WO2004013315A1

WO2004013315A1 - Method for the preparation, purification and differentiation of neurospheres from mammalian stem cells

Info

Publication number: WO2004013315A1
Application number: PCT/GB2003/003395
Authority: WO
Inventors: Stefan Alexander Przyborski
Original assignee: University Of Durham
Priority date: 2002-08-02
Filing date: 2003-08-04
Publication date: 2004-02-12
Also published as: EP1565549A1; AU2003255750A1; GB0217981D0

Abstract

Method for the preparation of a neurosphere in the form of a cellular aggregate of neural progenitors capable of terminal differentiation to form neurons and optionally additionally glial cells comprising culturing a mammalian pluripotent stem cell line in the presence of differentiating agent to form one or more cell neurospheres and isolating intact neurospheres from single cells, small cell clumps and cell debris, characterised in that cells are isolated as intact neurospheres of purity in excess of 99%; 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 a method for screening using the neurospheres, dissociated cells or composition.

Description

METHOD FOR PREPARATION, PURIFICATION AND

DIFFERENTIATION OF NEUROSPHERES FROM MAMMALIAN

STEM CELLS

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. More particularly 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. However the majority of existing stem cell cultures suffer from the drawback that they are impure, are of relatively low propensity to differentiate, or display undesired differentiation and developmental behaviour and this limits their usefulness in the more precise techniques such as drug screening and cell replacement therapy, in which they deliver non-uniform or unpredictable results. 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.

Several decades of accumulated evidence shows that ES cells and EC stem cells are closely related (Andrews PW, Przyborski SA & Thomson JA. (2001), 'Embryonal Carcinoma Cells as Embryonic Stem Cells' In: Marshak DR, Gardner, Gottlieb D, eds. Stem Cell Biology, New York: Cold Spring Harbor Press, Monograph 40.2001:231-266).

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-

2) cells, which were cloned from the teratocarcinoma line TERA-2 present a caricature of uncommitted stem cells from the early human embryo: they express a typical human EC cell phenotype, which is distinct from that of murine EC cells, and they closely resemble human embryonic stem (ES) cells

(Andrews PW, Przyborski SA & Thomson JA. (2001), 'Embryonal

Carcinoma Cells as Embryonic Stem Cells' In: Marshak DR, Gardner,

Gottlieb D, eds. Stem Cell Biology, New York: Cold Spring Harbor Press, Monograph 40.2001:231-266). When cultured in the presence of al\-trans- retinoic acid (RA), they differentiate into a variety of cell types that include an amount of well-developed, post-mitotic CNS neurons. P.W. Andrews "Developmental Biology", 103, 285-293 (1984) discloses a method by which cells were seeded and cultured in the presence of RA for up to four to six weeks in different concentrations of RA, forming monolayers of differentiated cells including neuron clusters connected by extended networks of axon bundles. 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.

In a subsequent publication, S. J. Pleasure, C. Page, and C. M. Y. Lee, Journal of Neuroscience, May 1992, 12(5): 1802-1815, 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.

However, 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). 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. Although 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.

It is therefore an object of the invention to provide new methods for the isolation and differentiation of pluripotent stem cell lines to produce populations of differentiated cell aggregates preferably of neurons and optionally additionally glial cells, and purification of such aggregates for use in the study of particular pathways of mammalian, in particular human or murine, development; in vitro research, modelling in tissue culture, pharmaceutical development, drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like. We have now found that 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.

In the broadest aspect of the invention there is provided 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. Preferably neurospheres are of purity in excess of 95%. In a further aspect of the invention there is provided a method for dissociating one or more neurospheres to produce a population of single neural progenitor cells, preferably in the form of monolayers of adherent neural cells, more preferably comprising neurons and optionally additionally glia.

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.

In a particular advantage of the invention 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.

In a particular advantage therefore 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. Preferably 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. 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.

Preferably 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.

In an embodiment A, suspension, 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.

In embodiment B, monolayer induction, 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. We have surprisingly found that by this monolayer method, 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).

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.

Preferably 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. Preferably pluripotent stem cells are cultured for a period of 2-6 weeks generating neurospheres in a single or multiple seedings. In embodiment A as hereinbefore defined cells are cultured in the presence of differentiation agent for a period of 2-3 weeks, with subsequent purification of neurospheres as hereinbefore defined. In 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.

In a particular advantage, treatment with mitotic inhibitors promotes the production of 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 (fϊbroblast 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.

Preferably differentiation agent, such as RA for example, is introduced in an amount of 10^"4 - 10^"9M, more preferably 10^"5 - 10^"7M, 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 lxlO⁵ - lxl0⁷M, 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.

It will be appreciated that traditional techniques for increasing purity of differentiated cell lines in fact forms neurospheres and then uses enzymes to break up interactions between cells thereby forming single cell suspensions allowing cell selection for example as described by Lee above. The method of the invention in contrast provides neurospheres and isolates these intact to provide substantially pure populations of neural progenitors for the first time.

Neurosphere purification may be by any suitable isolation technique and is preferably by size selection as hereinbefore defined. Preferably 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.

Preferably 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. Preferably a sieve comprises a sterile nylon mesh with desired gauge of 75 to

125 micron, more preferably 75 to 100 micron, most preferably with two sieves which may be separate or in series having first and second maximum and minimum gauge of respectively 500 micron and 75 micron, preferably 400 micron and 100 micron, purifying neurospheres having a particular size within these ranges. 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.

Preferably 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.

Alternatively 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-

12, most preferably pH 10.5. Preferably chemical disruption is conducted together with physical dirsuption for example by means of gentle aspiration, preferably x 2 to 10, more preferably x6, after one or two periods of incubation, for example after 2.5 and 5 minutes incubation. Preferably the method comprises subsequently additionally neutralising the alkaline media with additional media, preferably to pH in the range 5-8, more preferably pH

6.5 followed by further mechanical disruption, preferably by means of aspiration, for example x6.

Preferably 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.

It is a particular advantage of the combined chemical and physical dissociation method of the invention that in excess of 50%, more preferably in excess of 95% preferably substantially all 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. We have found that viability is far in excess of that obtained using dissociation techniques as known in the art such as mechanical and enzymatic disruption.

In a further aspect of the invention there is provided 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.

Preferably 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).

In a further aspect of the invention there is provided a composition 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. Preferably 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. Preferably the composition is for use in research, drug screening or therapy.

Preferably the undifferentiated composition of the invention comprises cells which maintain an undifferentiated state when cultured in the absence of a differentiating signal. Preferably 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.

Preferably 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). Preferably the undifferentiated composition comprises neural progenitor cells. Preferably 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.

Preferably cells of the composition express or show an increase in expression of certain antigens indicative of cell differentiation. For example 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.

Preferably the composition comprises cell which are capable of responding to external agents such as drugs and pharmaceuticals for screening. Preferably the composition is capable or establishing a graft in a recipient host brain. Preferably the composition is capable of migrating along host brain pathways and is capable of widespread distribution in host brain. Preferably the composition of the invention comprises cells which are responsive to host environmental signals.

In a further aspect of the invention there is provided the use of a 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. It is a particular advantage of the high purity of the composition obtained with the process of the invention that it is useful for pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation. Specifically 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.

In a further aspect of the invention there is provided 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. In a particular advantage 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.

Preferably 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.

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.

Suitably 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.

Preferably 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. Preferably 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.

In a further aspect of the invention there is provided 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.

It is known in the art that 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. It is generally recognised that 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.

We have found that 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. In addition 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.

Survival of 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). 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.

The invention is now illustrated in non limiting manner with reference to the following figures and examples.

Brief description of the Figures:

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

(E) were subsequently grown as confluent homogenous monolayers independent of feeder cells after 3 passages as shown in image (F). Scale bars: lOOμm (A,B); 25 μm (C,D,F); 120μm (E); Figure 2 shows the results of immunofluorescent flow cytometry, indicating antigen expression of the clonal pluripotent stem cell line and differentiated derivatives Expression of cell surface antigens by TERA2.cl.SP-12 EC cells and their differentiated derivatives is shown after 14 days exposure to retinoic acid. Nalues represent mean ± SEM from three replicates; Figure 3 shows the results of northern analysis for differential expression of the stem cell marker, Pou5Fl, in the clonal pluripotent stem cell line and its differentiated derivatives. Lanes: (I) TERA2 EC cells; (2) ΝTERA2.cl.Dl EC cells; (3) TERA2.cl.SP-12 EC cells; (4,5,6) TERA2.cl.SP-12 cells after 2, 4 and 7 days exposure to retinoic acid, respectively. GAPDH was used as a loading control;

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)

TERA2.cl.SP-12 cells after 28 days exposure to retinoic acid. R-actin was used as loading control;

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. In 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. In 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. Neurospheres are purified by filtration and plated onto glass coverslips for staining.

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.

MATERIALS & METHODS

Cells

Human cell lines, TERA2 and NTERA2.cl.Dl, were generously provided by P. Andrews, University of Sheffield, UK. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies Ltd, Paisley, Scotland) as described [Przyborski SA, Morton IE, Wood A et al. Developmental regulation of neurogenesis in the pluripotent human embryonal carcinoma cell line NTERA2, Eur J Neurosci 2000; 12:3521-3528]. Cells were induced to differentiate by seeding 1.5xl0⁶ cells per 75-cm² tissue culture flask (Nalge Nunc International, Roskilde Denmark) in DMEM containing lOμM retinoic acid (Sigma- Aldrich Company Ltd, Poole, UK) as described [Przyborski SA, Eur J Neurosci 2000 above].

Antibodies

Primary monoclonal antibodies 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. For example, 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 .

Isolation and cloning of new human pluripotent stem cell lines Confluent TERA2 EC cells (passage 15, earliest available) were briefly treated with 0.25% trypsin (Life Technologies) / 2mM EDTA (Sigma-Aldrich) in phosphate buffered saline (PBS) for 2-3 min to produce a suspension of single cells. Suspended TERA2 cells were diluted to 10⁷ cells/ml and incubated with stage specific embryonic antigen-3 (SSEA-3) antibody (diluted 1:5), a marker of pluripotent stem cells [Andrews PW, Stem Cell Biology, 2001], in wash buffer (WB): PBS plus 5% v/v fetal calf serum (Life Technologies)). Preliminary studies indicate that maximal numbers of cells bind the SSEA-3 antibody after 45 min incubation at 4°C (data not shown). Cells immunoreactive for SSEA-3 were isolated using direct positive magnetic separation according to manufacturers instructions (BioMag goat anti-mouse

IgM, Polysciences Europe GmbH, Postfach 1130, 69208 Eppelheim, Germany

(www.polysciences.de)). BioMag^® magnetic particles are approximately lμm and because of their non-uniform shape provide an increased surface area (>100m²/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.

Immunocytochemistry Cell cultures were fixed in ice-cold methanol (5 min), washed three times in PBS and incubated with primary antibody for 60 min at 4°C. After three PBS washes, cells were incubated with either fluorescein isothiocyanate (FITC)- conjugated goat anti-mouse IgG or IgM (ICN Pharmaceuticals, Inc., Aurora, OH) as appropriate for a further 60 min at 4°C, washed three times in PBS, and examined by fluorescence microscopy.

Differentiation and determination of antigen expression by flow cytometry Cell surface antigen expression was determined by indirect immunofluorescence using a Coulter (Fullerton, CA) EPICS XL cytometer in a manner similar to that previously reported [Przyborski S A, Eur J Neurosci 2000]. Markers of human pluripotent stem cells were highly expressed in each of the newly derived stem cell lineages, indicating strong enrichment of a sub-population of stem cells with a high level of purity. Each of the newly derived pluripotent clones down-regulated their expression of each pluripotent stem cell marker after retinoic acid-induced differentiation. In a reciprocal fashion, antigens expressed during differentiation, notably A2B5 and VIN-IS-

56, were up-regulated. Clone TERA2.cl.SP-12 was selected for further evaluation on the basis that it consistently displayed the highest levels of stem cell markers in its undifferentiated state and showed the strongest expression of A2B5 and VIN-IS-56 in response to retinoic acid (Figure 2). Figure 2 shows the results of antigen expression of the clonal pluripotent stem cell line and the differentiated derivatives thereof.

Northern analysis Poly[A⁺] 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. POU5F1 mRNA was detected at greatest concentrations in NTERA2.cl.Dl and TERA2.cl.SP-12 EC cells but at notably lower levels in the TERA2 parent lineage (Figure 3). TERA2.cl.SP-12 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.

Western analysis

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;

Chemicon Temecula, CA, MAB324; 1:2000), growth-associated protein 43

(GAP43; Sigma- Aldrich, clone 7B10; 1:4000), glial fibrillary acidic protein

(GFAP; Sigma- Aldrich, clone GA-5; 1:5000) and β-ACTLN (Sigma- Aldrich, clone AC-15; 1:5,000) were localized with IgG-horse radish peroyidase (HRP) secondary antibody (Amersham, Piscantaisay, N-J, 1:1,000) in preparation for chemiluminescent detection (Amersham). The reactivity of A2B5 and VIN-IS-

56 has 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).

Discussion

Earlier studies describing the isolation and cloning of human EC cells from primary explant/stock cultures have recognised that the parent material probably consists of multiple cell types and suggest that pluripotent EC cells represent a fraction of the total parent cell population. The current data clearly demonstrate that SSEA-3 and SSEA-4 positive EC cells correspond to a minor component of the parent TERA2 line, which correlates favourably with the lower expression of POU5F1 in TERA2 cultures compared to two of its clonal EC derivatives tested in this study. In earlier work, clonal lines established directly from intact stock cultures of the TERA2 parent lineage showed variability, frequently did not express SSEA-3 or SSEA-4, and often displayed limited capacity for differentiation. Human 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% C0₂. In preparation for differentiation, cultures of pluripotent stem cells were briefly treated with 0.25% trypsin / 2mM EDTA in phosphate buffered saline (PBS) for 2-3 min to produce a suspension of single cells. To produce human neurospheres, pluripotent stem cells were induced to differentiate by exposure to lOμM all-tr ns-retinoic acid (RA) using two different procedures. 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:

1.0 Pluripotent TERA2.cl.SP12 EC cells suspended in DMEMFG were seeded at 5x10 cells into sterile 90mm diameter petri dishes normally used for bacterial cultures.

1.1 Suspension cultures were maintained for 1 day before the addition of RA to a final concentration of lOμM.

1.2 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.

Purification 2.0 Suspended cell neurospheres (Fig A2) were passed over a sterile nylon mesh of pore size lOOμm to remove single cells, small cell clumps and cell debris.

2.1 Cell neurospheres >100μm isolated by the mesh were gently washed whilst on the mesh for a further x3 times with DMEMFG. 2.2 To remove neurospheres from the mesh, the mesh was backwashed with DMEMFG into a sterile bacteriological dish. At this stage, 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 2xl0⁴ cells per cm² in tissue culture flask (equivalent of 1.5xl0⁶ cells per T75 tissue culture flask) in DMEMFG containing lOμM RA. (Fig A/B 1) 1.1 Induction media was changed every 3-4 days.

1.2 After 21 days exposure to RA, cultures were dissociated using 0.25% trypsin / 2mM EDTA in PBS for 10 min at room temperature (RT).

1.3 Suspensions of differentiated cells were split 1 :3 and seeded into fresh tissue culture flasks containing DMEMFG without RA. 1.4 After 1-2 days, the media was replaced with DMEMFG containing mitotic inhibitors (MI) as follows: 0.1 μM cytosine arabinosine (for the first 10 days only); 3μM fluorodeoxyuridine; 5μM uridine.

1.5 Cultures were maintained for a further 21 days during which the medium and inhibitors were replaced every 3-4 days. During this period, large aggregations of cells appeared throughout the culture and formed prominent dome-like structures often linked to one another by connecting fibers. (Fig B2)

Purification

2.0 After 21 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).

2.1 The cell suspension was passed over a sterile nylon mesh with a pore size of lOOμm to remove single cells, small cell clumps and cell debris.

2.2 Neurospheres >100μm isolated by the mesh were gently washed whilst on the mesh for a further x3 times with DMEMFG.

2.3 To remove neurospheres from the mesh, the mesh was backwashed with DMEMFG into a sterile bacteriological dish. At this stage, 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 (lOμg/ml) and human placental laminin (lOμg/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: lμM cytosine arabinosine for the first 7 days only; lOμM fluorodeoxyuridine; lOμM uridine. 3.2 Cells were prepared for immunocytochemical analysis using standard procedures. (Fig A 3-5, Fig B3,4) Example 3 - Dissociation of Neurospheres:

4.0 Individual or groups of neurospheres obtained from the method of Embodiment A or B above were collected by pipette and washed in DMEMFG. Neurospheres were exposed to alkaline DMEMFG media (pH 10.5) for 5 minutes. The cells were aspirated (raised up and down a pipette) x6 after 2.5 and 5 minutes incubation in alkaline DMEMFG media.

4.1 Alkaline medium was neutralised using acidic DMEMFG media (pH 6.5) and the cells were aspirated a further 6 times. 4.2 Suspensions of dissociated cells were seeded onto glass coverslips pre- coated with either poly-D-lysine (lOμg/ml) and human placental laminin (lOμg/ml) or both.

4.3 Cells grown on coverslips were maintained in DMEMFG in the absence of RA but in the presence of mitotic inhibitors (lμM cytosine arabinosine (for the first 7 days only); lOμM fluorodeoxyuridine; lOμM uridine) (see Figure 5).

Results

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. Almost all (>99%) differentiated cells produced by the method of Embodiment A stained positive for B tubulin III indicating the neuronal phenotype but showed no significant expression of GFAP, a marker of astrocytes. Alternatively, neurospheres prepared by the method of

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.

Conclusions

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.

Example 4a: neurite outgrowth assay

Neurospheres derived from TERA2.cl.SP12 pluripotent stem cells as described hereinbefore were exposed to various concentrations (lOpg to lOng) of the following compounds: glial derived neurotrophic factor (GDNF); brain derived neurotrophic factor; neurotrophin-3 (NT-3) and neurotrophin-4 (NT-

4), to assess their effect on neural process outgrowth (formation of such neurites is known in the art as neuritogenesis).

Twenty-four hours after plating, vehicle (water), GDNF, BDNF, NT-3 or NT- 4 were added to neurospheres derived from human pluripotent stem cells grown in 6 well plates (5-10μl per well). On days 4 and 7, the culture medium was removed and replaced with fresh medium and the test compound. On the

10^th day of culture, the medium was removed and the cells were washed with

Dulbecco's phosphate-buffered saline (DPBS) and 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 β-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. Densitometric values were used as arbitrary measures of protein concentration. Values for MAP2 concentration were normalised against their corresponding value for β-actin. Data represents mean ± SEM, n=3. Through measuring the levels of MAP2 protein, a component of the neuronal cytoskeleton that is up-regulated during the growth of a nerve process, this assay showed that each of the compounds tested influenced normal nerve outgrowth. Results from experiments with BDNF showed the greatest significant effect (see Figure 6) whilst data from other compounds tested showed similar trends (data not shown). These experiments demonstrate the usefulness of neurospheres derived using the hereinbefore invention for screening the activity of compounds.

Claims

1. 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.

2. Method of Claim 1 wherein a neurosphere is a cellular aggregate of neural progenitors capable of terminal differentiation to form neurons and optionally additionally glial cells and optionally other neuronal subtypes.

3. Method of any of Claims 1 to 2 which comprises culturing cells selected from mammalian embryonic stem (ES), fetal, developing or adult stem cells or embryonal carcinoma (EC) cells.

4. Method of any of Claims 1 to 3 which comprises culturing human, primate, rat or murine stem cells.

5. Method of any of Claims 1 to 4 which comprises culturing human pluripotent ES or EC cell lines.

6. Method of any of Claims 1 to 5 wherein a mammalian pluripotent stem cell line is a clonal cell line.

7. Method of any of Claims 1 to 6 which 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.

8. Method of any of Claims 1 to 7 wherein neurospheres are isolated in purity in excess of 90%

9. Method of any of Claims 1 to 8 which is a suspension method, comprising culturing a cell line in suspension in a vessel to which cells do not adhere and forming neurospheres in situ.

10. Method of any of Claims 1 to 9 which is a monolayer induction method, comprising culturing a cell line in suspension in a vessel to which cells adhere to produce monolayers of differentiated cells, optionally contacting with a mitotic inhibitor (MI), and culturing for a further period in absence of differentiation agent to form neurospheres.

11. Method of any of Claims 1 to 10 which comprises culturing the cell line for a period and under conditions corresponding to a desired degree of aggregation thereby determining the size range of product neurospheres.

12. Method of Claim 11 which comprises culturing pluripotent stem cells for a period of 2-6 weeks leading to formation of neurospheres in a single or multiple seedings.

13. Method of any of Claims 1 to 12 wherein isolating neurospheres is by size selection, preferably by filtration, with selection of filter or sieve aperture for a particular maximum neurosphere size which it is desired to purify.

14. Method of Claim 13 wherein isolation is with two filters of different aperture size enabling purification of neurospheres of particular size which is greater than the smaller filter aperture and smaller than the larger filter aperture.

15. Method of any of Claims 1 to 14 wherein isolation is by passing the cell suspension through a filter or sieve with desired gauge up to 500 micron, more preferably up to 400 micron.

16. Method of any of Claims 1 to 15 wherein purified neurospheres are transferred to a surface for further use or investigation, for example tissue culture flask, a sterile bacteriological dish, or are transferred to a sealed container and stored for example by freezing, for subsequent use.

17. Method of any of Claims 1 to 16 comprising in a further stage dissociating one or more neurospheres by chemical and physical disruption of the neurosphere structure to produce a suspension or monolayer of single neural progenitor cells.

18. Method of Claim 17 comprising contacting one or more isolated neurospheres with alkaline medium of pH in the range 9 - 12, and thereafter neutralising thereby dissociating the neurosphere to obtain a collection of homogeneous or heterogeneous neural cells.

24. Method for differentiation of one or more neurospheres or of single dissociated neural progenitor cells obtained with the method of any of Claims 1 to 23 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.

25. A neurosphere or dissociated cells or composition or derivatives thereof obtained with the method of any of Claims 1 to 24 in the form of substantially pure homogeneous or heterogeneous neurospheres, dissociated cells and/or their derivatives of purity in excess of 90% and of same or mixed homology.

26. Use of a neurosphere or dissociated cells obtained by the method of any of Claims 1 to 24 in in vitro differentiation, in vitro neural research, modelling in tissue culture, pharmaceutical development including compound screening, toxicological testing or therapeutic cell replacement strategies including but not limited to transplantation.

27. 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 obtained with the method as hereinbefore defined in any of Claims 1 to 24, 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.

28. Method for therapy comprising introducing a composition, neurospheres, dissociated cells, and or their derivatives obtained with the method as defined in any of Claims 1 to 24 into a mammalian host.

29. Method, composition or population or cell line or use thereof substantially as herein described or illustrated in the description, the examples and/or the figures.