WO2003040353A1 - Nouvelle cellule souche presentant un potentiel de croissance a long terme, procedes de cultures et animaux obtenus - Google Patents

Nouvelle cellule souche presentant un potentiel de croissance a long terme, procedes de cultures et animaux obtenus Download PDF

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WO2003040353A1
WO2003040353A1 PCT/AU2002/001533 AU0201533W WO03040353A1 WO 2003040353 A1 WO2003040353 A1 WO 2003040353A1 AU 0201533 W AU0201533 W AU 0201533W WO 03040353 A1 WO03040353 A1 WO 03040353A1
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cells
cell
embryo
neural stem
fns
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PCT/AU2002/001533
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John Roderick Morrison
Shahnaz Fida
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Copyrat Pty Ltd
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Priority claimed from AUPR8780A external-priority patent/AUPR878001A0/en
Priority claimed from AUPS0204A external-priority patent/AUPS020402A0/en
Application filed by Copyrat Pty Ltd filed Critical Copyrat Pty Ltd
Publication of WO2003040353A1 publication Critical patent/WO2003040353A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0271—Chimeric animals, e.g. comprising exogenous cells
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    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
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    • 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
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • the present invention relates generally to the area of cell culturing.
  • the invention relates to methods of preparing a cellular composition comprising cells which may be expanded long term in culture and preferably maintain a high degree of plasticity.
  • the invention also extends to cells having this characteristic.
  • the invention relates to providing a substantially homogeneous neural stem cell-like composition having these characteristics.
  • the invention also relates to a cell composition prepared by the methods as well as uses of the cells from the composition.
  • neural stem cells The characterisation and isolation of neural stem cells is useful to understand and treat neurological disorders in mammals.
  • cell lines based on neural stem cells may be suitable for the generation of chimeric animals, gene targeting and gene knockout experiments and for nuclear transfer experiments to produce genetically modified animals. Further these cells may be used to generate "markers" (eg monoclonal antibodies) that may identify these cells or RNA may be extracted from these cells to generate gene expression profiles.
  • markers eg monoclonal antibodies
  • neural stem cells are believed to have a greater degree of developmental plasticity and therefore have the ability to generate neural lineages and haematopoietic lineages etc. Therefore, due to the multipotent phenotype of neural stem cells and their ability to readily multiply in a suitable culture they are useful for gene targeting and gene knockout experiments.
  • somatic cells have a limited life span and enter senescence after a limited number of cell divisions. Because the successful integration or deletion of a DNA sequence in cells in culture requires a relatively large number of cellular divisions, this limit on cell proliferation represents an obstacle to the genetic manipulation of the donor cell nuclei and, ultimately, to the production of genetically modified animals by nuclear transfer. The production of somatic cells capable of long term growth in culture and their application to nuclear transfer would represent a major step towards the production of such genetically modified animals.
  • neural stem cell line derived from foetal tissue with long-term growth potential.
  • the neural stem population isolated at this later stage of development has a different phenotype and characteristics to embryonic stem cells.
  • a cellular composition comprising one or more cells having a property characteristic of a precursor or neural stem cell-like cell and which are substantially nestin negative.
  • the cells are derived in the presence of NT3 or equivalent thereof and require NT3 or equivalent thereof to maintain their high rate of proliferation potential.
  • NT3 cells represent a primordial neural stem cell.
  • the NT3 cells have the ability to generate neural stem cells, which may in turn differentiate in to neurons or glial cells.
  • a method of preparing a cellular composition comprising a substantially homogeneous population of cells having a property characteristic of a precursor or neural stem cell-like cell and wherein said cell is substantially nestin negative said method comprising: obtaining a source of neural stem cells; preparing a suspension of cells from the source; contacting the suspension of cells with a medium containing NT3 or equivalent thereof; and culturing the cells including passaging and propagation of cells.
  • a media suitable for culturing NT3 cells wherein said media includes NT3 or equivalent thereof and at least one mitogenic factor in said media.
  • a method of culturing NT3 cells in long- term culture and wherein said cell has a property characteristic of a precursor or neural stem cell-like cell comprising culturing the cells in the presence of NT3 or equivalent thereof and at least one mitogenic factor.
  • a genetically modified NT3 cell said cell comprising a foreign gene which has been introduced into an FNS or NT3 cell.
  • a genetically modified NT3 cell in another aspect of the present invention, there is provided a genetically modified NT3 cell, said cell having a destroyed, modified or deleted gene.
  • Such genetically modified NT3 cells are useful in gene targeting and gene knockout experiments.
  • a method of producing a non-human animal comprising introducing one or more donor NT3 cells or nuclei from a donor NT3 cell into an oocyte or embryo and allowing the resulting embryo to mature and to preferably develop to a foetus or an adult animal.
  • a method of producing a cell line that may be expanded from an embryo to produce cloned cells of an embryo comprising introducing a donor NT3 cell or nucleus from a NT3 cell, into an oocyte or embryo; culturing the oocyte or embryo to an advanced cleavage stage embryo; separating and cloning the cleaved cells of the embryo; and optionally culturing the cloned cells.
  • a non-human animal produced by the methods of the present invention.
  • the animal is a genetically modified animal, preferably the genetically modified animal is a knockout animal.
  • a method of preparing a genetically modified animal comprising introducing a genetically modified NT3 cell or a nucleus from a NT3 cell into an oocyte or embryo and allowing the resulting embryo to mature to a foetus or animal.
  • a method of treating a neurological disorder comprising introducing a NT3 cell into a host animal to correct the disorder wherein the NT3 cell is capable of replacing neural cells affected by the neurological disorder.
  • the genetically modified NT3 cells are transfected with exogenous nucleic acid or are genetically modified by destroying, modifying or deleting genes.
  • Selected foreign nucleic acid may be introduced and/or recombinantly expressed in the cells of the present invention through the use of conventional techniques or the genes may be modified, destroyed or deleted by methods such as point or random mutations.
  • the present invention further includes NT3 cells and FNS-NT3 isolated by the methods hereinbefore described which are genetically modified by chemicals to induce random mutations.
  • the generation of random mutations in NT3 cells using a variety of chemical mutagens offers the potential to artificially generate genetic diversity in cell lines that may consequently be utilised to make cloned animals.
  • Figure 1 shows typical microscopic appearance of NT3-FNS cells. Phase contrast images of FNS-NT3 cells either a) floating in the media as clusters or b) plated to a tissue culture dish.
  • Figure 2 shows typical microscopic appearance of NT3-FNS cells generated in the presence of BNDF. Phase contrast images of FNS cells grown in the presence of BNDF (10 ng/ml) - NT3-like clusters are beginning to emerge from the plated cells.
  • Figure 3 shows typical microscopic appearance of NT3-FNS cells generated in the presence of NGF. Phase contrast images of FNS cells grown in the presence of NGF (10ng/ml) - NT3-like clusters are beginning to emerge from the plated cells.
  • Figure 4 shows immunofluorescence of nestin in FNS and FNS-NT3 cells.
  • DAPI 49,6-diamidino-2-phenylindole dihydrochloride
  • Figure 5 shows immunofluorescence of vimentin in FNS and FNS-NT3 cells.
  • Figure 6 shows A) Phase contrast image of adult neural stem cells; B) phase contrast image of NT3-like cells emerging from adult neural stem cells in the presence of NT3 (10 ng/ml).
  • Figure 7 shows staining of chimeric rat embryos for ⁇ -galactosidase activity. Blastocysts were injected with rat FNS-NT3 cells and transferred to the uterus of a pseudo-pregnant rat. On day 12-14 embryos were collected and stained for ⁇ -galactosidase activity that stains blue for both intrinsic mammalian enzyme activity as well as the introduced bacterial transgene. Hence a variety of tissues will stain positively in control tissue the most notable being the small intestine.
  • a listing of tissues with intrinsic levels of ⁇ -galactosidase is available [1].
  • a cellular composition comprising one or more cells having a property characteristic of a precursor or neural stem cell-like cell and wherein said cell is substantially nestin negative.
  • the cells Preferably have a property characteristic of a primordial foetal neural stem cell but has the capacity to retain a high degree of plasticity and is capable of long term culture.
  • the present invention provides a substantially homogeneous population of multipotential precursor cells which may be derived from foetal neural stem (FNS) cells, compositions containing FNS cells, adult neural stem (NS) cells, neural tissue, or any source which contains NS cells. These cells may be expanded indefinitely in culture while maintaining their multipotential capacity to differentiate into neurons and glial cells. This characteristic is understood to be a "primordial characteristic” or “primordial state” as used herein. These cells display a high plasticity and as such may represent a precursor to the standard polymorphic FNS cells that are isolated using established protocols. These cells represent an important tool for many cell-based and gene-based therapies for neurological disorders.
  • FNS foetal neural stem
  • NS adult neural stem
  • neural tissue or any source which contains NS cells.
  • the cells also may have application in the areas of mammalian cloning, generating chimeric animals and transdifferentiation.
  • the cells may be used to generate antibodies (monoclonal or polyclonal), used in gene expression studies (eg microarrays or proteomic applications) or used in mutagenesis studies (eg N-ethyl-N-nitrosourea (ENU)).
  • gene expression studies eg microarrays or proteomic applications
  • mutagenesis studies eg N-ethyl-N-nitrosourea (ENU)
  • a cell composition comprising cells that grow rapidly in culture (doubling time approximately 24 h) and appear to have long term culture potential.
  • these cells may represent precursor cells to FNS cells and as such represent a cell-type with a predictably high ability to transdifferentiate.
  • the precursor or neural stem celllike cells of the present invention have the capacity to develop into any particular cell type or tissue but will maintain the primordial phenotype or state until an appropriate differentiating condition is presented.
  • substantially nestin negative is a relative term compared against a nestin reaction of FNS cells. "Nestin negative” includes low and almost undetectable expression when compared against FNS cells run in parallel.
  • long term culture means an ability to grow long term such that the cell may be passaged to new cultures preferably maintaining a primordial state which is typically a stage prior to the foetal neural stem (FNS) cell stage.
  • long term culture means at least 3 months, more preferably 6 months.
  • NT3 cells For convenience, the nestin negative cells of the present invention will be designated generally as “NT3 cells” and will be referred to as such throughout this application.
  • NT3 cells is used generally herein to include all neural stem cells that are in the primordial state and have either derived directly from FNS, compositions containing FNS, adult NS cells, neural tissue or any source of NS cells.
  • FNS-NT3 cells are a preferred form of the NT3 cells and is a foetal neural stem cell subtype of the NT3 cell population.
  • the FNS-NT3 cells include NT3 responsive FNS cells and are also responsive to alternate growth factors that act on the same cellular signalling pathway as NT3. Such growth factors may include brain derived neurotrophic factor (BDNF) or neurotrophic growth factor (NGF).
  • BDNF brain derived neurotrophic factor
  • NGF neurotrophic growth factor
  • these cells can be FNS cells which have de- differentiated to a state of NT3 responsiveness and maintain the primordial state which is a stage prior to a standard FNS cell stage when exposed to NT3, or an alternate growth factor as described above, such as but not limited to BDNF or NGF.
  • FNS-NT3 cells whether they are derived from FNS by exposure to NT3 or to alternate growth factors such as BDNF and NGF.
  • NT3, BDNF, NGF Neutrotrophins
  • NT3 activates the TrkC pathway and to a lesser degree TrkA and B, and has high affinity for TrkC [3].
  • TrkC Through TrkC, NT3 signals a number of trophic effects, ranging from mitogenesis, promotion of survival, or differentiation depending on the developmental stages of the target cells [3].
  • BDNF belongs to the neurotrophin growth factor family and acts via a high affinity TrkB receptor [3].
  • NGF belongs to the neurotrophin growth factor family and acts via a high affinity TrkA receptor [3]. All three of these act through the tyrosine receptor kinase pathway initiating a cascade of intracellular cytoplasmic signals, produce dimers and activate tyrsine residues that in turn activate the MAP kinase pathway.
  • the NT3 cells of the present invention may be characterised by their nestin negative staining and preferably also by their ability to grow long term in tissue culture without undergoing transformation and retain some degree of developmental plasticity.
  • the phenotype of the NT3 cells do not change over long term culturing and the plasticity of the NT3 cells make them suitable for nuclear transfer experiments and various other applications such as gene knockout experiments.
  • these NT3 cells and preferably FNS-NT3 cells have the capacity to differentiate into one or more different types of cells when placed in differentiating conditions.
  • the types of cells, which may result from differentiation, include haematopoietic stem cells and their lineages and neural stem cells and their lineages.
  • the NT3-cells, and preferably the FNS-NT3 cells have the capacity to grow indefinitely in tissue culture and this means that they can remain undifferentiated.
  • the degree of plasticity means that these cells have the ability to generate multiple cell types and the cells of the present invention may be identified by these characteristics.
  • they are more plastic than neural stem cells and FNS cells and hence have a primordial phenotype which affords greater diversity to the resultant cell lines which ultimately differentiate from these cells.
  • the NT3 cells preferably FNS-NT3 cells, require the presence of at least one growth factor, preferably epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) for cell division.
  • the NT3 cells preferably the FNS-NT3 cells are however, dependent on NT3 or equivalent thereof for some of their properties, including robust growth rates. Removal of EGF, NT3 or equivalents thereof, or bFGF from the medium stops cell division in the cells and induces quiescence of the cells. Absence of a growth factor does not kill the cells. Depending on the passage number of the cells, the reintroduction of a growth factor may stimulate the cells to re-enter the cell cycle.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • Each bud comprises a plurality of cells which may be cultured to provide an isolated and purified population of the NT3 cells, or the FNS-NT3 cells.
  • the cells may be identified by an inability to be detected or be poorly detected by neural stem cell markers such as nestin. Hence, they are substantially nestin negative. Applicants have found that the neural stem cell marker, nestin, fails to detect (or poorly detects) the NT3 cell. However these NT3 cells may differentiate to become FNS cells and start to express Nestin. This characteristic is important for distinguishing these cells over other cells which have progressed along the differentiation pathway.
  • neural stem cell markers may therefore be utilized with other neural stem cell markers by identifying cell lines before and after the application of differentiation signals.
  • neural stem cell markers become useful as the cell progresses through to differentiation.
  • any neural stem cell marker may be used as a negative marker and confirmed by positive identification after differentiation.
  • Methods of identifying the cells which have the characteristics of neural stem cells may be any method known to the skilled addressee for detecting the properties listed above. For instance for detecting cell markers, antibodies (monoclonal or polyclonal) are available to identify them.
  • Methods of isolation may be employed based on the methods of identification. Since the cells may be identified by negative expression of the neural stem cell markers such as nestin, methods of isolation may also utilize the markers for neural stem cells in a negative manner and identify and isolate those cells which do not express a neural stem cell marker. For instance, antibodies may be used to select those neural stem cells and foetal neural stem cells having the appropriate markers, alternatively suitable cell culture conditions may be used to obtain cells with the morphology of the neural stem cells of the present invention and therefore select out for those cells which do not express these markers.
  • the neural stem cell markers such as nestin
  • methods of isolation may also utilize the markers for neural stem cells in a negative manner and identify and isolate those cells which do not express a neural stem cell marker. For instance, antibodies may be used to select those neural stem cells and foetal neural stem cells having the appropriate markers, alternatively suitable cell culture conditions may be used to obtain cells with the morphology of the neural stem cells of the present invention and therefore select out for those cells which do not express these markers.
  • a method of preparing an isolated cellular composition comprising cells having a property characteristic of a precursor or neural stem cell-like cell and wherein said neural stem cell-like cell is substantially nestin negative said method comprising: obtaining a source of neural stem cells; preparing a suspension of cells from the source; contacting the suspension of cells with a medium containing NT3 or equivalent thereof; and culturing the cells including passaging and propagation of the cells.
  • the source of neural stem cells includes adult neural stem cells, FNS cells and primordial neural stem cells. More preferably the source includes primordial neural stem cells having the properties as described above. The primordial cells will retain their primordial state in the presence of NT3 or equivalent thereof.
  • the neural stem cells may also include standard foetal neural stem cells that are not in their primordial state. Applicants have found that exposure of NT3, or alternate growth factors that act on the same cellular signalling pathway as NT3, to standard FNS cells can revert the cells to a primordial state characteristic of the NT3 cells. These alternate growth factors may include BDNF and NGF.
  • the source of the neural stem cells may derive from any animal that has a nervous system.
  • the animal is a mammal including but not limited to human, rodent, bovine, ovine, porcine, equine, feline, simian, endangered species, live stock or may derive from marsupials including kangaroos, wombats. More preferably, the animal is a rodent, more preferably the animal is a rat.
  • Neural stem cells such as adult NS cells, FNS cells, and primordial neural stem cells may be collected from any stage of development. More preferably the source of these cells is from a foetus which is differentiated at a stage after the embryonic stage. The whole foetus or a part thereof containing these cells may be used as a source of the neural stem cells. Preferably the head or spinal cord of the foetus provides the source of neural stem cells. More preferably, the head of a foetus or animal is used as a source of neural stem cells including adult neural stem cells, foetal neural stem cells, and preferably primordial neural stem cells.
  • the cells are obtained from rat foetuses and more preferably from the head of a rat foetus. It has been found that foetus obtained from Sprague- Dawley rats provides a reliable source of primordial neural stem cells and foetal neural stem cells.
  • FNS cells may be obtained by standard methods available to the skilled addressee. These FNS cells may have been prepared in the absence of NT3 and may also have undergone several passages prior to exposure to NT3 or alternate growth factors such as BDNF or NGF. NT3 cells and FNS-NT3 cells may be created as a result of exposure of FNS cells to NT3 or equivalent thereof such as alternate growth factors as described above.
  • membranes from foetuses may be removed and their heads separated from their bodies.
  • the pooled foetal heads may be placed into a petri dish and the tissue minced with a blunt object such as the tip of a syringe until homogeneous in size.
  • a syringe may be used to aspirate the minced tissue which may be transferred into a tube.
  • the dish can be washed with 5-10 ml PBS and then aspirated into a syringe and pooled into a tube containing tissue.
  • the minced tissue may be spun down and resuspended in a small volume of media.
  • the cells may be placed onto fibronection + poly- -Ornithine pre-coated plates at a density of approximately 2.5 x 10 5 to 5.0 x 10 5 cells/cm 2 and incubated in 5% CO 2 at 37°C. From this cell suspension, neural stem cells may be obtained for further culturing under appropriate conditions.
  • the isolation technique employed to obtain the neural stem cells may be conducted in the presence or absence of NT3 or equivalent thereof.
  • NT3-cells prepared directly from neural tissue will generally include primordial neural stem cells and primordial FNS cells.
  • the method of preparing the cellular composition requires exposure or contacting the various neural stem cell types including primordial neural stem cells, primordial FNS cells and standard polymorphic FNS cells, to NT3 or equivalent thereof.
  • the cells may therefore be prepared directly from neural tissue and are immediately responsive to NT3 or equivalent thereof (and have never progressed to a standard FNS state) or they may never have been exposed to NT3 and progressed to a standard FNS state, but show responsiveness to NT3 upon exposure to NT3 thereby manifesting an ability for long term culture and primordial state characteristics (NT3 responsive FNS cells).
  • the exposure or contacting of the NT3 may be through the preparative stages of isolating neural stem cells or it may occur at the stage of culturing the cells.
  • a media suitable for culturing NT3 cells wherein said media includes NT3 or equivalent thereof and at least one mitogenic factor.
  • NT3 or equivalent thereof is important for maintaining the cells in a primordial state.
  • the term "equivalent thereof as applied to NT3 means any natural or synthetic compound which behaves in the same manner as NT3. These equivalents may include alternative growth factors that act on the same signalling pathway as NT3 and include, but are not limited to BDNF and NGF.
  • NT3 has the characteristics of, but not limited to, enhancing the propagation of neural stem cells but has not previously been associated with the ability to allow cells to take on more primordial characteristics.
  • a suitable medium to maintain the cells in culture is a medium which can perpetuate the cultured NT3 cells as herein described, most preferably they are cultured indefinitely.
  • a suitable media may comprise Neurobasal-A media® (Life Technologies), containing Insulin-Transferrin-Selenium (Life Technologies) approximately 1 :100; EGF (Life Technologies) 2 to 20ng/ml; bFGF (Life Technologies) 2 to 20ng/ml; human NT3 (R&D) 2 to 20 ng/ml; Chemically defined lipid concentrate (Life Technologies) approximately 1 :100; N-2 supplement (Life Technologies) 1 :100; B-27 supplement (Life technologies) approximately 1 :100, L-glutamine 1 to 2 mM; 200 U/ml Penicillin, 200 ⁇ g/ml Streptomycin.
  • a further preferred cell medium suitable for the present invention comprises Dulbecco-modified Eagle's medium (DMEM) comprising 15 mM 4-(2-hydroxy- ethyl)-1-piperazine-ethanesulfonic acid, 4.5g/l glucose, 1.2g/l Bicarbonate, 200 U/ml Penicillin, 200 ⁇ g/ml Streptomycin; the following additional components are added prior to use of the media including Bovine insulin (10 ⁇ g/ml), Human transferrin (25 ⁇ g/ml), human NT3 (2-50 ng/ml) Mouse EGF (2-20 ng/ml), Sodium selenite 2-20nM, and Human HDL (freshly isolated) 5-50 ⁇ g/ml.
  • the EGF growth factor may be substituted with bFGF (FGF-2) or any other suitable mitogenic growth factors.
  • the media may contain known components that in combination, support the growth of the cultured NT3 cells or preferably the FNS-NT3 cells.
  • the media may include other nutrients, buffers, hormones, salts, antibiotics, proteins, growth factors and enzymes.
  • a suitable medium contains at least a combination of one or more mitogenic factors and lipids.
  • Suitable mitogenic factors may be selected from the group including, but not limited to, bFGF, EGF and PDGF. These factors may be used alone or in combination with the lipids providing both lipids and mitogenic factors are included in the media along with NT3 or equivalent thereof. EGF and/or bFGF are mostly preferred as mitogenic factors in the media along with NT3 or equivalent thereof.
  • Some components may be substituted for others (eg insulin-like growth factors for insulin; transforming growth factor alpha for epidermal growth factor; bovine serum albumin containing lipids; polylysine for fibronectin; and iron salts for transferrin).
  • other factors might be added to the culture medium, such as tumour promoters, additional hormones and/or growth factors, bovine serum albumin, low concentrations of serum or plasma, or modified plasma preparations with reduced inhibitory activity.
  • Fibronectin might be eliminated from the culture medium formulation to obtain anchorage-independent growth of the present cell lines. Alteration of culture medium components may also allow derivation of sublines of the non-tumorigenic cell lines of the present invention or their equivalent.
  • the media contains at least a cell survival factor, such as transferrin, insulin, growth factors such as EGF, bFGF (FGF-2) or PDGF, lipids and selenium.
  • a cell survival factor such as transferrin, insulin, growth factors such as EGF, bFGF (FGF-2) or PDGF, lipids and selenium.
  • a method of culturing NT3 cells comprising culturing the cells in the presence of NT3 and at least one mitogenic factor.
  • NT3 or an equivalent thereof is essential for maintaining the primordial primordial state and allows the cells to be cultured, preferably in long term culture without differentiating whilst maintaining a high degree of plasticity.
  • the cells may be cultured and passaged for propagation.
  • the neural stem cells of the present invention have the characteristic of being able to "bud off into the media. These can be seen with the naked eye.
  • the buds may be collected and spun down.
  • the buds may be disaggregated by any method available to the skilled addressee. However, vigorous pipetting can disaggregate the buds to provide separate cells. Prolonged use of trypsin is discouraged as the cells may be sensitive to trypsin.
  • the cells Once disaggregated, the cells may be inoculated into a fresh medium, preferably in a media described above. Therefore the present invention also relates to the long-term clonal expansion or propagation of NT3 cells, preferably FNS-NT3 cells having a primordial characteristic.
  • the cells may be passaged using trypsin for a short period.
  • Cells are first washed with PBS to remove media.
  • the cells may be loosened from the plate using a trypsin solution for a minimal period at 37°C, usually less than 2 min.
  • the cells Preferably the cells be free of the tissue culture plate. However, they do not need to be totally disaggregated.
  • the trypsin may be neutralised using soyabean trypsin inhibitor, preferably at 1 mg/ml made up in the suitable media being used to culture cells added 1:1 (v/v) to the trypsin solution.
  • the cells may be spun down at low speed in a centrifuge, the media removed and the cells resuspended in fresh media and plated in new fibronectin-treated tissue culture plates.
  • the cells may be split approximately 1 :4.
  • Preferably the cells are maintained at a minimum plating density such as 2.5 x 10 5 to 5.0 x 10 5 cells/cm 2 .
  • NT3 cells have a tendency to differentiate when plated at low density.
  • the cells may be frozen preferably in Neurobasal A Media containing 7.5% DMSO or by any methods available to the skilled addressee which would be suitable for freezing cells.
  • the NT3 cells preferably the FNS-NT3 cells of the present invention have the capacity to grow long term without undergoing transformation and retain a degree of plasticity. This can be achieved by culturing and propagating the cells as described above.
  • the present invention also provides an isolated NT3 cell prepared by the methods described above.
  • the cell is a FNS-NT3 cell which is a FNS cell which shows responsiveness to NT3. More preferably the cell is a primordial neural stem cell or a primordial FNS cell.
  • a genetically modified NT3 cell said cell comprising a foreign gene which has been introduced into the NT3 cell.
  • a genetically modified NT3 cell in another aspect of the present invention, there is provided a genetically modified NT3 cell, said cell having a destroyed, modified or deleted gene.
  • Such genetically modified NT3 cells are useful in gene targeting and gene knockout experiments.
  • a genetically modified NT3 cell refers to a cell into which a foreign (ie non- naturally occurring) nucleic acid, eg, DNA, has been introduced.
  • the foreign nucleic acid may be introduced by a variety of techniques, including, but not limited to, calcium-phosphate-mediated transfection DEAE-mediated transfection, microinjection, retroviral transformation, electroporation, immunoporation, protoplast fusion and lipofection.
  • the genetically modified cell may express the foreign nucleic acid in either a transient or long-term manner. In general, transient expression occurs when foreign DNA does not stably integrate into the chromosomal DNA of the transfected cell. In contrast, long- term expression of foreign DNA occurs when the foreign DNA has been stably integrated into the chromosomal DNA of the transfected cell.
  • Foreign (heterologous) nucleic acid may be introduced or transfected into the FNS cell which can de-differentiate to an FNS-NT3 cell or NT3 cell.
  • An NT3 cell which harbours foreign DNA is said to be a genetically modified cell.
  • the foreign DNA may be introduced using a variety of techniques.
  • foreign DNA is introduced into the NT3 or FNS-NT3 cells using the technique of retroviral transfection.
  • Recombinant retroviruses harbouring the gene(s) of interest are used to introduce into NT3 or FNS-NT3 cells using the technique of retroviral transfection.
  • Recombinant retroviruses harbouring the gene(s) of interest are used to introduce marker genes, such as but not limited to ⁇ galactosidase (lacZ) gene, or oncogenes.
  • the recombinant retroviruses are produced in packaging cell lines to produce culture supernatants having a high titre of virus particles (generally 10 5 to 10 6 pfu/ml).
  • the recombinant viral particles are used to infect cultures of the NT3 or FNS cells or their progeny by incubating the cell cultures with medium containing the viral particles and 8. ⁇ .g/ml polybrene for three hours. Following retroviral infection, the cells may be rinsed and cultured in standard medium. The infected cells may be then analysed for the uptake and expression of the foreign DNA.
  • the cells may be subjected to selective conditions which select for cells that have taken up and expressed a selectable marker gene.
  • the present invention accordingly includes NT3 cells and preferably FNS-NT3 cells isolated by the methods hereinbefore described which are transfected with exogenous nucleic acid. Selected foreign nucleic acid may be introduced and/or recombinantly expressed in the cells of the present invention through the use of conventional techniques.
  • a method of preparing a genetically modified animal comprising introducing one or more NT3 cells or cell nuclei from NT3 cells into an oocyte or embryo and allowing the resulting embryo to mature to a foetus or animal.
  • the NT3 cell is a genetically modified neural stem cell as described above having a gene inserted, deleted or destroyed.
  • the foreign gene may be a gene encoding a desired product preferably to induce a desired characteristic in the genetically modified animal or to generate a gene knockout model wherein the gene is absent.
  • the present invention also provides knockout animals which are useful for research in gene function, diseases, drug therapies and gene development of animal strains having knockout genes prepared as described above.
  • the genetically modified animals may be useful for research purposes at any stage of development, preferably adult knockout animals are obtained. However animals at any stage of development may be used.
  • the animal is a mammal including but not limited to murine, bovine, ovine, porcine, equine, feline, simian, endangered species, live stock or may derive from marsupials including kangaroos, wombats.
  • the animal is a rodent. Most preferably the animal is a rat.
  • a method of preparing a genetically modified animal comprising introducing one or more genetically modified NT3 cells or cell nuclei into an oocyte or embryo and allowing the resulting embryo to mature to a foetus or animal.
  • a method of producing an animal comprising introducing one or more continuously growing donor NT3 cells or cell nuclei from a continuously growing donor NT3 cell into an oocyte or embryo and allowing the resulting embryo to mature and to preferably develop to a foetus or an adult animal.
  • a donor cell or cells which have the ability to grow continuously in culture.
  • Some cells have the limitation of being short lived and they stop dividing in a very short period. Accordingly there is little time for genetic manipulation of these cells and this is often a major limitation in genetic modification or knockout studies.
  • Some cell lines which are naturally continuously growing (ie neuronal stem cells) and which do not require further genetic manipulation, may also be used. From these cells, the nucleus may also be extracted and used in the present invention. The nucleus may be extracted from neural stem cells described above and preferably grown under conditions utilizing the media as described above.
  • the genetically modified NT3-cell or FNS- NT3 cell or cells may be merged into an embryo (eg a morula or blastocyst) to generate a chimeric animal.
  • the genetically modified NT3 cell or FNS-NT3 cell or cells would be expected to be incorporated into all cell lineages (eg endoderm, mesoderm and ectoderm) of the developing embryo, allowing the study of the mutation to be studied in an in vivo setting. More preferably this chimeric animal will incorporate the genetically modified cells into the germ-line of the animal allowing the genetic modification to be transmitted to offspring.
  • the genetically modified NT3 cell or cells or FNS-NT3 cell or cells are used as the nuclear donors in nuclear transfer or cloning experiments.
  • the donor cell is a genetically modified continuously growing NT3 cell.
  • the nucleus may be derived from a genetically modified NT3 cell which is continuously growing.
  • the nucleus is from a NT3 cell as described above wherein the cell is capable of long term culture and hence is continuously growing and maintaining a high degree of plasticity.
  • the donor cell nucleus is derived from a non-transformed cell line. Manipulation or genetic modification of the cell line by any method that immortalizes the cell line may be used. More preferably, the nucleus is from a NT3 cell line. More preferably, it is from a FNS-NT3 cell line.
  • Oocytes may be obtained from any source. For example, they may be of bovine, ovine, porcine, rodent, caprine, simian, amphibian, equine or of a wild animal origin.
  • the oocyte is a rodent oocyte. More preferably it is a rat oocyte.
  • PCT/AU97/00868 The entire contents of PCT/AU97/00868 are hereby incorporated and referred to in this description particularly with respect to the oocytes suitable for this invention and of the enucleation of suitable oocytes.
  • an embryo wherein said embryo results from introducing a donor cell nucleus from a donor cell into an oocyte or embryo prepared by the method described herein.
  • the embryo is preferably a transplantation embryo.
  • the donor cells and the nucleus may be as described above.
  • the transplantation embryos produced by the methods of the present invention may be used to produce genetically identical or similar animals by transplantation into a recipient female, preferably a synchronised female.
  • the recipient female is synchronised using fertility drugs, steroids or prostaglandins. Methods for transfer of embryos to recipient females are known to those skilled in the art.
  • a genetically modified animal may include the addition of foreign genes capable of identification by the presence of marker genes which have been introduced into a donor cell or nucleus. Suitable marker genes may include fluorescently labelled genes which may facilitate identification of genetically modified animals. A genetically modified animal may include a transgenic animal.
  • Genetically modified animals may also include knockout animals having genes targeted, destroyed and/or modified so that an animal is developed without the gene. Genes may be modified by removal from the genome or by point or random mutations in a gene.
  • a method of producing a cell line that may be expanded from an embryo to produce cloned cells of an embryo, said method comprising introducing a donor NT3 cell or nucleus from a NT3 cell, into an oocyte or embryo; culturing the oocyte or embryo to an advanced cleavage stage embryo; separating and cloning the cleaved cells of the embryo; and optionally culturing the cloned cells.
  • the donor cells and the nucleus may be as described above.
  • the cell lines may be used to generate genetically identical lines and animals. This technique may be particularly useful for non- murine models such as monkeys to develop genetically identical animals.
  • the cells of such a nuclear transplantation embryo may be recycled to provide donor cells for further cycles of nuclear transfer, as described in Australian patent 687422, the entire disclosure of which is incorporated herein by reference.
  • the present invention provides a cell line expanded from an embryo as prepared by the methods described herein.
  • an animal produced by the methods of the present invention is a genetically modified animal; preferably the genetically modified animal is a knockout animal.
  • a method of treating a neurological disorder comprising introducing a NT3 cell into a host animal to correct the disorder wherein the NT3 cell is capable of replacing neural cells affected by the neurological disorder.
  • the NT3 cell is preferably an FNS-NT3 cell as described above.
  • the NT3 cells may be capable of regenerating the neural tissue.
  • the genetically modified NT3 cell may be introduced into the patient in need of regeneration and treatment of the neurological disorder.
  • the neurological disorder is Parkinsons disease.
  • the present invention also includes the use of NT3 cells, preferably FNS-NT3 cells in a wide range of applications including but not limited to transplantation, nuclear transfer and gene targeting and gene knockout experiments, the generation of transgenic animals and the construction of animal models.
  • the genetically modified NT3 cells and preferably FNS-NT3 cells are transfected with exogenous nucleic acid or are genetically modified by destroying, modifying or deleting genes.
  • Selected foreign nucleic acid may be introduced and/or recombinantly expressed in the cells of the present invention through the use of conventional techniques or the genes may be modified, destroyed or deleted by methods such as point or random mutations.
  • the present invention further includes NT3 cells, preferably FNS-NT3 cells isolated by the methods hereinbefore described which are genetically modified by chemicals to induce random mutations.
  • chemical agents include, but are not limited to ENU, or ethylmethanesulphonate (EMS).
  • ENU can generate very high mutation rates in cells (and gametes) approaching one hit per 200 cells on studies using the Hprt locus (Chen Y., et al (2000) Nat Genetics 24: 314-317).
  • ENU tends to induce point mutations similar to many of the variants found within the human genome. Sequencing relevant regions can identify cells carrying mutations in genes of interest.
  • the advantage of this approach is that the cell lines generated from ENU treatment carry many point mutations, possibly considerably more than 1 ,000 per cell. Thus not only have cell lines carrying a variety of point mutations in the gene of interest been identified, each cell line carries it own unique genetic diversity. Hence this random mutagenesis approach offers the potential of generating animal models for studying drug metabolism with a potentially multigenic phenotype.
  • the NT3 or FNS-NT3 cells may be screened directly for responsiveness to drugs in tissue culture. Alternately the NT3 or FNS-NT3 cells may be differentiated into a desired cell type, such as neurons, for drug screening.
  • Tissue culture plates were pre-coated with fibronectin at 1 ⁇ g/ml and poly- - Ornithine at 15 ⁇ g/ml in DMEM for 2-24 hours at 37°C; 5% CO 2 . (Enough volume was used to cover the surface). The fibronectin/ poly- -Ornithine was aspirated and plates washed with DMEM. This preparation can be stored at room temp for several days.
  • a pregnant rat eg. Sprague-Dawley was humanely killed at 9.5-16.5 days gestation by CO 2 asphyxiation. More preferably the foetuses are obtained at 12.5-14.5 days of gestation. Foetuses were removed and placed into a tube with PBS containing penicillin/streptomycin.
  • Membranes from the foetuses were removed and their heads were separated from their bodies.
  • the pooled foetal heads were placed into a 100mm Petri dish and the tissue was minced with a blunt object (the tip of a syringe) until it was homogeneous in size.
  • a syringe was used to aspirate the minced tissue which was then transferred into a tube.
  • the dish was washed with 5-10 ml PBS and then aspirated into the syringe and pooled into the tube containing the tissue.
  • the minced tissue was spun down and resuspended in a small volume of media.
  • the cells were placed onto fibronectin + poly- L -Ornithine pre-coated plates at a density of approximately 1.5 x 10 5 cells/cm 2 and incubated in 5% CO 2 at 37°C.
  • the cells are passaged every 3-4 days and by passage 3-5 the cells are placed under the NT3 media (see below). Passage 4-5 was found to be optimal for the generation of NT3 cells from FNS cells.
  • the NT3 cells appear after approximately 7 days in culture and initially look like spherical buds (refer to Figure 1). These spherical cells are collected and plated onto fibronectin/ornithine-coated plates.
  • Example 2 Preferred defined medium for culturing of NT3 cells
  • Neurobasal-A media® (Life Technologies), containing Insulin-Transferrin- Selenium (Life Technologies) - 1 :100; EGF (Life Technologies) 10ng/ml; bFGF (Life Technologies) 20ng/ml; human NT3 (R&D) 10 ng/ml; Chemically defined lipid concentrate (Life Technologies) - 1 :100; N-2 supplement (Life Technologies) 1:100; B-27 supplement (Life technologies) 1:100, L-glutamine 1 mM; 200 U/ml Penicillin, 200 ⁇ g/ml Streptomycin.
  • Example 3 Alternate Defined Medium for culturing NT3 cells
  • the FNS cell medium suitable for the present invention comprises Dulbecco- modified Eagle's medium (DMEM) comprising 15 mM 4-(2-hydroxy-ethyl)-1- piperazine-ethanesulfonic acid, 4.5g/l glucose, 1.2g/l Bicarbonate, 200 U/ml Penicillin, 200 ⁇ g/ml Streptomycin; the following additional components are added prior to use of the media:
  • DMEM Dulbecco- modified Eagle's medium
  • Bovine insulin (10 ⁇ g/ml), Human transferrin (25 ⁇ g/ml), human NT3 (2-50 ng/ml) Mouse EGF (2-20 ng/ml), Sodium selenite 10 nM, and Human HDL (freshly isolated) 25 ⁇ g/ml.
  • the EGF growth factor may be substituted with bFGF (FGF- 2) or any other suitable mitogenic growth factors.
  • Example 4 Preferred method for culturing and passaging of NT3 cells
  • a complete change of media was performed daily until cells reached approximately 80% confluency.
  • the media was then aspirated and a small volume of Hanks Buffered Saline Solution (HBSS - Life Technologies) was added to the flask.
  • Cells were harvested with a cell-scraper and transferred to a tube for centrifugation at 800g for 5 minutes.
  • the cell pellet was resuspended in a small volume of Neurobasal A media (Example 2) and live cell number estimated using a haemocytomer and staining of the cells with Trypan Blue.
  • the cells were placed onto fibronectin + poly- L -Ornithine pre-coated plates at a density of approximately 2.5 x 10 5 to 5.0 x 10 5 cells/cm 2 with a suitable volume of preferred defined culture medium (Example 3).
  • Example 5 Alternate method for culturing and passaging of NT3 cells.
  • the media containing the spheres was pipetted off into a tube.
  • the adherent cells were harvested in HBSS with a cell-scraper and transferred to the same tube.
  • the cells were centrifuged at 800 x g for 5 minutes and resuspended in a small volume of Neurobasal A medium (Example 2).
  • live cell number was estimated with a haemocytomer and staining of the cells with Trypan Blue.
  • the cells were then plated into fresh flasks at a density of 2.5 x 10 5 - 5.0 x 10 5 cells/cm 2 with a suitable volume of preferred defined culture medium (Example 3).
  • Example 6 Isolation FNS-NTS-like cells using alternate growth factors.
  • FNS-NT3-like cells can be isolated from sources of FNS cells (as per the previous Example 1) using alternate growth factors that act on the same cellular signalling pathway as NT3.
  • the FNS or FNS-NT3 cells may be cultured as per the previous Examples replacing NT3 in the media with other growth factors.
  • BDNF Brain derived neurotrophic factor
  • NGF Neurotrophic Growth Factor
  • Figures 1b, 2 and 3 show FNS-NT3 cells maintained in media containing NT3, BDNF or NGF respectively.
  • NT3, BDNF and NGF produced FNS-NT3-like balls.
  • BDNF enhanced cluster formation and both neurons and astrocytes were observed.
  • NGF produced more elaborate structures in addition to FNS-NT3-like cells, including some astrocyte-like cells.
  • FNS-NT3 cultures had more neuronal cells and lots of balls and fewer clusters in the presence of NT3.
  • NT3 In contrast to NT3, BDNF and NGF, other neural growth factors were not able to support the culture of FNS-NT3-like cells, e.g. TGF- ⁇ and CNTF.
  • Example 7 Production of NT3 cells from adult rat FNS cells
  • FNS-NT3-like cells can also be derived from adult rat neural stem cells (Adult- FNS cells or ANS cells) using the procedures described in Examples 1 to 5.
  • An adult rat (eg. Sprague-Dawley) was killed by CO 2 asphyxiation.
  • the head is removed and opened revealing the brain.
  • the brain is dissected out into PBS and kept cold on ice.
  • the hippocampus and cerebellum are removed and the grey matter discarded.
  • the tissue is mashed into small pieces with the blunt end of a syringe.
  • the tissues are washed twice by spinning through 10 ml of NSA media (Euroclone, Scotland UK).
  • the ANS cells are then plated onto 6 well culture dishes in the presence of 0.5% Hyclone serum (GibCo) in NSA media. The following day the media is exchanged for 100% NSA media.
  • Rat adult FNS cells when treated with 5-10ng/ml NT3 as per the previous examples remain similar to control cultures up to passage 2 but by passage 3 these cells appear more granular to control cells. There are more connections and structures being formed in the treated cells compared to the control cells. By passage 4 these cells produce NT3 balls in about 3-4 days and then rosette like clusters.
  • the FNS-NT3 balls when plated grow as long cells and become confluent in 3 days, higher cell proliferation than age-matched rFNS cells (Figure 6b). These when passaged produced clusters. The cells present in the clusters could not be propagated under our culture conditions.
  • FNS-NT3 balls did not grow when NT3 was removed but continued to proliferate in presence of NT3.
  • FACS analysis of FNS-NT3 cell revealed 6 times more G ⁇ / 0 and G 2 cells in culture maintain in the presence of NT3 (10ng/ml) compared to cultures lacking NT3.
  • Figure 6b shows NT3-like cells isolated from adult rat neural stem cells.
  • FNS-NT3 cells can be distinguished from FNS cells through the analysis of expression of Nestin, a neural precursor cell marker, their differentiation potential and their ability to contribute to all tissues in chimeric embryos produced following the injection of FNS-NT3 cells into rat blastocysts (see Example 9).
  • Slides were prepared by cytospin (50,000 cells per well) or by allowing cells to plate (10,000 cells per well) and fixed using 100% ethanol. 50 ⁇ l of 1% heat inactivated fetal calf serum was added to each slide and incubated 20-30 minutes at room temperature. 40 ⁇ l primary antibody was added to appropriate wells and incubated overnight at 37°C (see dilutions, below). The following day, 3x3 minute washes were performed using phosphate buffered saline. Undered by the addition of 30 ⁇ l 1% secondary antibody, which was incubated for 1 hour at 37 °C. Finally the slides weer washed 3x3 minute in phosphate buffered saline. Slides mounted using Vectrashield with DAPI. Primary Antibody dilutions
  • Figures 2 and 3 show nestin and vimentin immunofluorescence staining, respectively.
  • FNS-NT3 cells were used and prepared as in the previous examples.
  • the cell is still poorly characterized for molecular markers but its most notable feature is that unlike the neural stem cells these cells are nestin negative.
  • the cells are produced as loose clusters of spherical cells that are capable of attaching to matrix-coated plates (eg fibronectin).
  • Neurotrophin 3 (NT3) is a necessary requirement for the derivation of these cells hence the designation FNS-NT3 cells (see Figure 1 and previous Examples).
  • the cells have recently been used for the generation of chimeric rats by injection into the blastocoel cavity, analogous to the current ES cell based approach to generating chimeric mice ( Figure 7).
  • FNS-NT3 cells marked with LacZ were apparently able to undergo dedifferentiation and to contribute most cell types within the body (see Figure7). This is in marked contrast to experiments of a similar nature carried out using FNS cells [4].
  • the blastocyst-stage embryos are flushed from the uterine horns using 26 1 /2 gauge needles with 0.5 ml handling medium (mR1 ECM-Hepes)[6]into a Petri dish, recovered by mouth sucker with a fine "hand-pulled” glass pipette, washed twice in rat embryo culture medium (mR ECM) [6] and subsequently placed in rat embryo culture wells containing a preformed feeder layer and 400 ⁇ l of culture medium overlaid with paraffin oil. The embryos are then cultured for 24 to 48 hrs at 37.5 ° C under 5% C0 2 .
  • mR1 ECM-Hepes 0.5 ml handling medium
  • mR ECM rat embryo culture medium
  • a single cell suspension of FNS-NT3 cells are prepared on the injection day. A few hundred cells are transferred into the injection chamber. Following the introduction of the pre-prepared blastocysts into the drop, the blastocyst injections are performed. Firstly, the injection pipette is inserted into the blastocoel cavity followed by the gentle expulsion of approximately 10 FNS-NT3 cells. Following withdrawal of the injection pipette, the blastocyst collapses which results in the cells being brought into contact with the surface of the inner cell mass. The injected blastocysts are removed and cultured in 10 mm wells containing 400 ⁇ l of culture medium overlaid with paraffin oil at 37.5 ° C under 5%
  • the rat embryos are collected at the morula-stage of development either from in vitro development or after in utero development. These are generally equivalent to E3.0-E3.5 in the rat.
  • the zona pellucidas are removed by proteolytic enzymes (eg 0.5% pronase solution (g/ml) in M2 medium (reference)).
  • the embryos are kept in a humidified incubator with 5.0% CO 2 at 37°C until the zona are removed, which varies from 3-10 minutes.
  • FNS-NT3 cells may be aggregated with the morula stage embryos lacking the zona pellucida. The aggregated embryos are culture for a further 24-36 hours until the blastocyst stage before being transferred to recipient animals as above.
  • mice were inoculated with 5x 10 5 PC12 (rat phaeochromocytoma cells), 2 SCID mice were inoculated subcutaneously with 5x 10 5 rat FNS-NT3 cells. Animals were observed weekly. Nineteen days later, mice inoculated with PC12 cells were humanely killed; these had large lesions at all injection sites. At 20 weeks mice inoculated with rat FNS-NT3 cells show no lesion at the injection site and remain healthy.
  • Example 11 Generation of adult neural stem cells from neural tissue
  • Tissue culture plates were pre-coated with fibronectin at 1 ⁇ g/ml and poly- L - Ornithine at 15 ⁇ g/ml in DMEM for 2-24 hours at 37°C; 5% CO 2 . (Enough volume was used to cover the surface). The fibronectin/ poly- -Ornithine was aspirated and plates washed with DMEM. This preparation can be stored at room temp for several days.
  • tissue was then dissociated by blunt end of a 5ml syringe few times, then titurated by 1ml pipette tip and collected in 15ml tissue culture tube.
  • the cells were centrifuged at 400 rpm for 10 minutes and cell debri aspirated and the cell pellet resuspended in 5 ml of NSA culture medium (Euroclone, Irvine, Scotland) containing 2mM L-glutamine (GIBCO BRL), 0.6% D-Glucose, 1 :100 N2 supplement (GIBCO BRL), 1 :100 B27 supplement GIBCO BRL) and 50 units/ml penicillin and 50ug/ml streptomycin (GIBCO BRL).
  • NSA culture medium Euroclone, Irvine, Scotland
  • the cells were washed and resuspended in fresh NSA culture media containing 20ng/ml mouse EGF (GIBCO BRL), 20ng/ml human bFGF (GIBCO BRL). The total viable cell number was determined by haemocytometer and tryphan blue exclusion. Approximately 0.5-1 million cells were plated in a 6 well (9.6cm2) culture dish in 2 ml of NSA culture media in presence of 0.5% Hyclone sera (GIBCO BRL) and maintained at 37 0 C with 5% CO2. For comparison same number of cells were plated onto 6 well culture dishes pre-coated with poly-L- ornithine and fibronectin, similar cell yield was obtained with both methods of plating. Next day the medium was replaced with serum free NSA medium. Medium was changed every 3-4 days until the cultures were confluent enough to be passaged usually 6-8 days.
  • Cells were scraped and replated at 4-5 x10 4 cells/cm2. Cells could be frozen in liquid nitrogen in NSA medium with 7.5% DMSO (dimethy sulfoxide).
  • the cells were thawed quickly at 37 0 C added to NSA medium, centrifuged to remove DMSO and resuspended in fresh NSA medium and plated.
  • the embryos are washed twice (briefly) with PBS/3% DMSO and refixed over-night at 4°C.
  • the embryos are embedded in paraffin. 5 ⁇ sections were cut and mounted on glass slides.

Abstract

L'invention concerne une population sensiblement homogène de cellules précurseurs multipotentielles pouvant provenir de cellules souches neurales foetales (FNS), des compositions contenant ces cellules FNS, des cellules souches neurales adultes (NS), du tissu neural ou toute source contenant ces cellules NS. Ces cellules peuvent démontrer une croissance à long terme dans des cultures, tout en conservant leur capacité multipotentielle à se différencier en neurones et en cellules gliales. Ces cellules présentent une plasticité élevée. Elles représentent un outil de valeur pour de nombreuses thérapies cellulaires et géniques visant à traiter des maladies neurologiques. De ce fait, l'invention concerne une composition cellulaire contenant une ou plusieurs cellules possédant une propriété caractéristique d'une cellule souche précurseur ou neurale et dans laquelle ladite cellule est pratiquement nestine négative.
PCT/AU2002/001533 2001-11-09 2002-11-11 Nouvelle cellule souche presentant un potentiel de croissance a long terme, procedes de cultures et animaux obtenus WO2003040353A1 (fr)

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