that have neuronal properties, i.e., are committed neuroblasts, or properties of nonneuronal, embryonic neural crest-derived cells; and (b) I-type cells can generate multipotent I-type progeny, indicating their capacity for self-renewal, a feature of stem cells. We report here that I-type cells, derived from four different human neuroblastoma cell lines and experimentally induced to differentiate, give rise to cells with distinct N or S cell phenotypes, indicative of I cell multipotentiality. Experiments with a large panel of I-type subclones, isolated from clonal I-type BE(2)-C cells and exposed to retinoic acid to induce neuronal differentiation or 5-bromo-2'-deoxyuridine to obtain S-type cells, demonstrated that differentiation occurs via induction and selection and not by selection of spontaneously arising variants. The differentiation phenotype was stable. We conclude that human neuroblastoma I-type cells are multipotent embryonic precursor cells of the peripheral nervous system, capable of either neuronal or nonneuronal neural crest cell differentiation.

Immoralized neural crest stem cells and methods of making

Abstract: The invention includes mammalian multipotent neural stem cells and their progeny and methods for the isolation and clonal propagation of such cells. At the clonal level the stem cells are capable of self regeneration and asymmetrical division. Lineage restriction is demonstrated within developing clones which are sensitive to the local environment. The invention also includes such cells which are transfected with foreign nucleic acid, e.g., to produce an immortalized neural stem cell. The invention further includes transplantation assays which allow for the identification of mammalian multipotent neural stem cells from various tissues and methods for transplanting mammalian neural stem cells and/or neural or glial progenitors into mammals. A novel method for detecting antibodies to neural cell surface markers is disclosed as well as a monoclonal antibody to mouse LNGFR.

Neural transplantation using proliferated multipotent neural stem cells and their progeny

Abstract: The invention provides methods of transplanting multipotent neural stem cell progeny to a host by obtaining a population of cells derived from mammalian neural tissue containing at least one multipotent CNS multipotent neural stem cell; culturing the neural stem cell in a culture medium containing one or more growth factors which induce multipotent neural stem cell proliferation; inducing proliferation of the multipotent neural stem cell to produce neural stem cell progeny which includes multipotent neural stem cell progeny cells; and transplanting the multipotent neural stem cell progeny to the host. Also provided are methods of transplanting neural stem cell progeny to a host by obtaining an in vitro cell culture containing CNS neural stem cells where one or more cells in the culture (i) proliferates in a culture medium supplemented with one or more mitrogens, (ii) retains the capacity for renewed proliferation, and (iii) maintains the multipotential capacity, under suitable culture conditions, to differentiate into neurons, astrocytes, and oligodendrocytes; and transplanting the one or more cells to the hose.

Growth factor-induced proliferation of neural precursor cells in vivo

Abstract: A method is described for inducing in vivo proliferation of precursor cells located in mammalian neural tissue by administering to the mammal a fibroblast growth factor and at least one additional growth factor selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin. The method can be used to replace damaged or missing neurons and/or glia. Another method is described for transplanting multipotent neural stem cell progeny into a mammal. The method comprises the steps of administering growth factors to a mammal to induce in vivo proliferation of neural precursor cells, removing the precursor cell progeny from the mammal, culturing the removed cells in vitro in the presence of one or more growth factors that induces multipotent neural stem cell proliferation, and implanting the multipotent neural stem cell progeny into the mammal.

In vitro induction of dopaminergic cells

Abstract: The expression of thyrosine hydroxylase in neural cells in vitro can be induced by contacting neural cells with a culture medium comprising at least one member of the fibroblast growth factor family in combination with conditioned medium or molecules of the transforming growth factor beta family. The method induces neural cells obtained from normally non-dopaminergic neural tissue such as the striatum and the cortex to express tyrosine hydroxylase. The cells can be used to treat neurological disorders in patients requiring dopaminergic cells.

Epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and basic fibroblast growth factor (bFGF) differentially influence neural precursor cells of mouse embryonic mesencephalon.

Abstract: Growth factors are key elements in the process of neural cell differentiation. We examined the effects of classical mitogens on neural precursor cells, by culturing mouse cells of the embryonic (13.5 days postcoitum) mesencephalon and treating them with epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and transforming growth factor-beta (TGF-beta). Our initial results show that EGF, TGF-alpha, or bFGF, but not NGF or TGF-beta, induced general proliferation of the cultured cells, followed by formation of colonies. Combinations of these three growth factors suggest that most cells with the capacity to form colonies responded to EGF, TGF-alpha, or bFGF. The number of colonies increased significantly when EGF, but not TGF-alpha, was used in combination with bFGF. Furthermore, a population responding only to EGF + bFGF was detected in the dorsal mesencephalon. The colony-forming activity of bFGF was dependent on insulin, but bFGF and insulin cooperation was indirect since we could not observe colony formation in subcultures of cells derived from colonies, even in the presence of insulin. Cells obtained from our colonies displayed neuronal and glial morphology and expressed markers of both neurons and astrocytes; nestin, a marker of neural precursor cells, was also expressed in the majority of colonies. Growth factors also influenced neuronal maturation; the best neurite outgrowth was obtained from cells derived from bFGF-induced colonies cultured in the presence of EGF + bFGF. These data indicate the existence of neural precursor cells in the embryonic mesencephalon that respond differentially to growth factors.

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro

Abstract: In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated in vitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS.

Isolation and Characterization of Human Epidermal Stem Cells

Abstract: 1. The keratinocytes in human epidermis are constantly turned over and replaced by a population of stem cells located in the basal epidermal layer. Until recently there were no markers allowing the isolation of viable epidermal stem cells. However, it has now been shown that epidermal stem cells can be isolated both in vitro and direct from the epidermis as they express high levels of functional β 1 integrin family receptors for extracellular matrix proteins. 2. The evidence for integrins as stem cell markers and the insights that have been gained into stem cell behaviour are reviewed.

In vitro models of cns function and dysfunction

Abstract: Proliferating or proliferated multipotent neural stem cells and their progeny are used to produce a CNS model system for the study of neural development and function and for determining the CNS effects of novel therapeutic and other biological agents. The neural stem cells are obtained from small amounts of either normal or diseased CNS tissue from pre- and post natal individuals. The invention allows for large amounts of tissue, which may be clonally derived to limit variability, to be generated from a relatively small amount of CNS tissue. The invention describes a CNS model system whereby the differentiated progeny of the neural stem cells include multiple types of CNS cells, including neurons, astrocytes and oligodendrocytes. Screening for the effects of neurological or other biological agents and the analysis of gene expression in the multipotent neural stem cells and in the stem-cell derived progeny of a normal or a diseased donor may be undertaken using this model system.

Method for promoting neural growth comprising administering a soluble neural cell adhesion molecule

Abstract: The invention features a method for promoting neural growth in vivo in the mammalian central nervous system by administering a neural cell adhesion molecule which can overcome inhibitory molecular cues found on glial cells and myelin to promote neural growth. Also featured active fragments, cognates, congeners, mimics, analogs, secreting cells and soluble molecules thereof, as well as antibodies thereto, and DNA molecules, vectors and transformed cells capable of expressing them. The invention also includes transgenic mouse lines expressing a neural adhesion molecule in differentiated astrocytes, and cells and tissues derived therefrom. The expression of the neural adhesion molecule enhances neurite outgrowth on central nervous system tissue derived from these transgenic mice. The invention also features methods for enhancing neuronal outgrowth of CNS neurons, for enhancing memory and for increasing synaptic efficacy. Also featured are methods of testing drugs which modulate the effects of the neural adhesion molecule, and assay systems suitable for such methods.

Neural cells from dogfish embryos express the same subtype-specific antigens as mammalian neural cells in vivo and in vitro.

Abstract: Neural cells are classically identified in vivo and in vitro by a combination of morphological and immunocytochemical criteria. Here, we demonstrate that antibodies used to identify mammalian oligodendrocytes, neurons, and astrocytes recognize these cell types in the developing spiny dogfish central nervous system and in cultures prepared from this tissue. Oligodendrocyte-lineage-specific antibodies O1, O4, and R-mAb labeled cells in the 9 cm dogfish brain stem's medial longitudinal fascicle (MLF) and in areas lateral to it. Process-bearing cells, cultured from the dogfish brain stem, were also labeled with these antibodies. An anti-lamprey neurofilament antibody (LCM), which recognized 60 and 150 kDa proteins in dogfish brain stem homogenates, labeled axons and neurons in the brain stem and axons in the cerebellum of the dogfish embryo. It also labeled cell bodies and/or processes of some cultured cerebellar cells. An anti-bovine glial fibrillary acidic protein antibody, which recognized 42-44 kDa protein(s) in dogfish brain stem homogenates, labeled astrocyte-like processes in the brain stem and cerebellum of the dogfish embryo and numerous large and small flat cells in the cerebellar cultures. These results demonstrate that dogfish oligodendrocytes, neurons, and astrocytes express antigens that are conserved in mammalian neural cells. The ability to culture and identify neural cell types from cartilaginous fish sets the stage for studies to determine if proliferation, migration, and differentiation of these cell types are regulated in a similar fashion to mammalian cells.

An Immortalized Mouse Neuroepithelial Cell Line with Neuronal and Glial Phenotypes

Abstract: Evidence from retroviral marking techniques and immortalized cell lines indicates that multipotential stem cells exist in many areas of the developing central nervous system. However, the factors that influence the commitment of these stem cells into distinct neuronal or glial lineages are not known. We have created an immortalized hypothalamic cell line derived from embryonic day 14 hypothalamic cells with a replication-defective retroviral construct containing a temperature-sensitive allele (tsA58) of the large T antigen of the simian virus 40. The clonality of this cell line, which we have named VI, was established by single cell cloning and by Southern blot analysis. VI cells exhibit two different morphologies: the vast majority of cells are flat and stellate, and a smaller number are phase-bright round cells with processes. VI cells express nestin and neural-cell adhesion molecule, typical of proliferating neuroepithelial cells. They also express glial fibrillary acidic protein and S100 as well as the low molecular weight neurofilament protein. In addition, the phase-bright, process-bearing VI cells stain intensely for many typical neuronal proteins, such as low, medium and high molecular weight neurofilament proteins, tau protein, microtubule-associated protein-2, and neuron-specific enolase. The phase-bright cells also have condensed chromatin and display mitotic spindles, indicating that they are in mitosis. When VI cells are transferred from the permissive temperature (33 °C) to the restrictive temperature (39 °C), there is a decrease in expression of NF-L and an increase in expression of NF-H and glial fibrillary acidic protein in the flat VI cells. The enhanced expression of neuronal antigens in mitotically active VI cells is novel and may represent a more general property of the differentiation process. We suggest that VI cells arise from a mixed neural/glial neuroepithelial progenitor cell that expresses both neuronal- and glial-specific proteins in the developing hypothalamus.

Methods for differentiating neural stem cells to glial cells using neuregulins

Abstract: Method for producing a population of mammalian glial cells comprising contacting at least one mammalian neural stem cell with a culture medium containing a neuregulin and detecting the differentiation of stem cell to a population of glial cells.

Multipotent neural stem cell compositions

Abstract: The invention provides in vitro cell culture compositions consisting of neurospheres and culture medium, wherein the neurospheres consist of undifferentiated cells that are nestin + , glial fibrillary acid protein (GFAP) − , neurofilament (NF) − , and myelin basic protein (MBP) − and are not nestin − .

Separate Control of the Survival, the Self-renewal and the Differentiation of Hemopoietic Stem Cells

Abstract: Hemopoietic stem cells adhere to hemopoietic supportive (MS-5) cells, but not to non-supportive (MS-K) cells. Although a soluble stem cell factor (SCF) was produced by both of these cell lines, little activity was detectable in the supernatant from the cultures of either of these cells, indicating that SCF might be compartmentalized within the extracellular matrix (ECM), and transferred directly to the stem cells via the ECM (44). To probe this possibility, we studied the transfer of SCF from the ECM and the subsequent support of the survival of the hemopoietic stem cells. A stem cell-enriched bone marrow cell fraction was overlaid on SCF-containing ECM. The stem cells survived and proliferated for some days without differentiation under these conditions, whereas stem cells overlaid on ECM without SCF died within a few days. Addition of interleukin-3 (IL-3) to the ECM that contains SCF, induced differentiation of the stem cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced further differentiation of the stem cells, which was accompanied by a decrease in the number of colony-forming unit in spleen (CFU-S). These observations verified the above hypothesis, and indicated that the survival, the self-renewal, and the differentiation of hemopoietic stem cells can be separately controlled at least in vitro.

Cultivation of neural EGF-responsive precursor cells.

Abstract: We demonstrated that proliferation of dissociated neural E14 and E19 rat precursor cells could be induced by Long-EGF. Dividing EGF-responsive neural progenitor cells stimulated by Long-EGF formed spherical multicellular clusters (neurospheres) which may reach macroscopical size. We have studied the inner organization of cells in semithin sections and revealed that the cell population within the neurosphere is not uniform. These are original findings as other researchers did not process neurospheres histologically. Cells located in a central area possessed neuron-like morphology whereas peripheral cells where differentiated to a lesser degree. Generally, we have observed several apoptotic cells or apoptotic bodies per section. Moreover, the central portion of the neurosphere contained degenerating cells that probably die from worsened nutrition conditions in the large neurosphere. After plating the neurosphere in serum-supplemented medium, cells began to migrate radially from the edge of the neurosphere and differentiate. The cells lying in the vicinity to the cluster mimicked radial glia whereas the cells located at the periphery of the colony took morphology of astroglial cells. These observations suggest EGF-responsive neural precursor cells retained their ability to produce both neuronal and glial phenotypes after prolonged cultivation period.

Neural chest stem cell assay

Abstract: The invention includes mammalian multipotent neural stem cells and their progeny and methods for the isolation and clonal propagation of such cells. At the clonal level the stem cells are capable of self regeneration and asymmetrical division. Lineage restriction is demonstrated within developing clones which are sensitive to the local environment. The invention also includes such cells which are transfected with foreign nucleic acid, e.g., to produce an immortalized neural stem cell. The invention further includes transplantation assays which allow for the identification of mammalian multipotent neural stem cells from various tissues and methods for transplanting mammalian neural stem cells and/or neural or glial progenitors into mammals. A novel method for detecting antibodies to neural cell surface markers is disclosed as well as a monoclonal antibody to mouse LNGFR.

Remyelination of neurons using multipotent neural stem cell progeny

Abstract: The invention provides methods for producing myelin forming cells from multipotent self-renewing central nervous system neural stem cells as well as methods of using one or more cells from a multipotent self-renewing central nervous system neural stem cell population to form patches of myelin. Also provided are cell culture systems for forming patches of myelin and methods of treating demyelination diseases in a mammal.

Expression of gap junctions in neural cells derived from P19 embryonal carcinoma cells

Abstract: The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Using this cell line, we examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes following retinoic acid treatment. Normal, untreated P19 cells expressed two gap junction proteins, connexins 26 and 43. Neurons, the first neural cell type to develop expressed connexin26 which was detected at sites of cell-cell contact. Astrocytes developed later in culture and were detected by their expression of glial fibrillary acidic protein (GFAP). Astrocytes co-expressed GFAP and connexin43 in a similar pattern to that of primary astrocytes. These results suggest that differentiation of neural cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

Random catecholaminergic differentiation of mesencephalic neural precursors.

Abstract: We investigated how several factors influence the catecholaminergic phenotype establishment from embryonic mesencephalic neural precursors in culture. Using a semiquantitative RT-PCR procedure we found no significant effect of several growth factors or conditioned media on tyrosine hydroxylase (TH) mRNA levels. Nevertheless, neural precursor cells expanded by epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed the ability to express TH mRNA. Subcultures of EGF expanded neural precursor cells expressed TH mRNA, but not all individual secondary colonies obtained had this characteristic. Preferential dopaminergic differentiation was observed in our culture conditions. Our results suggest that EGF stimulates the proliferation of neural precursor cells that have the potential but differentiate randomly to catecholaminergic cells.