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WO2002014479A2 - Cellules souches neuronales isolees de mammiferes et procedes de fabrication et d'utilisation de ces cellules - Google Patents

Cellules souches neuronales isolees de mammiferes et procedes de fabrication et d'utilisation de ces cellules Download PDF

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WO2002014479A2
WO2002014479A2 PCT/US2001/025645 US0125645W WO0214479A2 WO 2002014479 A2 WO2002014479 A2 WO 2002014479A2 US 0125645 W US0125645 W US 0125645W WO 0214479 A2 WO0214479 A2 WO 0214479A2
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cells
cell
stem
astrocyte
type
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WO2002014479B1 (fr
WO2002014479A3 (fr
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Eric D. Laywell
Varely G. Kukekov
L. Brannon Thomas
Dennis A. Steindler
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University of Tennessee Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system

Definitions

  • This invention relates generally to novel mammalian brain cell types and methods of culturing such cells.
  • the methods of the instant invention which utilize suspension cultures and factors that limit cell contacts, result in an amplification of the production of neural stem and progenitor cells, and clones of such cells, from the adult mammalian brain, including the human brain and from tissue with significant ⁇ e.g. 1 day up to at least 5 days) postmortem intervals.
  • Propagation of neural stem and progenitor cells is relevant to the large-scale production of glial and neuronal cells, and clones of such cells, as well as self-repair ofthe brain in neurological disease.
  • Brain-derived stem cells have only recently been a major focus of attention, using a variety of lineage tracing and culture methodologies. See, Gage et al, 1995; Svendsen et al, 1995, Alvarez-Buylla et al, 1995, Steindler et al, 1996 and Housele et al, 1995.
  • ECM extracellular matrix
  • SEZ subependymal zone
  • Glycoproteins such as tenascin-C (TN) such as the chondroitin sulfate-containing proteoglycans (CSPG) are levels in the young brain, where they seem to have a role in forming glycoconjugate-rich boundaries around different functional groups of neurons, such as the somatosensory whisker barrel fields and striosomes in the striatum.
  • TN tenascin-C
  • CSPG chondroitin sulfate-containing proteoglycans
  • ECM molecules regulate cell proliferation, differentiation, migration, and survival through cell-cell and cell-ECM interactions.
  • Stem cells have been described in embryonic and postnatal mouse brain and in proliferative "neurospheres" that can be harvested and cultured from different brain areas, including the developing subventricular zone. See, Cattaneo et al, 1990; Richards et al, 1992; Reynolds et al, 1992; Reynolds et al, 1992; Reynolds et al, 1996; Nescovi et al, 1993; Kirschenbaum et al, 1994; Kirschenbaum et al, 1995; Fillmore et al, 1996; and Gritti et tf/., 1996.
  • the present invention discloses an advancement in the biological arts in which previously unknown brain stem cells are cultured and isolated.
  • the multipotent brain stem cells are characterized in that the daughter cells of the brain stem cells differentiate into neurons and glia and, therefore, are useful in neuroregeneration cell biology, and CNS drug-effects and drug-discovery studies.
  • a novel method of using such cells comprises culturing dissociated adult mammalian brain or postnatal tissue obtained from humans upt to two years of age in conditions that affect cell-substrate and cell-cell contacts. The cultured aggregates survive transplantation to the adult mammalian brain.
  • the daughter cells of the transplanted stem cells differentiate into other cell types, including but not limited to glia, such as astrocytes and oligodendrocytes and neurons, thus allowing for replacement of cells damaged by injury or disease.
  • glia such as astrocytes and oligodendrocytes and neurons
  • glia such as astrocytes and oligodendrocytes and neurons
  • glia such as astrocytes and oligodendrocytes and neurons
  • NSC's neural stem cells
  • NSC's neural stem cells
  • SEZ periventricular subependymal zone
  • CE Ciliated ependymal
  • CE cells might be capable of generating new neurons in adult mammals (Altaian, 1962) is supported by studies showing the production of neurons by ependymal cells in models of CNS injury in lower vertebrates (Anderson and Waxman, 1985; Molowny et al, 1995). Recent studies addressing the role of CE cells in mammalian neurogenesis have reached conflicting conclusions. It has been reported (Johansson et al, 1999) that individual CE cells of rodents are NSC's since they form neurospheres when cultured in isolation, and since some CE cells appear to divide in response to injury.
  • the in vitro manipulation of these and related molecules affects cellular adhesity to other cells or substrates, and affects the growth of neural stem and progenitor cells, as described below.
  • dissociated cells ftom the adult brain were cultured in factors that interfere with protein-protein interactions, or in gelatinous organic substances ⁇ e.g. methylcellulose) that also discourage cell contacts and allow the isolation of clonally-related colonies (spheres) of cells.
  • Such cells are suitable for studies of drug-discovery and testing using clones of glial and neuronal cells as well as for cell replacement therapies in a variety of brain structures e.g., in the brain or spinal cord for regeneration or space-occupation, as in spinal cord syrinx injuries, or stroke cavities, arteriovenous malformations, epileptic foci, or peripheral nerve neuromas.
  • Stem cells appear to make up 0.001-0.01% of an entire population of cells in renewing or potentially renewing tissues such as bone and "brain marrow" (Thomas et al., 1996).
  • any method that assures a large scale production of differentiated cells from a small number of the most primitive stem/precursor cells is extremely useful for self-repair (autologous cell replacement) therapies following traumatic or degenerative disease.
  • the invention harvests brain cells from a variety of sources including, but not limited to, mammalian brain specimens, human brain biopsy, and postmortem mammalian brain tissue.
  • the invention method includes isolation and amplification of neural cells as a therapeutic self-repair approach for neurological disease, particularly human disease.
  • the present invention uses a novel tissue culture approach to maximize the isolation of stem cells, and to assure a maximal number of transplantable cells from a very small number of stem cells, thus assuring the least risk for complications from a targeted and small biopsy site.
  • the culture method offers the possibility for large-scale cell production from a very limited number of extracted stem cells.
  • this cell type is more amenable to central nervous system (CNS) transplantation and integration, as well as gene transduction approaches.
  • the new cell type exhibits clonogenic properties and has the attributes of a multipotent astrocyte stem/progenitor cell.
  • the isolated cells of this invention can be transduced with genes coding for desired functions.
  • vectors containing genes coding for adhesion molecules can be transduced into these cells to increase cellular adhesivity. This would be useful, for example, to produce glia with increased adhesivity for cavity-filling approaches.
  • the isolated cells of the present invention can be transduced with genes coding for growth factors and directing neuronal phenotype, including neurotransmitter associated genes to produce neurons capable of increased neurotrasmitter production.
  • the cells of the present invention are multipotent, they are extremely well suited for cell-grafting approaches.
  • FIG. 1A, FIG1B, FIG. IC, FIG. ID, FIG. IE and FIG. IF show phase contrast and electron microscopic images of type I, II, and III clones.
  • FIG. 1A, FIG. IC, and FIG. IE are phase contrast images of type I, II and III clones of cultured adult brain cells, respectively.
  • FIG. IB, FIG. ID, and FIG. IF show type I, II and III spheres counterstained with propidium iodide, respectively.
  • FIG.2 depicts the types of spheres found in the culture paradigm ofthe invention, and the generation conditions for the appearance and evolution of sphere types from brain.
  • FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D show phase and electron microscopic images of type II (FIG. 3A and FIG. 3B) and type III (FIG. 3C and FIG. 3D) spheres.
  • Scale bars for FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D are 10, 5, 15, and 2 microns, respectively.
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H, FIG. 41 and FIG. 4J show immunostaining of early and late type II and type III spheres.
  • Scale bars for FIG. 4A, FIG. 4G and FIG. 4 are 10 microns
  • FIG. 4B and FIG. 4C are 15 microns
  • FIG. 4E and FIG. 4F are 30 microns
  • FIG. 4H is 20 microns
  • FIG. 41 is 100 microns.
  • FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D show the evolution and proliferation of type II (FIG. 5A and FIG. 5C), and type III (FIG. 5B and FIG. 5D) spheres.
  • Scale bars for FIG. 5 A and FIG. 5B are 25 microns
  • for FIG. 5C and FIG. 5D are 10 microns
  • FIG. 6A and FIG. 6B show type II and type III spheres from ROSA-26 transgenic mice. Scale bars for FIG. 6A is 50 microns, and FIG. 6B is 30 microns.
  • FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D show phase and electron microscopy of a type II adult mouse and type II adult human sphere.
  • the adult mouse sphere is approximately 100 microns in diameter, while the adult human sphere is approximately 200 microns in diameter.
  • FIG. 8A, FIG. 8B and FIG. 8C show astrocyte monolayers from postnatal day 2 mouse cerebral cortex generate spherical clones.
  • FITC immunofluorescence for Mac-1
  • PI propidium iodide
  • FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, and FIG. 9E shows astrocyte-derived neurospheres are proliferative and express neuronal and glial antigens.
  • FIG. 9B shows immunolabeling of a single P7 cerebellar astrocyte-derived neurosphere showing MAP-2 positive cells, indicating the presence of neurons within these clones.
  • the inset in FIG. 9B shows a higher magnification of a single MAP-2 positive immature neuron within the neurosphere.
  • Scale bars 30 ⁇ m in FIG. 9B, and lO ⁇ m in the inset.
  • FIG. 9C shows GFAP and ⁇ -III tubulin, PI counterstained, double immunolabeling of a neurosphere derived from a PI cerebral cortex astrocyte monolayer. Note the beta-Ill tubulin immunopositive neurites arborizing around Pi-stained cells, and GFAP astrocytic processes within the lower portion of the neurosphere. Scale bar - lO ⁇ m.
  • FIG. 9D shows ⁇ -III tubulin immunolabeling of a neurosphere derived from a PI spinal cord astrocyte monolayer.
  • FIG. 10A, FIG. 10B, FIG. IOC, FIG. 10D, FIG. 10E, FIG. 10F, and FIG. 10G show alkaline phosphatase (AP) enzyme histochemistry and immunocytochemistry of cells and neurospheres derived from Gtv-a transgenic mouse astrocytes infected with RCAS-alkaline phosphatase, showing cells expressing both AP and neuronal phenotype markers.
  • FIG. 10A shows a P2 astrocyte monolayer after infection with the avian leukosisvirus expressing the AP reporter gene, showing infected astrocytes with varying morphologies (e.g. arrow).
  • FIG. 9B shows a neurosphere derived from a Gtv-a astrocyte monolayer. AP histochemistry reveals cells of this neurosphere expressing the RCAS-AP gene, thus indicating their derivation from a single, infected astrocyte.
  • Upper right inset shows an example of a neuron derived from such a neurosphere, immunofluorescence for ⁇ -III tubulin (FITC).
  • FIG. IOC and FIG. 10D show a single RCAS-AP infected neuron that has migrated and differentiated away from its neurosphere.
  • FITC ⁇ -III tubulin immunofluorescence
  • FITC tubulin positive neurons
  • FIG. 11 is a summary of the neurosphere-generating potential of astrocyte monolayers derived from different CNS regions, from animals of different ages. Note an apparent critical period at PI 0/11 when astrocytes derived from the cerebral cortex, cerebellum and spinal cord cease to generate neurospheres, while astrocytes from the subependymal zone do not observe this critical period and retain the ability to generate neurospheres well into adulthood.
  • FIG. 12A, FIG. 12B and FIG. 12C show fibroblast monolayers, derived from mouse muscle, form spherical clones when replated in suspension culture supplemented with EGF and FGF2 (arrowheads in FIG. 12A).
  • fibroblast-derived clones When attached and allowed to differentiate, fibroblast-derived clones give rise only to cells with fibroblast-like morphology (FIG. 12B), and these cells are never im unopositive for neural-specific antigens.
  • Monolayers of microglia (FIG. 12C) fail to generate spherical clones when replated in suspension culture supplemented with EGF and FGF2.
  • FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F and FIG. 13G show brain tumor-derived clones.
  • stem/progenitor cells are harvested and cultured from an adult human brain anaplastic astrocytoma (this example, but also from astrocytomas and glioblastomas) both astrocytes (immunofluorescence for astrocytic glial fibrillary acidic, GFAP, protein) and neurons (immunofluorescence for neuronal ⁇ -III tubulin) arise from multipotent neurospheres in suspension culture (with semi-solid, i.e.
  • FIG. 13A are two attached clones from this anaplastic astrocytoma seem to merge, with neurons (bright cells) and astrocytes (darker stained cells) within and surrounding the clones.
  • FIG. 13 B are two clones immunostained for just neuronal ⁇ -III tubulin and shows many neurons (brightly immunofluorescent cells) that arise from such glial tumors.
  • FIG. 13C are two different clones, from the same tumor, again stained for the same astrocytic (dark) and neuronal (bright cells) markers.
  • FIG. 13A are two attached clones from this anaplastic astrocytoma seem to merge, with neurons (bright cells) and astrocytes (darker stained cells) within and surrounding the clones.
  • FIG. 13 B are two clones immunostained for just neuronal ⁇ -III tubulin and shows many neurons (brightly immunofluorescent cells) that arise from such glial tumors.
  • FIG. 13C are
  • FIG. 13D is a single, beta III tubulin positive neuron that is outside of the attached clone, and exhibits unusual somatodendritic morphology.
  • FIG. 13E shows higher magnification of neurons and astrocytes with apparently normal morphologies.
  • FIG. 13F and FIG. 13G show the same field of cells immunostained for GFAP (FIG. 13F) and neuronal ⁇ -III tubulin (FIG. 13G). It can be seen that there are single cells expressing both neuronal and glial antigens, which is not normally seen within clones derived from non-tumor stem/progenitor cells.
  • the present invention describes methods which can be used to isolate, amplify, and grow stem/precursor cells from the mammalian brain. Some of these stem/progenitor cell are multipotent astrocytes or tumor cells. By manipulating specific aspects of cell separation and cell adhesion, the invention methods described herein can be used to specifically isolate and culture type I, type II and type III precursor cells. It should be noted that in a recent paper by Kukekov et al. ⁇ alia, 1997 the type II and type III cells of the instant invention are referred to as type I and type II cells, respectively, for convenience. Types II and III appear to be more mature stages of type I cells.
  • type I, II or III stem cells of the instant invention are the cells which generate type I, II, and III clones/aggregates/neurospheres, respectively.
  • the term clone or aggregate will be used to refer to these cell aggregates generated from a single cell.
  • Cell separation and cell adhesion can be manipulated using a variety of contact- limiting and contact-inhibiting factors. For example, chemical-separating agents such as mercaptoethanol, physical separating agents such as methylcellulose, and anti-adhesives such as poly 2-hydroxyethyl methacrylate are used to deter cell-cell and cell-substrate associates during the initial isolation of stem/precursor cells from the newly-dissociated brain.
  • agents such as mercaptoethan are always used in the first stage of isolation of type I and II clones to help deter the survival of the more mature cellular elements by deterring their clustering.
  • agents such as mercaptoethanol may have certain growth-promoting actions on the single stem/precursor cells that eventually proliferate to form these early sphere types.
  • contact-inhibiting (or contact-limiting) factors as mercaptoethanol are eventually removed from the culture medium for the evolution or differentiation of type II and type III spheres.
  • the differentiation of type III spheres requires other additional factors, including growth factors such as beta fibroblast growth factor, epidermal growth factor, or similar factors that are also contained within pituitary extract. Such additional factors are described in the type III culture media discussed below, see Example 3.
  • brain stem/progenitor cells may be produced on a large scale and can be genetically altered using a variety of transfection methods.
  • the multipotential brain cells ofthe instant invention can be directed to particular neuronal or glial lineages.
  • the culturing methods of the instant invention can be used to produce large numbers of clonally related cells of a specific cell type.
  • the methods of producing such large populations of cells as well as the cells themselves are specifically useful in replacing cells that are lost due to disease processes. For example, the production of large quantities of specific glial cells by the methods of the present invention and subsequent transplantation of these glia into the brains of multiple sclerosis patients, would be particularly beneficial to such patients.
  • neurons generated from the stenvprecursor cells of the instant invention, transplantation of these neurons, and the integration of the activities of the replaced cells into established brain circuitries is particularly useu in brains where neuronal cell loss has occurred.
  • Cells generated using the methods of the instant invention can also be used in the brain or spinal cord for regeneration or space-occupation, as in spinal cord syrinx injuries, or stroke cavities, arteriovenous malformations, epileptic foci, or peripheral nerve neuromas to fill cavities. Large or small scale production of the cells using the invention methods are also useful for drug-discovery and testing.
  • clones of type I appear as phase-bright, very small dense bodies. It is not possible to discern individual cells within the clones using phase microscopy. However, when the cells are counterstained with propidium iodide (PI) (FIG. IB), type I clones exhibit areas of very small, punctate staining interspersed with regions that lack staining. Type I clones do not attach to either plastic or laminin-coated substrates, and cells of individual clones are not separable by trypsinization. Furthermore, type I clones are immunonegative for all ofthe cell-specific markers tested so far.
  • PI propidium iodide
  • Some exemplary contact limiting factors include, but are not limited to, mercaptoethanol, poly 2-hydroxyethyl methacrylate, and methylcellulose.
  • Mercaptoethanol functions as a chemical-separating agent as it breaks disuff ⁇ de linkages of proteins which are involved in cell-cell and cell- substrate interactions.
  • Methylcellulose functions as a physical separator; it is a viscous bioorganic solution which, by its viscous nature, limits contacts between cells and between cells and a substrate, and allows clonal analyses.
  • Poly-2-hydroxyethyl methacrylate functions as an anti-adhesive for substrate coating. This prevents cell contact with adhesive surfaces and also prevents differentiation. Other compounds or procedures which function to limit or inhibit cell-cell and cell-substrate interactions can also be used with this invention. Culturing as a suspension also deters cell-cell and cell- substrate contacts with higher yields of spheres, but culturing in methylcellulose again, allows clonal analyses.
  • type II clones are phase-dark, spherical bodies that become larger with time (FIG. IC and FIG. 3 A). Electron Microscopy (EM) revealed that type II clones consist of rings of small, tightly apposed cells that surround a core of flocculent, non-cellular material having many characteristics of extracellular matrix (FIG. ID and FIG. 3B).
  • the type II cell has many organelles, including endoplasmic reticulum, Golgi apparatus, dense bodies, and mitochondria.
  • the closed arrow in FIG. IC points to a multisphere aggregate as shown by propidium iodide counterstaining, a DNA stain for cell nuclei, (FIG. ID).
  • the closed arrow in FIG. IE points to a type II sphere residing near a type III sphere, which is marked by an open arrow.
  • Type II clones do not attach to either plastic or laminin-coated substrates. Immediately after they appear in culture, type II clones are immunonegative for cell- specific markers, including GFAP (glial fibrillary acidic protein, which labels more mature and reactive astrocytes), nestin (RAT-401, which stains neurepithelial stem cells as well as radial glia), and TuJI (which stains class III ⁇ -tubulin that is expressed in recently postmitotic (committed) as well as mature neurons in the CNS).
  • GFAP glial fibrillary acidic protein, which labels more mature and reactive astrocytes
  • nestin RAT-401, which stains neurepithelial stem cells as well as radial glia
  • TuJI which stains class III ⁇ -tubulin that is expressed in recently postmitotic (committed) as well as mature neurons in the CNS.
  • some cells of type II clones (late type II, or early type III) become immunopositive for nestin, but remain immuno
  • type II clones will convert to type III clones.
  • type III clones when plated on plastic or laminin- coated substrates, attach readily, and produce a number of process-bearing cells that migrate away from the sphere to form a single layer of cells.
  • Type III cells are immunopositive for a variety of cell-specific markers, including GFAP, nestin, LI (a marker of an adhesion molecules that is on the surface of neurons and their processes), and TuJI (FIG. 4). Some cells exhibit very light, punctate staining using an antibody to the 04 antigen (marker of oligodendrocytes).
  • the primary cultures of the instant invention also appear to initially contain differentiated neurons and glia. However, in the presence of contact-inhibiting factors, neurons and astrocytes were not immunologically detected after 1 week; they presumably cannot survive under these culture conditions.
  • Type II and type III stem/precursor cells are generated from a dissociated adult mammalian brain.
  • Type II cells appear 2-3 weeks after mercaptoethanol, or other cell contact limiting factor, is added to the culture medium.
  • mercaptoethanol, or other contact-limiting factor is subsequently removed from the medium after the appearance of type II spheres, the type II spheres increase in size and, after an additional 10-14 days, convert to type III spheres.
  • type III clones can also appear spontaneously in suspension cultures grown in the absence of contact limiting factors.
  • FIG. 3 shows the phase and electron microscopic images of type 11 (FIG. 3 A and FIG. 3B) and III (FIG. 3C and FIG. 3D) spheres.
  • type II clones take on characteristics of type III clones.
  • the size of the type II clones increases dramatically, and their color changes from phase-dark (FIG. IC and FIG. 3 A) to phase-bright (FIG. IE and FIG. 3C).
  • large cells appear at the edges of the type III clones.
  • FIG. 3C shows type III spheres which are generally larger and phase-brighter than type II spheres.
  • the open arrow points to a late type II or early type III sphere, and the closed arrow points to a large cellular protrusion on the periphery of a late type III sphere which appears brighter than the early type III, and has a more irregular border due to many protrusions.
  • FIG. 3D shows cells of type III spheres which look more mature than type II cells.
  • the open arrow points to a large cell, and the closed arrow marks a smaller cell with a darker nucleus.
  • Type III spheres will continue to increase in size and begin to display cellular heterogeneity when grown in suspension culture.
  • cells within suspended type III spheres begin to express markers of differentiation.
  • Such cells can eventually become neurons and glia when plated onto favorable substrata such as laminin/poly ornithine coated surfaces, polylysine, or plastic, or other matrix molecules, or transplanted into a host brain.
  • FIG. 4 shows the immunostaining of early and late type II and III spheres illustrating that the various characteristics of the type II and type III spheres can be readily distinguished from their immunostaining profiles and their phase-contrast, and EM images.
  • Early type II spheres are shown in FIG. 4A.
  • the open arrows point to two type II spheres that abut each other. These spheres are negative for the putative stem cell intermediate filament protein, nestin.
  • FIG. 4E A more mature type II sphere that is immunopositive for nestin is shown in FIG. 4E.
  • FIG. 4C shows an early type III sphere immunopositive for nestin, with some unlabeled cells also apparent.
  • FIG. 4A shows the immunostaining of early and late type II and III spheres illustrating that the various characteristics of the type II and type III spheres can be readily distinguished from their immunostaining profiles and their phase-contrast, and EM images.
  • Early type II spheres are shown in FIG. 4A.
  • FIG. 4D shows a phase contrast image of a late type III sphere (open arrow) showing processes after attaching to plastic.
  • FIG. 4E and FIG. 4F show a type III sphere, with cells beginning to disperse after attaching to plastic. This sphere is double immunostained for GFAP (FIG. 4E) and O-IH tubulin (FIG. 4F).
  • FIG. 4G depicts astrocytes and shows that dispersed cells of type III spheres are immunopositive for nestin.
  • FIG. 4H shows astrocytes from dispersed type III spheres also stain for GFAP.
  • the open arrow points to a morphologically different astrocyte than the large cell on the right.
  • FIG. 41 shows cells that are dispersed from an attached type III sphere and which are immunopositive for LI.
  • the arrow points to a long LI -positive process.
  • FIG. 4J shows a single cell immunopositive for ⁇ -tubulin, counterstained with propidium iodide, after attachment of a type III sphere.
  • type II and type III spheres are easily distinguishable from each other, not only by the differences in phase contrast, and EM images, but also by their distinct immunostaining profiles.
  • the evolution and proliferation of a type II sphere differentiating into a type III sphere can be seen in FIG. 5.
  • a single late type II sphere (arrow in FIG. 5 A) was followed in suspension culture with phase microscopy.
  • this type II sphere had increased in size and altered its morphology to become the type III sphere shown in FIG. 5B.
  • the open arrowhead points to a cellular protrusion on the edge of the sphere typical of the type III sphere.
  • Cellular debris around the type II sphere in FIG. 5A is absent from the type III sphere in FIG. 5B due to movement during feeding.
  • Heterogeneous cultures of spheres exposed to a 20 hour pulse of BrdU reveal proliferative cells in both type II (FIG. 5C) and type III (FIG. 5D) spheres.
  • Both the small sphere in FIG. 5C and the larger sphere in FIG. 5D contain labeled nuclei with a variety of sizes. This indicates that there may be different progenitor cells types proliferating in the spheres; some progenitor cell types giving rise to neurons, some giving rise to glia.
  • type II and type III growth media containing growth factors such as basic fibroblast growth factor (bFGF), or epidermal growth factor (EGF) encourages differentiation.
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glial derived neurotrphic factor
  • NT3 ciliary neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • Type II and type III clones were also generated from ROSA-26 mice, a strain that expresses the ⁇ -galactosidase transgene in all cell types, to perform transplant experiments.
  • ROSA-26 transgenic mice are commercially available from the Jackson Laboratories, Bar Harbor, Maine. The results show that following transplantation into adult mouse brain, type II and type III clones can survive and differentiate. (FIG. 6).
  • ⁇ -galactosidase positive type II and type III clones were transplanted to the strialum of adult ICR mice, small and large X-gal positive cells were found to survive up to two weeks.
  • immunocytochemistry with GFAP revealed the presence of labeled astrocytes and non-labeled larger cells (presumably neurons).
  • FIG. 6A shows a ⁇ - galactosidase positive late type III sphere (large open arrow), and unlabeled early (right open arrowhead) and late (left open arrowhead) type II spheres. The filled arrow points to unidentified-labeled elements out of the plane of focus.
  • FIG. 6B shows the combined bright field/fluorescence image of a transplant of type II and III spheres into the striatum of an adult ICR mouse. Surviving astrocytes marked with a filled arrow are counterstained for GFAP. Large GFAP-negative, ⁇ -galactosidase- positive cells, presumed to be neurons (open arrow), are also seen. Other transplanted cells can be seen within this host brain structure as well. Immunofluorescence is light in color; the ⁇ - galactosidase product is dark in color.
  • FIG. 7 shows phase and electron microscopic images of type II adult mouse and human spheres.
  • FIG. 7 A shows the phase microscopic image of a type II adult mouse sphere that looks similar to a sphere with a postmortem interval of 0 hours
  • FIG. 7B shows the phase microscopy of a type II sphere from an adult mouse with a postmortem interval of sixteen hours.
  • FIG. 7C shows the electron microscopic image of a type II sphere from an adult transgenic mouse (tenascin glycoprotein knockout mouse). A complex cellular aggregate can be seen.
  • FIG. 7D shows the electron microscopy of an adult human sphere (as shown in the inset, a phase microscopic image of a type II sphere). Similar to the electron microscopy of a type II sphere from an adult transgenic mouse (FIG. 7C), this human sphere (FIG. 7D) also shows a complex cellular aggregate. This human sphere is approximately 200 microns in diameter.
  • the identification and understanding of neuropoietic stem and progenitor cells based on a combination of morphology, gene marking, and unique biochemical features, such as that described herein, ensure reliability in the results.
  • the use of just one feature as an identification tool can occur, although it makes the recognition of the specific stem cell type rather tenuous.
  • neuropoiesis in the adult brain is probably a rather limited event, based on current knowledge of hematopoiesis and the presence of stem cells in other tissues, as well as the apparent existence of a quiescent population of so-called stem cells in the SEZ (see, Potten et al., 1990 and Morshead et al, 1994). For this reason, methods that amplify the ex vivo production of these cells, as described herein, are useu to generate large numbers of such cells for classification and transplantation. Furthermore, the methods of the instant invention can be applied to harvesting the small number of stem/precursor cells from adult brains, particularly adult brains with significantly long postmortem intervals ⁇ e.g. 1-5 days), allowing the "banking" of these cells for future studies and cell- replacement therapies.
  • novel approaches as described here that uncover either novel stem/precursor cells or aspects of their growth and differentiation that lead to classification of stages of adult brain neuropoiesis are also useful.
  • the described methods allow this process with the production of the most primitive stem/precursor cells, and facilitate the generation and analyses of a continuum of developing and differentiating brain stem/precursor cells.
  • the culture conditions ofthe present invention involve limiting cell-cell and cell- substrate interactions leading to the enhanced production of spheres of type II and III cells.
  • type II, and III clones two types of proliferating cells that form neurosphere-like aggregates from dissociated adult brain, termed type II, and III clones. While the type III clones in the culture paradigm of the invention most likely represent neural progenitor cells that have previously been described in the aforementioned papers (see, for example, Reynolds et al., 1992; Reynolds et al, 1996; and Weiss et al, 1996, all cited above, it has been heretofore unrecognized that such type III spheres are derived from the type II precursors of the instant invention. By isolating and amplifying type II spheres in specific culture conditions, type III spheres can be obtained. In addition, prior to the disclosure of the methods of the instant invention, type II and type III spheres have not been previously obtained from postmortem brain specimens.
  • prior art stem cells are not as primitive as the type II stem cells of the present invention as evidenced by the fact that the prior art stem cells are all nestin- positive.
  • the type II clones of the instant invention represent a truly unique, ontogenetically earlier form of stem progenitor than those previously described. This conclusion is supported by data showing that the type II clones are first initially nestin- negative, followed by a progression through a nestin-positive state to become a type III clone, and finally differentiation into neurons or glia (FIG. 2, FIG. 3 and FIG. 4).
  • mice Adult ICR or transgenic or mutant mice, or biopsy specimens ftorn human temporal lobe (for epilepsy surgery), or brain specimens with significant (i.e. at least one day) postmortem intervals, were used as tissue sources for dissociations.
  • the brains were dissociated and cultured follows. Extracted brain tissues were minced with a razor blade and washed in a mixture of ice-cold DMEM (Dulbecco's modified Eagle's medium, commercially available from a variety of vendors) and any antibiotic-antimycotic product such as Sigma Chemical Co. Catalogue #A5955 (lOOx). (Such antibiotic-antimycotic products are also commercially available from Gibco Bri, Grand Island, NY).
  • Brain pieces were transferred to a beaker containing 0.25% trypsin and EDTA (ethylenediarninetetraacetic acid) and mixed on a magnetic stir-plate for 15 minutes, triturated with a plastic pipette, filtered through sterile gauze, and collected in a 15 ml tube and centrifuged for 5 minutes at 1200 rpm.
  • Cells were resuspended in DMEM/F12 medium + Nl supplement (a standard tissue culture medium available from a variety of vendors) plus 5% FBS (fetal bovine serum) and grown in suspension cultures by plating at high density on a non-adhesive substrate (tissue culture plastic coated with poly 2- hydroxyethyl methacrylate, Sigma Chemicals). Cells were fed every 3-4 days by centrifugation and resuspension in fresh medium.
  • trypsin and EDTA ethylenediarninetetraacetic acid
  • the basic media for culturing type I cells comprises the following ingredients: Insulin (5 ⁇ g/mL), putrescine (100 ⁇ M), progesterone (20 ⁇ M), sodium selenite (30 ⁇ M), pituitary extract (20 ⁇ g/mL), transferring (100 ⁇ g/mL), and 5% fetal calf serum (FCS) in DMEM/F12 media.
  • Type I cells only appear in suspension cultures containing a non-adhesive substrate such as poly 2-hydroxyethyl methacrylate. Some type II cells are also present in these cultures. 5.2 EXAMPLE 2 - THE PRODUCTION OF TYPE II CLONES
  • Type II clones similar to type I clones, were generated from adult ICR, transgenic or mutant mice, or biopsy specimens from human temporal lobe (for epilepsy surgery). In addition, Type II clones were also generated from the adult human brain, and from dead animals with long post mortem intervals when the animals were kept at 4°C.
  • the brains were dissociated and cultured as previously described for the generation of Type I clones. Briefly, extracted brain tissues were minced, washed, and transferred to a beaker containing 0.25% trypsin and EDTA. After being mixed on a magnetic stir-plate for 15 minutes, the culture was triturated with a plastic pipette, filtered through sterile gauze, centrifuged, and resuspended in DMEM/FI2 medium + NI supplement, plus 5% FBS, plus 20 ⁇ g/mL pituitary extract (from Gibco) and grown in suspension cultures by plating at high density on a non-adhesive substrate.
  • the basic media described above (comprising insulin (5 ⁇ g/mL), putrescine (100 ⁇ M), progesterone (20 ⁇ M), sodium selenite (30 ⁇ M), pituitary extract (20 ⁇ g/mL), transferring (100 ⁇ g/mL), and 5% fetal calf serum (FCS) in DMEM F12 media) also contained 10 ng/mL basic fibroblast growth factor (bFGF), and 10 ng/mL epidermal growth factor (EGF).
  • the culture media additionally contained 100 ⁇ M mercaptoethanol as a contact-limiting factor that reduces disulfide bonds (see, Herrington, 1986). Cultures contained dense debris for 10-14 days. Mercaptoethanol was then removed from the medium after 10-14 days. Clones of type II were present in these cultures. Some type III clones were also present.
  • mice ICR, transgenic or mutant mice, or biopsy specimens from human temporal lobe, or from the adult human brain, or from dead animals with long post mortem intervals when the animals were kept at 4°C.
  • the brain source was dissociated as described in Example 1 and Example 2 above, and the cells were grown in the suspension culture described above either without contact inhibiting factors, or, more often with a contact-inhibiting factor such as mercaptoethanol.
  • the basic media for culturing type III cells was the same as that used for culturing type II cells.
  • the media comprised insulin (5 ⁇ g/mL), putrescine (100 ⁇ M), progesterone (20 ⁇ m), sodium selenite (30 ⁇ M), pituitary extract (20 ⁇ g/mL), transferring (100 ⁇ g/mL), 10 ⁇ g/mL basic fibroblast growth factor (bFGF), 10 ng/mL epidermal growth factor (EGF), and 5% fetal calf serum (FCS) in DMEM/F12 media.
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • FCS fetal calf serum
  • BDNF brain-derived neurotrophic factor
  • GDNF glial derived neurotrphic factor
  • CNTF ciliary neurotrophic factor
  • Cultures are fixed in one of two ways depending upon the cultivation paradigm.
  • Adherent clones (cultivated on plastic or laminin-coated plastic) are washed three times with room temperature Dulbecco's PBS (phosphate buffered saline) and then fixed either with 10% acetic acid in pure ethanol at -20°C, or ice cold 4% paraformaidehyde in PBS. After % to 1 hour, the fixative was removed and the cultures were washed three times with PBS.
  • PBS phosphate buffered saline
  • Clones cultivated as suspension cultures were collected in a 15-ml plastic tube and centrifuged to form a pellet.
  • Culture medium was aspirated and fixative (described above) was added to the pellet.
  • Clones were then triturated and kept in fixative for % to 1 hour, after which time the cells were again centrifuged and washed in PBS. Finally, the pellet was resuspended in a small volume of fresh PBS, and small aliquots of cells were placed on polylysine-coated coverslips and allowed to dry before the application of cellular stains or antibodies.
  • Clones grown in suspension cultures were prepared for ultrastructural analysis by fixation in sodium cacocodylate buffer comprising 2% glutaraldehyde, 2% paraformaldehyde, 0.5% acroline, and 5% sucrose.
  • the fixative was warmed to 37°C and gradually added to cultures until the fixative-to-medium ratio was 1:1.
  • Cells were then collected in 15-ml tubes and centrifuged to form a pellet which was then covered with 100% fixative for several hours before processing with standard embedding, staining, and sectioning protocols. Samples were viewed on a JEOL 2000 electron microscope.
  • EM of type II clones revealed rings of small, tightly apposed cells that often surround a core of flocculent, non-cellular material (FIG. 3B) having many of the characteristics of extracellular matrix.
  • the type II cell has many organelles, including endoplasmic reticalum, Golgi apparatus, dense bodies, and mitochondria.
  • EM of type III clones revealed cells that appear to be more differentiated than type II cells, as their cytoplasm is less dense than type IIs and their organelles appear to be more developed.
  • FIG. 7 shows EM of a clone from a transgenic mouse (tenascin knockout mouse) and a sphere (clone) from an adult human temporal lobectomy specimen.
  • Methylcellulose (StemCell Technologies, Vancouver, B.C.) was dissolved in medium (at a concentration of 1.6%, and Dulbecco/FI2 + NI supplement medium was added, with an equal volume of brain cells suspension, to a final concentration of 0.8% methylcellulose (see, Worton et al, 1969). Cells were fed every 2-3 days by the addition of small aliquots of medium without methylcellulose. Single clones were followed over time (FIG. 5), and observed to increase in size indicating cell proliferation and growth.
  • Type II clones are immunonegative for cell-specific markers, including GFAP, nestin, and TuJI. These are considered early type II clones. However, after approximately 10 days in vitro, some cells of type II clones become immunopositive for nestin but remain immunonegative for GFAP and TuJI.
  • Type III clones exhibit cell phenotype markers of more differentiated cells being immunopositive for nestin, GFAP, LI, and TuJI. This staining pattern shows the continuum of evolution of type II clones from early (immunonegative) to late (selectively immunopositive) and eventually to type III clones (fully immunopositive) (FIG. 5).
  • rtPCR can be used to reveal genes that are different between type II and type III clones. Not only will this further characterize the type II and type III clones, but it will also reveal novel genes in each clone type, as well as identify genes involved in different developmental stages of neuronal precursor cells. 5.8 EXAMPLE 8 - USING BRAINS FROM DEAD MAMMALS AS A SOURCE OF STEM PRECURSOR CELLS
  • type I, II and III spheres from acutely dissociated brain tissue
  • dissociated brain from adult mice with postmortem intervals from 16-24 hours have yielded normal type I, type II and type III spheres.
  • Pilot studies have indicated that it might be possible to harvest brain tissue (if stored at 4°C) from animals or human cadavers with postmortem intervals of up to 2-5 days.
  • EXAMPLE 9 - LIGHT AND ELECTRON MICROSCOPY OF CLONES FROM ADULT HUMAN BRAIN
  • Human brain also generates type II and III clones, as described for the adult rodent. Dissociating biopsy specimens from the adult human temporal lobe may be used to generate type II and II spheres using the same culture methods as described for isolating type II and type II spheres from rodent brain tissue.
  • human brain may also generate type I clones.
  • FIG. 7D shows human spheres at the light and electron microscopic level that have many ofthe same cyto logical feature as described in rodent spheres.
  • EXAMPLE 10 - TRANSPLANTATION (GRAFTING) OF TYPE I OR II CLONES FROM ROSA-26 ADULT MICE Type II or III clones, generated as described in Examples 2 and 3 (see, above) from adult ROSA-26 transgenic mice (Friedrich et al, 1991) were aspirated, with media, into a Hamilton microsyringe with an attached 31 gauge needle using a video stereomicroscope set-up. 1 ⁇ l was slowly injected into the adult ICR mouse striatum. For stereotaxic coordinates, as well as details for the histochemical detection of ⁇ - galactosidase activity (Gates et al, 1996).
  • Type II and III clones were also found to exhibit X-gal labeling in vitro (see, FIG. 6). Survival times of 7-14 days were observed thus far, with a 10-day survival shown in FIG. 6. It is possible that the transplants can survive longer times, simply carrying the experiment out for a longer time point will determine that. Preliminary studies also indicate that grafted type II and III spheres from adult human brains give rise to cells that survive in the mature brains of immunocompromised mice.
  • the transplants can differentiate into different types of resident neuronal populations.
  • some of the transplanted spheres from ⁇ - galactosidase-positive mice differentiate into astrocytes in the host ICR (non- ⁇ - galactosidase-positive brain); some transplanted spheres differentiate into neurons.
  • Immunocytochemistry with GFAP revealed the presence of labeled astrocytes and non- labeled larger cells that are presumed to be neurons.
  • in vitro cultures using feeder cells from different parts of the body ⁇ i.e. endothelial cells, lung endothelial cells, or kidney cells) change the gross morphology of the spheres generated.
  • Astrocyte monolayers were derived from the cerebral cortex, cerebellum, and spinal cords of early postnatal (postnatal day 1-10), late postnatal (postnatal day 11-20), and adult (>30 days) ICR or Gtv-a transgenic mice. Mice were deeply anesthetized and decapitated. Cerebral cortex, cerebellum, spinal cord, and SEZ were dissected and processed separately to generate astrocyte monolayers. The cerebral cortex dissection was performed in such a way as to exclude all cells of the ventricular or subependymal region. This was accomplished by first blocking the rostral forebrain in the coronal plane, and then shaving superficial slices of the cerebrum tangential to the pial surface.
  • BrdU (lO ⁇ g/mL) was added to suspension cultures for 24 hours prior to removal and plating of neurospheres. Detection of BrdU labeled cells followed these steps: fixation of neurospheres with paraformaldehyde as described below; incubation in 1:12X SSC:formamide for 2 hrs. at 65 ° C); incubation in 2N HCI for 30 min. at 37 ° C; equilibration in borate buffer for 10 min. at room temperature. Finally, immunofluorescence with monoclonal anti-BrdU was performed as described below.
  • Clones were removed from individual microwells, or methylcellulose suspension culture, and placed into a drop of N2 medium containing 1% FCS on glass coverslips that had been coated sequentially with poly-1-ornithine (lO ⁇ g/mL) and laminin (5 ⁇ g/mL).
  • GFAP glial fibrillary acidic protein
  • Immunon an astrocyte-specific intermediate filament protein
  • vimentin and nestin cytoskeletal markers of immature astrocytes, and neural stem cells (Developmental Hybridoma Bank)
  • ⁇ III-tubulin a neuron-specific intermediate filament protein (Promega or Sigma)
  • LI a cell adhesion molecule associated with neurons
  • MAP-2 a microtubule protein associated with neurons
  • A2B5 a surface protein associated with O2A progenitors capable of giving rise to either astrocytes or oligodendrocytes (Boehringer Mannheim).
  • astrocytes In order to examine the potential for astrocytes to display NSC characteristics, we first generated monolayer cultures of astrocytes from cerebral cortex, cerebellum, spinal cord, and SEZ from mice ranging in age from late embryonic to adult. Because of the close juxtaposition of the cerebellum and spinal cord to the ventricular system, we could not be certain of excluding all periventricular cells from the dissociation of these tissues. However, cerebral cortex dissociations followed a careful dissection procedure whereby, with the aid of a dissecting microscope, only the superficial cortical surface was isolated, excluding the possibility of contamination by ependymal or subependymal cells.
  • the primary fraction of cells used to generate astrocyte monolayers initially contains cells that are immunopositive for a variety of phenotypic antigens, including the oligodendrocyte membrane protein 04, and neuron specific ⁇ III-tubulin (data not shown); however, after 7-10 days in vitro, plated cells form a confluent monolayer (FIG. 8A), and these markers fail to label any cells. At this stage, a majority of cells in the monolayer are immunopositive for the astrocyte-specific intermediate filament protein GFAP (FIG.
  • astrocyte-derived spheres When removed from suspension culture and plated onto a favorable substrate (polyornithine/laminin coated glass), astrocyte-derived spheres readily attach, and the cells at the bottom begin to migrate, differentiate, and send out processes (FIG. 8B, inset and FIG. 9).
  • the largest percentage of cells derived from the sphere are immunopositive for the astrocyte-specific filament protein GFAP (FIG. 9A and inset); however, many cells are observed to be immunopositive for the oligodendrocyte-type 2 astrocyte progenitor cell (O-2A) marker A2B5 (FIG. 9A, lower inset), as well as the neuron- specific markers ⁇ III-tubulin, LI and MAP-2 (Fig. 9B-3).
  • O-2A oligodendrocyte-type 2 astrocyte progenitor cell
  • INFECTION OF GFAP-TVA ASTROCYTES, AND ALKALINE PHOSPHATASE HISTOCHEMISTRY Astrocyte monolayers were prepared as described above from the cerebral cortices of P2 Gtv-a transgenic mice.
  • typical infection rates in the astrocyte monolayers are 1-5%, with the best infection rate being 16%.
  • There is very little chance for successfully infecting a cell with immunologically undetectable levels of GFAP and all ofthe cells in the monolayer cultures express one or another astrocyte phenotypic marker ⁇ i.e. GFAP, nestin, vimentin, or S100B), and with the addition of small numbers of Mac-1 labeled microglial cells in the monolayer cultures, the totals equal 100% of propidium iodide- counterstained cells.
  • Gt -a transgenic mouse was constructed such that the gene encoding the TVA receptor is under control of the GFAP promoter (GFAP is an astrocyte-specific intermediate filament protein).
  • TVA expression is required for infection with avian leukosis virus.
  • Mouse cells not expressing GFAP are non-infectable with the avian leukosis virus.
  • Monolayers of astrocytes from the Gtv-a mouse were grown in the presence of an avian leukosis virus containing the alkaline phosphatase (AP) reporter gene in order to visualize infected cells.
  • AP alkaline phosphatase
  • infected cells divide, the virus is incorporated into the genome, and is therefore passed on the all progeny.
  • Expression of AP is not under the control of the GFAP promoter, but rather is under control of the viral LTR gene which results in constitutive expression of AP; this means that the progeny of an infected astrocyte will express AP even if the GFAP promoter is never active in this cell.
  • neurospheres were derived from monolayers of Gtv-a astrocytes (containing variable numbers of infected cells), some clones were AP-positive. Combining AP histochemistry with immunocytochemistry for neuronal antigens ⁇ i.e. ⁇ III-tubulin) showed that neurons expressed AP, and therefore must be the progeny of an infected astrocyte.
  • astrocyte monolayers The ability of astrocyte monolayers to produce spherical clones is discretely age- and region-related (FIG. 11).
  • region-related region-related
  • neurospheres are generated from astrocyte monolayers derived from cerebral cortex, cerebellum and spinal cord only when these monolayers originate from animals younger than postnatal day 11.
  • SEZ-derived astrocytes continue to produce clones even when generated from animals older than Pl l.
  • astrocyte monolayers maintain neurosphere-generating ability even after five passages, but there appears to be a gradual reduction in neurosphere yield with increasing passage.
  • Fibroblasts were obtained from mouse muscle. First postnatal week mice were anesthetized and decapitated. The limbs and torso were stripped of skin, and the thoracic, abdominal, and pelvic viscera were removed. The remaining carcass was minced with a razor blade, incubated in trypsin, and tirturated with a fire-polished Pasteur pipette. The resulting cell suspension was seeded in tissue culture flasks as described above to yield a highly enriched culture of fibroblasts. Microglia cultures were generated from primary monolayers derived from brain as described above for astrocyte cultures.
  • CSF colony stimulating factor
  • Cytodex dextran microbeads
  • FIG. 12 shows fibroblast monolayers, derived from mouse muscle, form spherical clones when replated in suspension culture supplemented with EGF and FGF2 (arrowheads in FIG. 12 A).
  • fibroblast-derived clones When attached and allowed to differentiate, fibroblast-derived clones give rise only to cells with fibroblast-like morphology (FIG. 12B), and these cells are never immunopositive for neural-specific antigens. Monolayers of microglia (FIG. 12C) fail to generate spherical clones when replated in suspension culture supplemented with EGF and FGF1.
  • astrocytes immunofluorescence for astrocytic glial fibrillary acidic, GFAP, protein
  • neurons immunofluorescence for neuronal beta III tubulin
  • FIG. 13 A Two attached clones from this anaplastic astrocytoma seem to merge, with neurons and astrocytes within and surrounding the clones.
  • FIG. 13B Two clones immunostained for just neuronal ⁇ III- tubulin shows many neurons that arise from such glial tumors.
  • FIG. 13C Two different clones, from the same tumor, again stained for the same astrocytic and neuronal markers.
  • FIG. 13D A single, ⁇ III-tubulin positive neuron that is outside of the attached clone, and exhibits unusual somatodendritic morphology.
  • FIG. 13E Higher magnification of neurons and astrocytes with apparently normal morphologies.
  • FIG. 13F and FIG. 13G The same field of cells immunostained for GFAP (FIG. 13F) and neuronal ⁇ III-tubulin (FIG. 13G). It can be seen that there are single cells expressing both neuronal and glial antigens, which is not normally seen within clones derived from non-tumor stem/progenitor cells.
  • Neuron 13:1071-1082, 1994.
  • Potten and Loeffler "Stem cells: Attributes, cycles, spirals, pitfalls, and uncertainties.

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Abstract

Dans le système nerveux central (SNC) adulte ou en développement, les cellules souches/progénitrices peuvent former des neurosphères ou des clones multipotents lorsqu'elles sont cultivées en présence de facteurs de croissance. Des cellules souches/progénitrices multipotentes ont été isolées et cultivées à partir de spécimens cérébraux humains adultes après des intervalles post-mortem prolongés. On a découvert que les astrocytes cultivés comme des monocouches forment des neurosphères dans des cultures issues de nombreuses régions différentes du SNC, y compris le cortex cérébral, la zone sous-épendymaire et l'écorce cérébelleuse. De plus, les astrocytes conservent cette propriété même après de nombreux passages. Une pluralité d'immuno-marqueurs pour les astrocytes et les neurones produits à partir de neurosphères contiennent des cellules immunoréactives pour ces marqueurs. Or, il s'avère que les neurosphères présentent certaines caractéristiques propres aux cellules souches/progénitrices dans des conditions de culture de tissu particulières.
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US6897061B1 (en) 2000-06-16 2005-05-24 Spinal Cord Society Transdifferentiation of glial cells
EP1608728A2 (fr) * 2003-03-31 2005-12-28 University Of South Florida Research Foundation, Inc. Materiels et procedes pour reguler la formation de processus lors de la culture cellulaire
EP1682075A4 (fr) * 2003-11-07 2009-12-09 Univ Florida Mise en culture et differenciation de cellules precurseurs neuronales
US20170073640A1 (en) * 2007-07-12 2017-03-16 Discgenics, Inc. Compositions of adult disc stem cells and methods for the treatment of degenerative disc disease

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US6071889A (en) * 1991-07-08 2000-06-06 Neurospheres Holdings Ltd. In vivo genetic modification of growth factor-responsive neural precursor cells
US6638763B1 (en) * 1997-01-07 2003-10-28 University Of Tennessee Research Foundation Isolated mammalian neural stem cells, methods of making such cells

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6897061B1 (en) 2000-06-16 2005-05-24 Spinal Cord Society Transdifferentiation of glial cells
EP1608728A2 (fr) * 2003-03-31 2005-12-28 University Of South Florida Research Foundation, Inc. Materiels et procedes pour reguler la formation de processus lors de la culture cellulaire
US8252279B2 (en) 2003-03-31 2012-08-28 University Of South Florida Methods for cell therapy
EP1682075A4 (fr) * 2003-11-07 2009-12-09 Univ Florida Mise en culture et differenciation de cellules precurseurs neuronales
US20170073640A1 (en) * 2007-07-12 2017-03-16 Discgenics, Inc. Compositions of adult disc stem cells and methods for the treatment of degenerative disc disease
US11168305B2 (en) * 2007-07-12 2021-11-09 Discgenics, Inc. Methods for the treatment of degenerative disc disease

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