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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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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/06—Anti-neoplasic drugs, anti-retroviral drugs, e.g. azacytidine, cyclophosphamide
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
  • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

• the present invention relates to a method for in vitro neurodifferentiation of pluripotent stem cells producing aggregates of neural progenitor cells in the form of neurospheres and purification thereof; a method for dissociation of neurospheres thereby producing a population of single neural progenitor cells of high purity; substantially pure neurospheres and populations of single neural progenitor cells obtained thereby; compositions thereof; the use of neurospheres and single cell populations obtained by dissociation thereof or compositions thereof in in vitro differentiation, in in vitro research, modelling in tissue culture, pharmaceutical development including drug screening toxicological testing and therapeutic cell replacement strategies including transplantation, and the like; and methods for screening or therapy using the neurospheres, dissociated cells or composition.
• the invention relates to a method for in vitro neurodifferentiation of clonal pluripotent stem cell lines, more particularly clonal lines obtained by isolation of mammalian pluripotent stem cells and cloning thereof, producing neurospheres, and purification thereof; and to a method for dissociation of such neurospheres; substantially pure neurospheres and populations of single neural progenitor cells obtained thereby; the use thereof; and methods of screening or therapy with the use thereof.
• Pluripotent stem cells retain the capacity for unlimited cell proliferation but also retain the ability to differentiate into a multitude of somatic cell types.
• Embryonic stem (ES) cells are pluripotent stem cells isolated from pre- implantation embryos whereas embryonal carcinoma (EC) are pluripotent stem cells isolated from germ cell tumours and may be considered the malignant counterparts of ES stem cells.
• Uses of such ES cells include inter alia in vitro differentiation; in vitro research, modelling in tissue culture, drug screening, cell replacement therapy and the like.
• Derivative stem cell cultures are obtained by methods including forming aggregates of neural progenitor cells in the form of neurospheres, and dissociation thereof by enzyme digestion which is a means to produce large quantities of progenitor cells.
• the present invention relates to a novel direct approach to prepare and isolate neurospheres of high purity and of desired cell type that display desired differentiation and developmental behaviour and that may be used directly or dissociated in novel manner for use for pharmaceutical development, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like.
• Murine EC cells have been widely used to study cell differentiation in vitro for the investigation of murine embryogenesis.
• Corresponding experimental investigation of cellular differentiation in human teratocarcinomas has been limited by the lack of pluripotent stem cell lines with the capacity for extensive cellular differentiation into somatic derivatives in vitro.
• Early studies on EC cells of the human malignant testicular teratocarcinoma cell line TERA-2 formed well differentiated tumours when injected into athymic mice, but showed only limited spontaneous differentiation in vitro.
• NTERA-2 (NT-
• TERA-2 teratocarcinoma line
• Neuronal character was confirmed by reaction of cells with tetanus toxin and with monoclonal antibodies specific for the neurofilament protein. However non reactivity with monoclonal antibodies specific for the glial cell intermediate filament protein, GFAP, indicated absence of glial cells.
• NT-2 cells were differentiated with RA to yield more than 95% pure cultures of neuronal cells (NT2-N cells).
• This publication discloses a method of differential attachment to tissue culture plastic, comprising seeding cells in a flask and treating with RA for four weeks, replating cells in culture and periodically striking the cells to mechanically dislodge any neuronal cells, washing the dislodged floating cells and replating again, thereby separating neuronal cells from other differentiated cell types.
• This method traditionally used to enrich for neurons in primary cultures led to an enrichment of the neuronal cells, with a final mitotic inhibition to suppress the growth of remaining contaminating non-neuronal flat cells, after which about 95% of the cells were differentiated neurons.
• NT2-N cells were then finally removed enzymatically, from non-neuronal flat cells and replated at greater than 99% NT2-N cells. Investigation of astrocytic marker expression indicated once more that cells did not react with GFAP, indicating the absence of glial cells.
• TERA2.cl.SP12 neural differentiation by pluripotent stem cells is inefficient even when using the newly derived clonal sub-line, TERA2.cl.SP12, which appears to have a greater capacity for neural development (S.A. Przyborski, Stem Cells, 2001, 19, 500-504).
• TERA2.cl.SP12 When grown as adherent monolayers and exposed to retinoic acid, only 10-15% of TERA2.cl.SP12 cells form terminally differentiated neurons whilst the remainder of the culture differentiates into non-neuronal cell types.
• techniques have been developed to isolate and purify EC-derived neurons from other contaminating cell types (see above, S. J. Pleasure, C. Page, and C. M. Y. Lee, Journal of Neuroscience, May 1992, 12(5): 1802-1815), these methods are laborious, yield relatively few neurons per culture, and can take at least 6 weeks to perform.
• a particular preparation and purification method dictates the formation and differentiation of neurons and have by this means derived novel methods for differentiation of clonal pluripotent stem cell lines producing neural cell aggregates, hereinafter neurospheres and a method for purification and optional dissociation of neurospheres and differentiation thereof.
• a method for the preparation of a neurosphere comprising culturing a mammalian pluripotent stem cell line in the presence of differentiating agent to form one or more neurospheres and isolating intact neurospheres from single cells, small cell clumps and cell debris, characterised in that cells are isolated as intact neurospheres.
• neurospheres are of purity in excess of 95%.
• Reference herein to a neurosphere is to a cellular aggregate of neural progenitors capable of terminal differentiation to form neurons and optionally additionally glial cells and optionally other neuronal subtypes.
• Reference herein to purity is to presence of desired cell type without contaminating cell type or debris.
• Reference herein to homogeneous and heterogeneous cell populations is to populations of same or different cell type or subtype as known in the art.
• Reference herein to % homology is to common genetic make up, including genotype and phenotype, as known in the art.
• the method for preparation and purification provides for increased efficiency in terms of productivity and speed of generating neurospheres having regard to prior art methods, moreover neurospheres obtained by the method of the invention are suited for use directly as highly pure neurospheres or populations thereof, and also provide for increased productivity, diversity and efficiency of differentiation to neural cells and optionally additionally glia and/or subtypes.
• Mammalian pluripotent stem cells suitable for preparing a neurosphere as hereinbefore defined are preferably selected from mammalian pluripotent embryonic stem (ES), fetal, developing or adult stem cells or embryonal carcinoma (EC) cells, more preferably human, primate, rat or murine stem cells, more preferably selected from embryonal carcinoma," blastocyst, bone marrow, blood or skin tissue stem cells and the like, most preferably selected from heterogenous cultures of cells or surgically removed tissue specimens.
• ES mammalian pluripotent embryonic stem
• EC embryonal carcinoma
• the method comprises differentiation of clonal stem cell lines which stain positive for markers of pluripotent stem cell lines, more preferably cell lines obtained by cloning single cells, thereby possibly enhancing the purity of the neurosphere population.
• a clonal stem cell line is obtained by the method as described in Przyborski SA, Isolation of Human Embryonal Carcinoma Stem Cells by Immunomagnetic Sorting, Stem Cells 2001; 19:500-504, the contents of which are incorporated herein by reference, comprising isolating a population of marker-positive (SSEA-3 + , -4 + , ⁇ 1 + or TRA-l-60 + ) pluripotent stem cells from the parent lineage prior to deriving clonal lines.
• SSEA-3 + , -4 + , ⁇ 1 + or TRA-l-60 + markers of pluripotent stem cells from the parent lineage prior to deriving clonal lines.
• Clonal cell lines cloned from single cells are suitably obtained as described in Przyborski SA above, by incubating mammalian pluripotent stem cells with magnetically labelled antibody that is stage-specific for embryonic antigens including SSEA-3, SSEA-4 and SSEA- 1, TRA-1-60 and the like, and isolating cells immunoreactive for the antibody using direct positive magnetic isolation and retrieval, and subsequently culturing one or more single separated, positively recognised cells and producing one or more clonal lines.
• Direct positive magnetic isolation and retrieval is known in the art for isolation of cells from blood, and kits are available commercially (BioMag, Polysciences Europe GMBH) comprising antibodies labelled with 1 micron magnetic particles.
• the method may be applied to any desired cell type as defined above and is preferably a method for culturing mammalian clonal pluripotent stem cells optionally in the presence of one or more differentiating agent(s), to form neural progenitors in the form of neurospheres.
• Small or large neurospheres may be in the size range 75-500 micron, more preferably 100 - 600 micron, more preferably 100-400 micron.
• Neurospheres may be of any substantially 3-dimensional aggregate shape and are preferably substantially spherical or dome shaped.
• the method of the invention comprises culturing a cell line as hereinbefore defined to neurodifferentiate to form one or more cell types including neurospheres of substantially pure homogeneous populations of neurons or heterogeneous populations of neurons and glial cells, together with cell debris as hereinbefore defined and purification to isolate intact neurospheres, preferably of purity in excess of 90%, more preferably in the range 95-100%, most preferably in the range 99-100%. Purity may be determined by known means including microscopical examination and staining for cell type specific markers.
• the method comprises culturing a cell line in suspension in a suitable vessel to which cells do not adhere, and forming neurospheres in situ.
• a suitable vessel may be such as a bacteriological dish.
• a vessel may be coated or base-coated with a coating to discourage cell adhesion, such as agarose.
• This method facilitates aggregation of neural progenitor cells in suspension, in contrast to some prior art methods which provide monolayer differentiation initially or throughout.
• Neurospheres formed by the method may then be purified and used directly or differentiated to form a substantially pure population of neurons, which is preferably substantially homogeneous. Sampling of individual neurospheres allows determination of mono neurospheres and fused neurospheres (two or more neurospheres which have grown together) whereby neurospheres may be isolated which are of substantially 100% homology or are of mixed homology.
• the method comprises culturing a cell line in suspension in a suitable vessel to which cells adhere to produce monolayers of differentiated cells and optionally contacting with a mitotic inhibitor (MI) and culturing for a further period in absence of differentiation agent to form neurospheres.
• a suitable vessel to encourage adhesion may be a tissue culture flask. Increasing MI concentration suppresses formation of glial cells, and promotes formation of neurons, and vice versa.
• differentiated neurospheres are obtained which comprise neurons together with an amount of glial cells, such as astrocytes. Glial cells, once present, may be cultured to be present in any desired amount and may be encouraged by contacting with growth factors and the like.
• This method employs the known method of Lee et al above initially but allows cells to proliferate in monolayer rather than striking them to remove from vessel walls. We have found that this encourages neurosphere formation with a diverse content of neural progenitor cells including glia. Neurospheres formed by the method may then be purified and used directly or differentiated to form a substantially pure population of neurons and glia which is preferably substantially heterogeneous. Sampling of individual neurospheres allows determination of mono neurospheres and fused neurospheres (two or more neurospheres which have grown together) whereby neurospheres may be isolated which are of substantially 100% homologous genotype and mixed phenotype or are of non-homologous genotype and phenotype.
• Culturing is under suitable conditions, for example close to physiological conditions at pH 6 to 8, preferably pH 7 to 7.8 more preferably pH 7.4 and temperature in the range 30 - 40C, preferably 32 to 38C, more preferably 35 to 37C most preferably 37°C.
• Culture medium may be any known culture medium capable of supporting cell growth, including HEM, DMEM (Dulbecco's modified Eagles's medium), RPMI, F-12 and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamycin, and the like. In some cases the medium may contain serum from bovine, equine, chicken and the like. A defined culture medium is preferred if cells are to be used for transplantation purposes. A particularly preferred culture medium is a defined culture medium comprising DMEM or DMEMFG (DMEM supplemented with 10% fetal calf serum (FCS) and 2mM L-glutamine).
• HEM HEM
• DMEM Dulbecco's modified Eagles's medium
• RPMI fetal calf serum
• F-12 fetal calf serum
• Preparation of cellular aggregates suitably comprises culturing the desired cell line for a sufficient period, preferably 2 - 6 weeks, for example 2-3 or 3-4 weeks, corresponding to a desired degree of aggregation determining the size range of neurospheres for isolation. Culturing EC cells, which are tumouriogenic ensures the continued production of neurospheres in contrast to fetal cells for which the development period cannot be as readily controlled.
• cells are cultured for a sufficient period to produce neurospheres which are visible to the naked human eye, more preferably which are readily separated by size from single cells and cell debris.
• pluripotent stem cells are cultured for a period of 2-6 weeks generating neurospheres in a single or multiple seedings.
• cells are cultured in the presence of differentiation agent for a period of 2-3 weeks, with subsequent purification of neurospheres as hereinbefore defined.
• embodiment B as hereinbefore defined cells are cultured in the presence of differentiation agent for a period of 3-4 weeks with subsequent seeding into fresh culture, contact with mitotic inhibitors and subsequent purification of neurospheres as hereinbefore defined.
• treatment with mitotic inhibitors promotes the production of glial cells such as astrocytes which are supportive of neurons and may facilitate neurosphere growth.
• glial cells such as astrocytes which are supportive of neurons and may facilitate neurosphere growth.
• Combined neuron-glial neurospheres have additional advantages in subsequent investigations and uses for example facilitating adhering cells to surfaces for staining and the like.
• Differentiation agents which may be used in the method of the invention are selected from natural and synthetic retinoic acid, retinoids and derivatives thereof, preferably all-trans retinoic acid (RA), bone morphogenic proteins such as BMP-2, growth factors such as FGF (f ⁇ broblast growth factor), TGFbeta, NGF, PDGF and the like, trophic factors such as CNTF, TNFalpha (tumor necrosis factor alpha), macrophage inflammatory proteins such as MIP-1 alpha, MlP-lbeta, MIP-2 and the like, noggin, heparan sulfate, amphiregulin, interleukins and the like. It is within the scope of the present invention that other classes of differentiation agent may be found to be effective in differentiation of the composition of pluripotent stem cells of the invention.
• RA all-trans retinoic acid
• BMP-2 all-trans retinoic acid
• growth factors such as FGF (f ⁇ broblast growth factor), TGFbet
• Preferably differentiation agent such as RA for example, is introduced in an amount of 10 "4 - 10 "9 M, more preferably 10 "5 - 10 "7 M, for example in a final concentration in culture of 0.5-50 microM.
• Mitotic inhibitors which may be used in the method of the invention include any known inhibitors of cell mitosis, preferably selected from nucleotides such as cytosine arabinosine, fluorodeoxyuridine, uridine and the like. Mitotic inhibitors may be used in any effective inhibiting amount for example in the range lxlO 5 - lxl0 7 M, preferably 0.5-50 microM.
• the concentration of mitotic inhibitor and stage and duration of inhibition is thought to affect the concentration and ratio of neuron and glial cells in the product neurosphere.
• Morphology of cells may be determined by immunocytochemical analysis as known in the art with suitable pre-analysis preparation comprising culturing in the presence of mitotic inhibitors as hereinbefore defined.
• Neurosphere purification may be by any suitable isolation technique and is preferably by size selection as hereinbefore defined.
• isolating neurospheres is by filtration, with selection of filter aperture for a particular maximum neurosphere size which it is desired to purify. Successive filtration with two filters of different aperture size enables purification of neurospheres of particular size which is greater than the smaller filter aperture and smaller than the larger filter aperture.
• size selection is by passing the cell suspension through a sieve having a suitable gauge to retain and thereby separate neurospheres from single cells, small cell aggregates and cell debris as hereinbefore defined.
• a sieve comprises a sterile nylon mesh with desired gauge of 75 to
• Narrow gauge sieves are known in the art for isolating individual cells and modified sieves may therefore be prepared or commercially available which are suitable for separating neurospheres in comparable manner.
• isolated neurospheres are washed in the sieve in medium (for example DMEM or DMEMFG culture as hereinbefore defined completing purification thereof.
• Purified neurospheres may be removed from the mesh by back washing with culture medium such as DMEMFG into a suitable sterile container.
• Purified neurospheres may be transferred to a surface for further use or investigation, for example tissue culture flask, a sterile bacteriological dish, or may be transferred to a sealed container, for example a sterile vial, and stored for example by freezing, preferably in freezing media such as fetal bovine serum (FBS) and cryogenic agent such as DMSO, for subsequent use.
• a surface for further use or investigation for example tissue culture flask, a sterile bacteriological dish
• a sealed container for example a sterile vial
• freezing media such as fetal bovine serum (FBS) and cryogenic agent such as DMSO
• the method comprises additionally the dissociation of a neurosphere obtained with the preparation and purification method as hereinbefore defined, comprising chemical and physical disruption of the neurosphere structure to produce a suspension or monolayer of single neural progenitor cells preferably in the form of monolayers of adherent homogeneous or heterogeneous neural cells, more preferably comprising neurons and optionally additionally glia.
• Chemical and physical disruption may be by methods as known in the art but preferably and in a still further aspect of the invention the method comprises chemical disruption by means of incubating neurospheres in alkaline media, more preferably in the range pH 9-
• the method comprises subsequently additionally neutralising the alkaline media with additional media, preferably to pH in the range 5-8, more preferably pH
• alkaline media is any media in which cells remain viable, more preferably is cell culture such as DMEMFG and the like, as herenbefore defined, made alkaline by adjusting pH with alkali such as NaOH, preferably 3N NaOH.
• dissociated suspended cells from one or more neurospheres are viable and may be cultured on appropriate surfaces.
• These cells may be used to provide a population of neural progenitor cells of high purity, preferably in excess of 90%, more preferably in excess of 95%, as a homogeneous or heterogeneous suspension of single cells which are of same or mixed homology as hereinbefore defined for further use.
• viability is far in excess of that obtained using dissociation techniques as known in the art such as mechanical and enzymatic disruption.
• a method for differentiation of one or more neurospheres or of single dissociated neural progenitor cells as hereinbefore defined comprising seeding neurospheres or dissociated cells on coated surfaces and culturing in the absence or presence of differentiation agent and/or mitotic inhibitors to induce differentiation thereof to pure homogeneous or heterogeneous populations of differentiated derivatives of same or mixed homology.
• the method comprises seeding neurospheres or dissociated cells on surfaces coated with polymeric growth promoters such as poly-D-lysine and laminin, and culturing in the presence or absence of differentiation agent(s) and in the presence or absence of mitotic inhibitor(s).
• polymeric growth promoters such as poly-D-lysine and laminin
• compositions comprising one or more neurospheres, dissociated cells and / or their derivatives obtained with the purification or dissociation method of the invention as hereinbefore defined characterised in that the composition comprises substantially pure homogeneous or heterogeneous neurospheres, dissociated cells and/or their derivatives of purity in excess of 90% and of same or mixed homology.
• the composition or population comprises neurospheres and/or dissociated cells of purity in excess of 98%, more preferably in excess of 99%, most preferably is substantially pure.
• the composition is for use in research, drug screening or therapy.
• the undifferentiated composition of the invention comprises cells which maintain an undifferentiated state when cultured in the absence of a differentiating signal.
• the composition comprises cells which are capable of proliferation in vivo and differentiating to form neural progenitor cells, and differentiating into other lineages such as neurons and glia.
• the composition consistently displays excellent levels of neuronal cell markers of same or different type eg neurons and optionally additionally glia, in undifferentiated state indicative of the homogeneity or heterogeneity thereof and are capable of differentiation in response to differentiation agents for example retinoic acid (RA).
• RA retinoic acid
• the undifferentiated composition comprises neural progenitor cells.
• the differentiated composition comprises 20 to 50% more cells which show the appearance of morphologically identifiable neural cells or produce proteins indicative of neural cells, in particular neurons and glia and optionally differentiated subtypes, than would be obtained from the starting cell population.
• cells of the composition express or show an increase in expression of certain antigens indicative of cell differentiation.
• A2B5 or VIN-IS-56 is expressed in response to differentiation agents such as RA indicating the commitment of cells to form neural derivatives, and moreover ultimately show the appearance of morphologically identifiable neural cells.
• the composition comprises cell which are capable of responding to external agents such as drugs and pharmaceuticals for screening.
• the composition is capable or establishing a graft in a recipient host brain.
• the composition is capable of migrating along host brain pathways and is capable of widespread distribution in host brain.
• the composition of the invention comprises cells which are responsive to host environmental signals.
• composition neurosphere, dissociated cells and/or their derivatives as hereinbefore defined in research, including in vitro differentiation, in vitro neural research, modelling in tissue culture, pharmaceutical development including drug screening, toxicological testing or in vitro or in vivo therapeutic cell replacement strategies including but not limited to transplantation.
• pharmaceutical development including drug screening, toxicological testing or in vitro or in vivo therapeutic cell replacement strategies including but not limited to transplantation.
• the composition displays the ability to show increased variation in differentiation thereby enabling the development of a diverse heterogeneous differentiated cell population or to show a uniform differentiation thereby developing a homogeneous or limited diversity population, as desired and in response to appropriate stimuli.
• a heterogeneous population may show a more complete response or increased viability and a homogeneous or limited diversity population show a uniform response or specific viability when used for pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like. This provides a unique means to ensuring a higher success rate and a means for more specific cell selection in screening or therapy due to the greater reliability of the response detected, the greater expectation of viability or the more specific indication of viability.
• a method for screening compounds which affect proliferation, differentiation or survival of neurospheres or their dissociated cells comprising preparing a composition of neurospheres, dissociated cells and or their derivatives as hereinbefore defined, contacting the composition with at least one compound and determining if the compound has an effect on proliferation, differentiation or survival of the neurospheres, dissociated cells and/or their derivatives.
• the method may comprise determining the effect of the compound(s) on differentiation of cells comprised within the composition or may comprise inducing differentiation of cells within the composition prior to contacting with the compound(s), and monitoring the effect on proliferation or viability of cells.
• the method comprises selecting a composition of neurospheres or dissociated cells as hereinbefore defined comprising homogeneous or heterogeneous neuronal progenitor cells, contacting with compound(s) and subsequently monitoring the effects of the compound(s) on the ability of the progenitor cells to differentiate into neuronal cells, preferably by culturing in suspension or monolayer in the presence of differentiating agent in known manner.
• Compounds for screening may be selected from any drug which it is desired to test for medicinal or other therapeutic use, environmental or other agricultural use or the like, suitably selected from growth factors, trophic factors, regulatory factors, hormones, viruses, proteins, peptides, amino acids, lipids, carbohydrates, nucleic acids, nucleotides, drugs, pro-drugs, and other substances intended to have a harmful or beneficial effect on diseased or healthy cells respectively and the like.
• compounds for screening are selected from compounds which are intended to have a harmful or beneficial effect on neural cells and include neurological inhibitors or agents such as neuroblockers or neurotransmitters and receptors therefor, growth inhibitors of growth factors and receptors therefore and enzymes used in their synthesis, more preferably growth factors and neurotrophic factors and the like; more preferably selected from neurotrophic factor such as glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor; neurotrophin such as neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and the like.
• neurological inhibitors or agents such as neuroblockers or neurotransmitters and receptors therefor, growth inhibitors of growth factors and receptors therefore and enzymes used in their synthesis, more preferably growth factors and neurotrophic factors and the like; more preferably selected from neurotrophic factor such as glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor; neurotrophin such as neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and the like.
• GDNF
• the effect on proliferation of the neurospheres and/or dissociated cells comprised within the composition suitably comprises generating a screening composition and a control composition from each composition, performing the screening method of the invention on the screening composition and culturing in parallel with the control composition and observing changes in the number of neurospheres and/or dissociated cells in the screening composition, compared to the control composition.
• Optionally compounds are additionally screened in parallel against compositions of diseased cells to determine differences in effect from healthy neurospheres and/or dissociated cells.
• the compound is provided in solution and compositions are contacted with solution in given concentration(s) and volume(s) in a single dose or at intervals providing a sustained concentration or variations in concentration with time, as compound is consumed.
• Concentration is suitably in the range of 1 femtogran to 1 milligram, in most cases this may be in the range 1 picagram to 100 nanogram, but depends on the active concentration of individual compounds being screened.
• Compositions are suitably transferred to sterilised wellplates or the like for contacting and contacting is in volumes of 1 to 100 microlitres per wellplate.
• Changes in proliferation or viability are suitably monitored in known manner, including monitoring rate of cell proliferation, or of cell progeny proliferation, monitoring nature of cell differentiation and ratio of differentiated cell types, for example ratio of neurons to glia or the like, monitoring cell death and the like, monitoring changes in cell development or morphology, monitoring changes in expressed phenotypes, amount or type of proteins expressed, and the like, monitoring changes in neuronal characteristics including electrophysiological properties such as resting membrane potential, evoked potential, direction and ionic nature of current flow, and dynamics of ion channels.
• Monitoring may be by techniques such as immunohistochemistry; or biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis, HPLC analysis, western blots, radioimmune assays, nucleic acid analysis such as northern blots and PCR and the like; or extracellular or intracellular voltage recording, voltage clamping and patch clamping, or using voltage sensitive dyes or ion sensitive electrodes.
• biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis, HPLC analysis, western blots, radioimmune assays, nucleic acid analysis such as northern blots and PCR and the like; or extracellular or intracellular voltage recording, voltage clamping and patch clamping, or using voltage sensitive dyes or ion sensitive electrodes.
• biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis
• the method is for monitoring changes in neural cell development from neural progenitor cells and comprises differentiating cells comprised within a composition of the invention to obtain a composition of neural progenitor cells of purity in excess of 90%, conducting the method for sceening as hereinbefore defined, culturing in the presence of differentiation agent and monitoring changes in neural development such as for example, to assess the effect of compound(s) on neural process outgrowth (formation of neurites, known in the art as neuritogenesis or neurite outgrowth) and the like.
• measuring neuritogenesis is by measuring levels of expression of proteins which are typically upregulated during normal outgrowth of nerve processes, in particular measuring expression of MAP2 protein.
• a method for therapy comprising introducing a composition or neurospheres, dissociated cells, and/or their derivatives as hereinbefore defined into a mammalian host, preferably a human, primate, rat or murine host.
• transplantation of tissue into the CNS is a potential route to treatment of neurodegenerative disorders and CNS damage due to injury.
• Transplantation of new cells into the damaged CNS has the potential to repair damaged circuitries and provide neurotransmitters thereby restoring neurological function.
• the absence of suitable cells prevents the full potential of this procedure being met.
• selecting stem cells for transplantation offers the highest success rate, allowing cells to be obtained in large numbers, capable of surviving indefinitely but stop growing after transplantation to the brain for example, cells can be obtained from patients normal tissue lessening the chances of rejection, and are capable of differentiating to form normal neural connections and respond to neurological signals.
• Stem cell therapy is well recognised and has yet to reach its full potential in terms of successful transplantation for which an improved source of cells having the required diversity or homogeneity and viability are required.
• the method of the invention provides a pure source of stem cells which is ideally suited for therapy, for transplantation by introduction into a host as hereinbefore defined.
• Suitably introduction is by grafting or injection of neurospheres or dissociated cells and/or their derivatives at the site of damage or therapy for example site of injury or disease or remote therefrom.
• Injection may be into the nervous system of a host using any known technique such as using a microinjector or syringe, and facilitates site specific introduction.
• Grafting is preferably by a stable graft established in the CNS or PNS or other organ such as the hematopoietic or hemal system, from where engrafted neurospheres or cells may release desired substances or may migrate and incorporate into the host. Migration via the hemal system may allow site specific delivery of released substances or cells to a site of damage or therapy.
• Transplantation may be to repair injury or to treat disease. Areas of disease can in some cases be visualised and transplantation directed to appropriate sites.
• Introduction of neurospheres or dissociated cells releasing substances may also be used to administer growth factors and other substances such as pharmaceuticals that will induce proliferation and differentiation of the grafted cells, site specifically to a graft established as hereinbefore defined.
• Introduction of cells may be with immunosuppression of a host as known in the art. It is an advantage of the invention that the neurospheres or dissociated cells of the invention may be well suited to acceptance by a host reducing or eliminating the requirement for immunosuppression or may allow the use of alternative techniques such as gene replacement or knockout, for the ablation of major histocompatibility MHC) genes.
• the neurospheres or dissociated cells of the invention may provide enhanced cell viability which will allow successful delivery of released substances or of cells to a target site and successful formation of a graft.
• Neural stem cell progeny introduced in known manner preferably form a neural graft in the particular neural region to which they are delivered, wherein neurons form normal neuronal or synaptic connections with neighbouring neurons and maintain contact with transplanted or existing glia, thereby reestablishing connections which have been damaged due to injury, disease or aging.
• a graft can be monitored in known manner using non-invasive scan such as computerised axial tomography (CAT) or the like, nuclear magnetic resonance (nmr) or magnetic resonance imaging (MRI).
• CAT computerised axial tomography
• nmr nuclear magnetic resonance
• MRI magnetic resonance imaging
• Functional integration of a graft into a hosts neural tissue can be assessed by examining restoration of various functions including tests for endocrine, motor, cognitive and sensory functions.
• Figure 1 shows the isolation and cloning of human pluripotent stem cells from the teratoma line, TERA2.
• Phase image and immuno-fluorescence localisation of SSEA-4 in cultures of TERA2 (pi 5) cells grown at low seeding density are shown in images (A) & (B) respectively.
• a tight colony of EC cells is indicated by ec.
• a small colony of TERA2.cl.SP-12 EC cells (p2) co-cultured with fibroblast feeders (fibro) is shown in image (C).
• the corresponding image (D) shows specific SSEA-3 immunoreactivity to TERA2.cl.SP-12 EC cells. Expanding colonies of TERA2.cl.SP-12 EC cells (p3) shown in image
• Figure 4 shows the results of western analysis for differential expression of neural proteins during differentiation of by the clonal pluripotent stem cell line and its differentiated derivatives. Lanes: (I) TERA2.cl.SP-12 EC cells; (2)
• Figure A/B 1 shows a culture of a human pluripotent stem cell line used in the differentiation-method of the invention, derived using the isolation method of Figure 1.
• Procedure A cells are grown in suspension for 14d in the presence of RA. Neurospheres are purified by filtration and plated onto glass coverslips for staining.
• Procedure B cells are grown as monolayers for 21d in the presence of RA. Cultures are split 1:3 and grown in the presence of mitotic inhibitor (MI) for a further 2 Id.
• MI mitotic inhibitor
• Figures A2 and B2 show differentiated cells, either by suspension method (Embodiment A) or monolayer method (Embodiment B)
• Figures A 3-5 show differentiating neurospheres obtained by suspension method (A) showing neurons (indicated by B tubulin III) marker of neurons) and the absence of glial derivatives (GFAP negative staining, marker of glia).
• Figures B 3-4 show differentiating neurospheres obtained by monolayer induction method (B) showing neurons (indicated by B tubulin III staining) and glial derivatives (GFAP staining).
• Figure 5 shows cultures of human cells derived from purified neurospheres of the invention that have been dissociated according to the invention.
• Neurospheres are dissociated using chemical and physical methods that maintain high cell viability resulting in a suspension of single cells. Seeding of cell suspensions onto coated plastic or glass surfaces produces adherent monolayer cultures of neural cell types as shown.
• Figure 6 shows the effect of Brain Derived Neurotrophic Factor (BDNF) on formation of neural processes by neurons derived from pluripotent stem cells.
• BDNF Brain Derived Neurotrophic Factor
• SSEA-3, SSEA-4, A2B5, VLN-IS-56 and TRA-1-60 were generously provided by P. Andrews, University of Sheffield, UK. These antibodies recognise specific cell surface antigens and show highly regulated expression profiles related to the differentiation of human pluripotent ES and EC cells.
• SSEA-3 which was originally raised against four-cell-stage mouse embryos, is expressed highly in human pluripotent ES and EC cells and not their differentiated derivatives.
• Antibodies were pretitered and diluted (1:2 to 1:5) in wash buffer (WB) to give maximal binding .
• BioMag ® magnetic particles are approximately l ⁇ m and because of their non-uniform shape provide an increased surface area (>100m 2 /g) of 20-30 times greater than that of uniform spherical particles allowing for a higher binding capacity while utilizing a lower amount of particle.
• the magnetic particles detach from the cell membrane automatically as the cell surface is turned over during subsequent culturing for up to 48 hours. Isolated cells were immediately re-suspended and washed x3 in WB, magnetically separated a second time and finally re-suspended in 10ml WB.
• Single cells were picked at random with a micropipette under a dissecting microscope and transferred to a drop of DMEM where the presence of a single cell was confirmed.
• a single cell was added to each well of a tissue culture plate (Nunc) containing irradiated ( ⁇ 10,000 rads) STO-transformed mouse feeder cells (12 wells were seeded in total). Feeder cells were maintained for the first three passages until the newly derived clones formed large enough colonies to establish clonal sublines and grow independently of feeder cells, as homogeneous monolayers at high confluency.
• Figure 1 shows the isolation and cloning of pluripotent EC stem cells from the human teratoma line TERA2.
• RNA was isolated from human pluripotent cells and retinoic acid- induced derivatives, and prepared for northern blotting as previously described [5].
• the blot was hybridised with a 2127bp BamHl-Xbal fragment of POU5F1 generously provided by F. Gandolfi, Institute of Anatomy, Milan, Italy and the hybrid signal detected by autoradiography.
• the octamer-binding transcription factor-4 (Oct-4) is encoded by the gene POU5F1 and is expressed in human pluripotent ES and EC stem cells.
• TERA2.cl.SP-12 EC cells showed decreased expression of POU5F1 in response to retinoic acid, indicating that the vast majority of pluripotent stem cells had committed to differentiate after 7 days exposure.
• Protein samples were prepared from human pluripotent stem cells and their differentiated derivatives. Samples were separated on SDS-polyacrylamide gels and immuno-blotted. Antibodies for neuron-specific enolase (NSE;
• TERA2.cl.SP-12 stem cells have previously been associated with neuro-ectodermal derivatives, and thus may indicate the ability of TERA2.cl.SP-12 stem cells to form neural derivatives in response to retinoic acid. Whilst TERA2.cl.SP-12 stem cells showed no expression of neural proteins, markers indicative of both neurons and glia were detected after 28 days exposure to retinoic acid ( Figure 4). In an identical experiment, NTERA2.cl.Dl EC cells reacted in response to retinoic acid to produce neurons but no glial markers were detected (data not shown).
• NTERA2.cl.Dl EC cells were originally cloned from a xenograft tumor of the TERA2 parent line whereas TERA2.cl.SP-12 cells have been isolated directly from the earliest available passage of TERA2. It is well recognised that NTERA2.cl.Dl EC cells produce neurons in vitro [Przyborski S A, Eur J Neurosci 2000] but there is no evidence that glial cells form in response to retinoic acid under the conditions described in this prior art study or by the same prior art conditions used by others. In contrast, differentiating pluripotent TERA2.cl.SP-12 cells produced proteins indicative of both neurons and glia.
• Example 1 Method for preparation of neurospheres and purification thereof Human pluripotent stem cells were maintained in the appropriate culture media such as Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2mM L-glutamine (DMEMFG) at 37°C in 5% C0 2 .
• DMEM Dulbecco's modified Eagle's medium
• FCS fetal calf serum
• DMEMFG 2mM L-glutamine
• Embodiment A is based on the differentiation of cells that are maintained in suspension, whilst in Embodiment B the cells differentiate as confluent monolayers adhered to tissue culture plastic. Both methods result in the formation of neurospheres and both procedures involve the purification of neurospheres by filtration and optional neurosphere dissociation.
• Embodiment A Suspension Preparation of Neurospheres:
• Retinoic acid induced suspension cultures were maintained for a further 2-3 weeks during which the RA-induction medium and bacteriological plate was replaced every 2-3 days. Coating the bottom of the bacteriological plate with 2% agarose further reduced the adherence of suspended cells to the surface of the culture vessel.
• neurospheres >100 ⁇ m isolated by the mesh were gently washed whilst on the mesh for a further x3 times with DMEMFG.
• DMEMFG fetal calf serum
• the mesh was backwashed with DMEMFG into a sterile bacteriological dish.
• neurospheres may be used for a variety of purposes, including growth in culture and research and development, drug screening and therapeutic applications in cell replacement and transplantation strategies.
• Embodiment B Monolayer Induction of Neurospheres 1.0 Suspended pluripotent TERA2.cl.SP12 stem cells were seeded at 2xl0 4 cells per cm 2 in tissue culture flask (equivalent of 1.5xl0 6 cells per T75 tissue culture flask) in DMEMFG containing lO ⁇ M RA. (Fig A/B 1) 1.1 Induction media was changed every 3-4 days.
• the medium from the cultures was removed and the cells were lifted into a cell suspension using trypsin (0.25% trypsin / 2mM EDTA in PBS for 10 min RT).
• the suspension consisted of single cells, flat sheets of cells (easily broken apart), and intact neurospheres previously described as dome-like structures (see 1.5 above).
• the cell suspension was passed over a sterile nylon mesh with a pore size of lOO ⁇ m to remove single cells, small cell clumps and cell debris.
• neurospheres may be used for a variety of purposes, including growth in culture and research and development, drug screening and therapeutic applications in cell replacement and transplantation strategies.
• Example 2 Differentiation of Neurospheres: 3.0 Individual or groups of neurospheres obtained from the method of Embodiment A or B above were collected by pipette and seeded onto glass coverslips pre-coated with either poly-D-lysine (lO ⁇ g/ml) and human placental laminin (lO ⁇ g/ml) or both ( Figures A2, B2). Collection can be either using the naked eye or using a dissecting microscope at low power. 3.1 Cells grown on coverslips were maintained in DMEMFG in the absence of RA but in the presence of mitotic inhibitors: l ⁇ M cytosine arabinosine for the first 7 days only; lO ⁇ M fluorodeoxyuridine; lO ⁇ M uridine. 3.2 Cells were prepared for immunocytochemical analysis using standard procedures. (Fig A 3-5, Fig B3,4) Example 3 - Dissociation of Neurospheres:
• TERA2.cl.SP12 stem cells grow as confluent monolayers. Induction of differentiation by exposure to RA and appropriate culture manipulation (Example 1) results in the production of pure suspensions of cell aggregations consisting of differentiated derivatives. Such aggregations develop neurites that stain for neural markers (e.g. B tubulin III) and form complex neural networks upon differentiation (see Figures) and hence these aggregations have been termed 'neurospheres'. Accordingly, these methods describe the production of neurospheres from pluripotent stem cells. The method of Embodiment A may be applied to produce neurospheres in less than 3 weeks, whilst the method of Embodiment B takes approximately twice as long. Initial investigations suggest that the neurospheres produced by these methods are not the same.
• Embodiment B showed high levels of both B tubulin III and GFAP, indicating the formation of both neurons and astrocytes.
• Neurospheres produced by either Embodiment were successfully dissociated using chemical and physical techniques that maintained cell viability and resulted in dispersed adherent monolayer cultures of neural cells.
• This invention describes methods by which human neurospheres and dispersed cultures of neural cells may be produced from existing and newly derived lines of pluripotent stem cells.
• the procedures involve established cell culture techniques and novel methods for the manipulation of differentiated derivatives. Collectively, these methods allow for the consistent and reproducible production of neurospheres and dispersed cell cultures from a pluripotent cell line and hence avoid the need to use fetal tissues that often show inconsistencies and variability. Moreover, this strategy avoids the ethical and moral issues associated with the use of fetal materials.
• the formation of human neural tissues from pluripotent stem cells allows for experimentation and research on human neural development, drug screening, toxicological testing and the potential for novel therapeutic applications.
• EXAMPLE 4 Method for screening activity of test compounds on Compositions derived using the process of the invention.
• GDNF GDNF
• BDNF BDNF
• NT-3 or NT- 4 neurospheres derived from human pluripotent stem cells grown in 6 well plates (5-10 ⁇ l per well). On days 4 and 7, the culture medium was removed and replaced with fresh medium and the test compound. On the
• DPBS Dulbecco's phosphate-buffered saline
• proteins harvested for western analysis. Protein samples were separated on SDS-polyacrylamide gels and immuno-blotted.
• Antibodies to microtubule-associated protein 2 (MAP2; Sigma- Aldrich, clone HM-2; 1:1000) and ⁇ -actin (Sigma- Aldrich, clone AC- 15; 1 : 5,000) were localized with IgG-horse radish peroxidase (HRP) secondary antibody (Amersham, 1:1,000) in preparation for chemiluminescent detection (Amersham). Densitometry was used to quantify the changes in banding pattern seen on photographic film exposed to the chemiluminescent signals.
• HRP IgG-horse radish peroxidase

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