EP1565550A1

EP1565550A1 - Method for isolation of pluripotent stem cells

Info

Publication number: EP1565550A1
Application number: EP03766487A
Authority: EP
Inventors: Stefan Alexander School of Biol. PRZYBORSKI
Original assignee: University of Durham
Current assignee: University of Durham
Priority date: 2002-08-02
Filing date: 2003-08-02
Publication date: 2005-08-24
Also published as: GB0405910D0; GB0217983D0; GB2395957A9; AU2003255751A1; WO2004013316A1; GB2395957A

Abstract

Method for preparing one or more compositions of clonal pluripotent stem cells derived from an individual mammalian pluripotent stem cell isolated from a cell population, comprising exposing a heterogeneous cell population to an amount of a tag which recognises and binds individual cells that express markers of mammalian pluripotent stem cells wherein the tag comprises additionally a retrieval means, the method further comprising retrieving the tag and the bound cells to obtain one or more individually selected cells, separation out of one or more single cells and cloning of single cells to obtain one or more cell lines and transferring the or each cell line to a container in culture medium or freezing thereby generating the or each composition characterised in that the or each composition or population comprises clonal pluripotent stem cells of purity or homogeneity or homology in excess of 90 %; the composition obtained thereby; the use thereof in pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like; and methods for screening or therapy therewith.

Description

METHOD FOR ISOLATION OF PLURIPOTENT STEM CELLS

The present invention relates to a method for the preparation of a composition of clonal pluripotent stem cells useful for pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like; the composition obtained thereby; the use thereof in pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like; and methods for screening or therapy therewith; more particularly the invention relates to a method for preparing a composition of clonal pluripotent stem cells comprising the isolation of mammalian pluripotent stem cells and single cell cloning thereof; the composition or population of clonal pluripotent stem cell lines obtained thereby; use thereof for in vitro differentiation; pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like; and methods for screening or therapy therewith.

Embryonic stem (ES) cells are pluripotent stem cells isolated from pre- implantation embryos. Cultures of mammalian ES cells have been established using existing technologies such as the indirect method of immuno-surgery

(Thomson et al. "Science", 282, 1145-1147 (1998)). 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 differing 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. The present invention relates to a novel direct approach to prepare compositions of pluripotent stem cells using immuno-magnetic selection that may be applied to generate additional lines of pluripotent cells which are substantially pure uncommitted cells which are capable of a high level of differentiation to neuronal progenitor cells and which display uniform differentiation and developmental behaviour for use for pharmaceutical development, toxicological testing and therapeutic cell replacement strategies including transplantation, and the like.

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. 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 embryogenesis in mice. 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 et al Cold Spring Harbor Press (2001) above). When cultured in the presence of all-trans-retmoic 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 two 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 neurofϊlament protein. However non-reactivity with monoclonal antibodies specific for the glial cell intermediate filament protein, GFAP, indicated absence of glial cells.

It is therefore an object of the invention to isolate new clonal pluripotent stem cell lines and identify clonal lines that show further variation in their ability to differentiate and have useful characteristics appropriate for the study of particular pathways of mammalian, in particular human or murine, development. It is a further object to provide new clonal lines which neurodifferentiate to produce populations of neurons and optionally glial cells, and methods for isolation thereof for novel applications, in particular for drug screening or therapy.

We have now developed a method for preparing a composition of pluripotent stem cells comprising isolating single mammalian pluripotent stem cells from a heterogeneous cell population using a modification of a known cell isolation method and kit and cloning thereof to produce clonal lines. We have by this means developed a method for preparing a composition of new clonal pluripotent cell lines which display the ability to differentiate including the ability to form populations of neurons and glia. In the broadest aspect of the invention there is therefore provided a method for preparing a composition of clonal pluripotent stem cells derived from an individual mammalian pluripotent stem cell isolated from a cell population comprising exposing a heterogeneous cell population to an amount of a tag which recognises and binds individual cells that express markers of mammalian pluripotent stem cells wherein the tag comprises additionally a retrieval means, the method further comprising retrieving the tag and the bound cells to obtain one or more individually selected cells, separation out of one or more single cells and cloning of single cells to obtain one or more cell lines and transferring the or each cell line to a container in culture medium or freezing thereby generating the or each composition .

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 population 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.

Pluripotent cells are cells which are uncommitted and are capable of differentiation into any tissue types and include human ES and EC cells. It is a particular advantage of the isolation and direct selection of individual pluripotent stem cells with the method of the invention that it is useful for creating new clonal compositions of pluripotent stem cells that can be used directly or as sources of stem and/or differentiated cells for pharmaceutical development including drug screening, toxicological testing and therapeutic cell replacement strategies including transplantation. Specifically the composition of clonal pluripotent stem cells 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.

The composition suitably comprises a population of cells or culture thereof or a cell line. Preferably the composition comprises clonal pluripotent stem cells of purity in excess of 95%, more preferably in excess of 99%. The uncommitted composition is substantially homogeneous and individual cells are of substantially identical homology.

Preferably the method comprises isolating individual mammalian embryonic stem (ES) cells, fetal, developing or adult stem cells or embryonal carcinoma (EC) cells; more preferably selected from human, primate, rat or murine pluripotent stem cells, most preferably human pluripotent stem cells.

Suitably the method comprises isolating cells from a heterogeneous population of cells which may be derived from tissue or culture, for example surgically isolated specimen, or taken from heterogeneous cell culture. Preferably a starting population of cells is derived from blastocyst, bone marrow, blood or other somatic tissues and the like, more preferably, derived from explanted tissue soon after surgical removal, representing the earliest available stage of such pluripotent stem cells, or cells derived from the human teratocarcinomas. The method of the invention employs a tag for binding to cells and means for retrieval, thereby isolating recognised cells.

Preferably a tag comprises antibody recognising and binding mammalian pluripotent stem cell specific cell surface antigens, whereby the method recognises single cells expressing mammalian pluripotent stem cell specific cell surface antigens.

More preferably, a tag comprises certain primary antibodies, including but not limited to human ES and EC cell specific primary antibody SSEA, -3, -4 and TRA-1-60, and murine ES and EC cell specific antibody SSEA-1, which recognise specific cell surface antigens and show highly regulated expression profiles relating to the differentiation of pluripotent stem cells, such as pluripotent ES and EC cells. Such antibodies are expressed highly in pluripotent stem cells and not the differentiated derivatives.

Preferably the method comprises incubating with magnetically labelled antibodies that are stage-specific for embryonic antigens including SSEA-3, SSEA-4 and SSEA-1, and antibodies including TRA-1-60, and the like as hereinbefore defined.

Retrieval means may be any means facilitating automatic retrieval such as a bulky component facilitating size sorting, a magnetic component, electrical component, immunogenic component or the like, for sorting by filter, which has gauge restraining passage of attachment means, sorting by magnetic or electrical field to attract, repel or move cells attached to a magnetic or ionic label, or sorting by immunomagnetic selection and the like.

The method therefore comprises isolating a population of cells identified as positive for markers of mammalian pluripotent stem cells, from a heterogeneous population of cells, and deriving clonal lines of single cell origin. This results in the majority of cells expressing and regulating cell surface antigens in a similar manner. More preferably the method comprises isolating a population of marker-positive (SSEA-3⁺, -4⁺, -1⁺ or TRA-l-60⁺) cells from the starting tissue prior to deriving clonal lines. For example, close examination of the TERA2 parent lineage enables the identification of morphologically different cell populations. Distinct colonies of cells displaying typical pluripotent stem cell morphology are occasionally observed, and such colonies stain positive for markers of human pluripotent stem cells, including TRA-1-60, SSEA-3 and SSEA-4, while surrounding cells of non- pluripotent stem cell structure do not express these antigens. The frequency of pluripotent stem cell marker-positive cells such as TRA-1-60, SSEA-3 , -4⁺ cells in TERA2 cultures may be determined by techniques such as flow cytometry and may represent only 2%-3% of the total population. The TERA2.cl.SP12 clonal line, was isolated from the TERA2 parent lineage and expressed high levels of pluripotent stem cell markers comparable with the well established NTERA2.cl.Dl human pluripotent EC line and human ES cells (see Example 1).

Preferably the method of the invention comprises immunomagnetic isolation and retrieval of one or more cells expressing the desired pluripotent stem cell antigen(s), and single cell separation as hereinbefore defined followed by single cell cloning to produce one or more clonal lines and generating one or more compositions therefrom. More preferably the method comprises incubating mammalian pluripotent stem cells with magnetically labelled antibody and isolating cells immunoreactive for the antibody using direct positive magnetic isolation and retrieval, optionally subsequently removing the label, and culturing one or more single separated, positively recognised cells and producing one or more clonal lines and generating one or more compositions therefrom. The method is illustrated in Scheme 1. Preferably the method comprises direct positive magnetic isolation and retrieval as known in the art for isolation of cells from blood, and for which kits are available commercially (BioMag, Polysciences Europe GMBH) comprising secondary antibodies labelled with 1 micron magnetic particles. Magnetic particles may be retrieved or preferably detach from the cell membrane automatically as the cell surface is turned over during subsequent culturing.

Culturing is preferably for up to 48 hours 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 2rnM L-glutamine).

Previous attempts to separate individual cells using fluorescence activated cell sorting has failed to isolate viable EC cells (PW Andrews, "Differentiation", 1985, 29, 127-135). Biomagnetic sorting is however direct, i.e. identifies and isolates cells recognised by biomagnetic markers, and is rapid which is an important factor in conserving viability of mammalian stem cells.

The method of the invention is a method for isolating pluripotent stem cells as hereinbefore defined, separation out of one or more single cells and cloning thereof. Separation out of single cells may be by manual or automated means. Manual means includes withdrawing single cells by pipette, capillary, or the like, under microscope. Automated means includes passing the population of cells to an automated cell sorter, which is capable of delivering single cells to a suitable vessel for cloning, for example to separate wells in a multiwell tissue culture dish.

The method may be operated with subsequent washing of collective individually selected cells and repeating the isolation method, as many times as required to get a distinct isolation. Repeat isolations may be carried out with the same or different tag, for example labelled antibody, to isolate cell classes or subclasses, for example recognising different cell surface antigens in each separation. Different clonal lines may be obtained by conducting several repeat isolations each with different tags.

Cloning is suitably by known means, for example culturing the or each selected separated cell(s), optionally in culture under condiitons and in media as hereinbefore defined and in the presence of non-dividing feeder cells. The method provides one or more clonal lines each cloned from a single stem cell, thereby providing one or more clonal lines of pluripotent stem cells capable of differentiating into a range of cell types. Preferably the method comprises cloning single cells and obtaining one or more cell lines with propensity to neurodifferentiate, preferably with greater propensity than prior art cell lines for example producing in the range 20 - 50% more neural cells. Preferably the or each cell line of cloned cells is transferred into a suitable sterile container and suspended in medium or frozen thereby generating the or each composition. Preferably medium is for example DMEM or DMEMFG culture as hereinbefore defined and completes derivation of the or each composition. Preferably freezing is in media such as for example fetal bovine serum (FBS) and cryogenic agent such as DMSO.

Compositions are thereafter sealed in the container, for example a sterile vial, and stored for example by freezing as hereinbefore defined for subsequent use in research, screening or therapy.

In a further aspect of the invention there is therefore provided a method for preparing a homogeneous of heterogeneous composition of differentiated clonal pluripotent stem cells in the form of progenitor cells of purity in excess of 90% comprising culturing the composition or population of the invention in the presence of differentiating agent and/or mitotic inhibitors. Cells within the composition are characterised by same genotype and same or mixed phenotype.

Preferably culturing in the presence of differentiating agent and/or mitotic inhibitors is by methods as known in the art including use of desired culturing period, period and sequence of contact with differentiating agent or mitotic inhibitor with optional replating of cells at intervals between culturing and contacting and the like. Preferably culturing is under conditions and with media as hereinbefore described.

Differentiation agents which may be used in differentiation of the cell lines obtained with the method of the invention are selected from naturally occurring compounds and synthetic differentiation reagents, for example retinoic acid, retinoids and derivatives thereof, preferably all-trans retinoic acid (RA), bone morphogenic proteins such as BMP-2, growth factors such as

FGF (fibroblast 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.

In a further aspect of the invention there is provided a composition comprising mammalian pluripotent clonal stem cells and/or their derivatives obtained with the method of the invention as hereinbefore defined characterised in that the composition and comprises clonal pluripotent stem cells and/or their derivatives of purity in excess of 90%. The composition is suitably substantially homogeneous, and the differentiated composition may be homogeneous or heterogeneous and the cells thereof have same genotype and same or mixed phenotype. Preferably the composition comprises clonal pluripotent stem cells of purity in excess of 95%, more preferably in excess of 99%. 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 cells of the composition of the invention consistently display excellent levels of stem cell markers in undifferentiated state and are capable of differentiation in response to differentiation agents for example retinoic acid (RA). Preferably the composition comprises a cell line or culture which indicates the commitment to form neural derivatives or which 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 of the invention express or show a 20 to 50% increase in expression of certain antigens, than with the starting population, indicative of cell differentiation. For example A2B5 or VLN-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 cells 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 obtained by the method of the invention in research, 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 or population obtained with the method 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 in screening or therapy due to the greater reliability of the response detected or the greater expectation of viability.

In a further aspect of the invention there is provided a method for screening compounds which affect proliferation, differentiation or survival of stem cells and/or their derivatives comprising preparing a composition of mammalian pluripotent stem cells according to the method 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 stem 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 differentiating a composition of mammalian pluripotent stem cells of the invention to generate a composition of 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 the method comprises culturing the uncommitted cells in suspension or monolayer in the presence of differentiating agent in known manner to generate a composition of aggregates of neural progenitor cells in the form of neurospheres as hereinbefore defined.

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 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 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 on healthy and diseased 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 femtogram to 1 milligram, in most cases this may be in the range 1 picogram 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 typically 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 moφhology, 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 of the invention as hereinbefore defined comprising isolated and cloned cells and/or their derivatives 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 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 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 stem 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 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 stem cell lines 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:

Scheme 1 shows a schematic procedure for the immunomagnetic isolation and cloning of human pluripotent stem cells; 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); 25μm (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. Values 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: (1) TERA2 EC cells; (2) NTERA2.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: (1) TERA2.cl.SP-12 EC cells; (2) TERA2.cl.SP-12 cells after 28 days exposure to retinoic acid. 5-actin was used as loading control;

Figure 5 shows the effect of Brain Derived Neurotrophic Factor (BDNF) on the formation of neural processes by neurons derived from pluripotent stem cells. Data represents mean ± SEM, n=3. MATERIALS & METHODS

Cells Human EC 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]. EC cells were induced to differentiate by seeding 1.5x10 cells per 75-cm tissue culture flask (Nalgen Nunc International, Roskilde Denmark) in DMEM containing lOμM retinoic acid (Sigma- Aldrich Company Ltd, Poole, UK) as previously reported [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 EC cells [Andrews PW, Przyborski SA, Thomson JA. 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]. For example, SSEA-3, which was originally raised against four-cell-stage mouse embryos, is expressed highly in EC stem cells and not their differentiated derivatives [Andrews PW, Stem Cell Biology above]. Antibodies were pretitered and diluted (1:2 to 1:5) in wash buffer (WB) to give maximal binding . EXAMPLE 1 - Isolation and cloning of human pluripotent 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 above; Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM. (1998), Embryonic stem cell lines derived from human blastocysts. Science, 282:1145-1147], 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 (>100m /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 three times 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 stem cells from the human teratoma line TERA2.

Immunocvtochemistry

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 detection of antigen expression using 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 SA, Eur J Neurosci 2000 above]. Markers of human pluripotent stem cells were highly expressed in each of the newly derived TERA2 subclones, indicating strong enrichment of a sub-population of stem cells with a high level of purity. Each of the newly derived TERA2 subclones down-regulated their expression of each pluripotent stem cell marker after retinoic acid-induced differentiation according to the method of Andrews above. In a reciprocal fashion, antigens expressed during differentiation, notably A2B5 and VLN-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 pluripotent stem cell markers in its undifferentiated state and showed the strongest expression of A2B5 and VLN- IS-56 in response to retinoic acid (Figure 2). Figure 2 shows the results of antigen expression of the clonal EC line and the differentiated derivatives. Northern analysis

Poly[A⁺] RNA was isolated from human EC cells and retinoic acid-induced derivatives, and prepared for northern blotting as previously described

[Przyborski SA, Eur J Neurosci 2000 above]. 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 stem cells. POU5F1 rnRNA 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 EC 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-ACTIN (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 VLN- IS-56 has previously been associated with neuro-ectodermal derivatives, and thus may indicate the ability of TERA2.cl.SP-12 pluripotent stem cells to form neural derivatives in response to retinoic acid. Whilst TERA2.cl.SP-12 pluripotent 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).

Formation of teratomas:

To further demonstrate the pluripotent nature of newly derived clonal lines of human EC cells, 5xl0⁶ TERA2.cl.SP12 stem cells were injected subcutaneously into immune-deficient mice. Xenograft tumours resulted consisting of multiple types of differentiated human tissues.

Notes relating to Example 1

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 stem cells represent a fraction of the total parent cell population. The current data clearly demonstrate that SSEA-3 and SSEA-4 positive pluripotent stem 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 Example 1. 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 using the present invention. It is well recognised that NTERA2.cl.Dl EC cells produce neurons in vitro [Przyborski SA, Eur J Neurosci 2000 above] 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 TERA2.cl.SP-12 cells produced proteins indicative of both neurons and glia.

EXAMPLE 2: Method for screening activity of test compounds on Compositions of the invention: neurite outgrowth assay

Neurons derived from TERA2.cl.SP12 pluripotent stem cells as described hereinbefore were exposed to various concentrations (10pg 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 neurons 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 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 5) whilst data from other compounds tested showed similar trends (data not shown). These experiments demonstrate the usefulness of cells derived using the hereinbefore technologies for screening the activity of compounds.

Claims

1. Method for preparing a composition of clonal pluripotent stem cells derived from an individual mammalian pluripotent stem cell isolated from a cell population comprising exposing a heterogeneous cell population to an amount of a tag which recognises and binds individual cells that express markers of mammalian pluripotent stem cells wherein the tag comprises additionally a retrieval means, the method further comprising retrieving the tag and the bound cells to obtain one or more individually selected cells, separation out of one or more single cells and cloning of single cells to obtain one or more cell lines and transferring the or each cell line to a container in culture medium or freezing thereby generating the or each composition .

2. Method of Claim 1 wherein a population of stem cells is selected from embryonal carcinoma (EC) stem cells, mammalian embryonic stem (ES) cells, fetal, developing or adult stem cells or neural stem cells.

3. Method of any of Claims 1 and 2 wherein a population of cells is selected from human, primate, rat or murine pluripotent stem cells.

4. Method of any of Claims 1 to 3 comprising isolating cells from cells from a heterogeneous population of cells which may be derived from tissue or culture, for example surgically isolated or taken from heterogeneous cell culture.

5. Method of any of Claims 1 to 4 wherein a tag comprises antibody recognising and binding mammalian pluripotent stem cell specific cell surface antigens, whereby the method recognises single cells expressing mammalian pluripotent stem cell specific cell surface antigens.

6. Method of any of Claims 1 to 5 wherein a tag is selected from pluripotent stem cell specific antibody such as SSEA-3, -4 and TRA-1-60, and murine EC cell specific antibody SSEA-1.

7. Method of any of Claims 1 to 6 wherein retrieval means is selected from a bulky component facilitating size sorting, a magnetic component, electrical component, an immunogenic component and a fluorescent component, for sorting by filter, which has gauge restraining passage of attachment means, sorting by magnetic or electrical field to attract, repel or move cells attached to a magnetic or ionic label or sorting by immunomagnetic selection or FACS (fluorescence activated cell sorting).

8. Method of any of Claims 1 to 7 which comprises incubating mammalian pluripotent stem cells with magnetically labelled antibody and isolating cells immunoreactive for the antibody using direct positive magnetic isolation and retrieval, optionally subsequently removing the label, and culturing one or more single separated, positively recognised cells and producing one or more clonal lines and generating one or more compositions therefrom.

9. Method of any of Claims 7 and 8 wherein magnetic component or antibody comprises magnetic particles which are released or detach from the cell membrane automatically as the cell surface is turned over during subsequent culturing for a period of up to 48 hours.

10. Method of any of Claims 1 to 9 wherein separation out of single cells is by manual or automated means.

11. Method of Claim 10 wherein manual means includes withdrawing single cells by pipette, capillary, or the like, under microscope, and wherein automated means includes passing the population of cells to an automated cell sorter, which is capable of delivering single cells to a suitable vessel for cloning.

12. Method of any of Claims 1 to 11 operated with subsequent washing of individually selected cells and repeating the isolation method one or more times prior to cloning.

13. Method of any of Claims 1 to 12 comprising cloning single cells and obtaining one or more cell lines with propensity to neurodifferentiate and generating one or more compositions.

14. Method of any of Claims 1 to 13 wherein the or each cell line of cloned cells is transferred into a suitable sterile container and suspended in culture or medium or frozen thereby generating the or each composition.

15. Method of any of Claims 1 to 14 wherein the one or more cell lines are differentiated by contacting with differentiation agent, and a composition is prepared comprising differentiated cells.

16. Method for preparing a composition of differentiated clonal pluripotent stem cells in the form of progenitor cells of purity in excess of 90% comprising culturing the composition of the invention in the presence of differentiating agent and/or mitotic inhibitors.

17. Method or method as claimed in any of Claims 15 and 16 wherein a differentiation agent for inducing differentiation of the cell line is selected from naturally occurring compounds and synthetic reagents such as retinoic acid, retinoids and derivatives thereof, bone morphogenic proteins, growth factors, trophic factors macrophage inflammatory proteins, noggin, heparan sulfate, amphiregulin, interleukins and the like.

18. A composition comprising mammalian pluripotent clonal stem cells and/or their derivatives obtained with the method of the invention as hereinbefore defined characterised in that the composition and comprises clonal pluripotent stem cells and/or their derivatives of purity in excess of 90%.

19. Composition of Claim 18 which comprises a cell line or culture which indicates the commitment to form neural derivatives or which comprises neural progenitor cells and / or differentiated neural cell types.

20. Composition or population obtained by the method of any of Claims 1 to 17 for use in compound testing or screening, eg in pharmaceutical development for activity or toxicological testing or therapeutic cell replacement strategies including but not limited to transplantation.

21. Composition as claimed in Claim 20 comprising cells which are capable of responding to external agents such as drugs and pharmaceuticals for screening, or which are capable or establishing a graft in a recipient host brain and are responsive to host environmental signals

22. Use of a composition or population or cell line obtained by the method of any of Claims 1 to 17 in compound testing or screening, eg in pharmaceutical development for activity or toxicological testing, comprising contacting a composition comprising sample population of the cell line with a compound to be tested and monitoring the development of cell growth and differentiation and determining compound efficacy or compound toxicology.

23. Method for screening compounds which affect proliferation, differentiation or survival of stem cells and / or differentiated derivatives comprising preparing a composition according to the method as hereinbefore defined, contacting the composition with at least one compound and monitoring the effect of the compound on proliferation, differentiation or survival of stem cells and / or differentiated derivatives comprised within the composition.

24. Method of Claim 23 comprising differentiating cells to generate a composition of neural cell types, contacting with compound(s) and subsequently monitoring the effects of the compound(s) on the ability of the progenitor cells to differentiate into neural cells.

25. Method of any of Claims 23 and 24 wherein compound(s) 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.

26. Method of any of Claims 23 to 25 wherein monitoring the effect on proliferation or viability includes 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.

27. 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.

28. 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.