EP1565550A1 - Procede d'isolation de cellules souches pluripotentes - Google Patents

Procede d'isolation de cellules souches pluripotentes

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Publication number
EP1565550A1
EP1565550A1 EP03766487A EP03766487A EP1565550A1 EP 1565550 A1 EP1565550 A1 EP 1565550A1 EP 03766487 A EP03766487 A EP 03766487A EP 03766487 A EP03766487 A EP 03766487A EP 1565550 A1 EP1565550 A1 EP 1565550A1
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European Patent Office
Prior art keywords
cells
cell
composition
pluripotent stem
stem cells
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EP03766487A
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German (de)
English (en)
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Stefan Alexander School of Biol. PRZYBORSKI
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University of Durham
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University of Durham
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]

Definitions

  • 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
  • ES cells include inter alia in vitro differentiation; in vitro research, modelling in tissue culture, drug screening, cell replacement therapy and the like.
  • 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.
  • 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 , Gottling 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
  • 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 neurof ⁇ lament protein.
  • monoclonal antibodies specific for the glial cell intermediate filament protein, GFAP indicated absence of glial cells.
  • 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.
  • 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.
  • 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.
  • ES mammalian embryonic stem
  • EC embryonal carcinoma
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SSEA-3 + , -4 + , -1 + or TRA-l-60 + marker-positive
  • 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).
  • 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.
  • 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).
  • HEM HEM
  • DMEM Dulbecco's modified Eagles's medium
  • RPMI fetal calf serum
  • F-12 fetal calf serum
  • 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.
  • 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.
  • 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.
  • medium is for example DMEM or DMEMFG culture as hereinbefore defined and completes derivation of the or each composition.
  • freezing is in media such as for example fetal bovine serum (FBS) and cryogenic agent such as DMSO.
  • FBS fetal bovine serum
  • DMSO cryogenic agent
  • 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.
  • 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.
  • 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.
  • 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 fibroblast growth factor
  • NGF fibroblast growth factor
  • PDGF vascular endothelial growth factor
  • 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.
  • 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.
  • TNFalpha tumor necrosis factor alpha
  • macrophage inflammatory proteins such as MIP-1 alpha, MlP-lbeta, MIP-2 and
  • compositions 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.
  • the composition comprises clonal pluripotent stem cells of purity in excess of 95%, more preferably in excess of 99%.
  • the composition is for use in research, drug screening or therapy.
  • the undifferentiated composition of the invention comprises cells which maintain an undifferentiated state when cultured in the absence of a differentiating signal.
  • the composition comprises cells which are capable of proliferation in vivo and differentiating to form neural progenitor cells, and differentiating into other lineages such as neurons and glia.
  • 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).
  • the composition comprises a cell line or culture which indicates the commitment to form neural derivatives or which comprises neural progenitor cells.
  • the differentiated composition comprises 20 to 50% more cells which show the appearance of morphologically identifiable neural cells or produce proteins indicative of neural cells, in particular neurons and glia and optionally differentiated subtypes, than would be obtained from the starting cell population.
  • cells of the composition 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.
  • 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.
  • the composition comprises cells which are capable of responding to external agents such as drugs and pharmaceuticals for screening.
  • the composition is capable or establishing a graft in a recipient host brain.
  • the composition is capable of migrating along host brain pathways and is capable of widespread distribution in host brain.
  • the composition of the invention comprises cells which are responsive to host environmental signals.
  • composition 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compounds for screening are selected from compounds which are intended to have a harmful or beneficial effect on neural cells and include neurological inhibitors or agents such as neuroblockers or neurotransmitters and receptors therefor, growth inhibitors of growth factors and receptors therefore and enzymes used in their synthesis, more preferably growth factors and neurotrophic factors and the like; more preferably selected from neurotrophic factor such as glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor; neurotrophin such as neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and the like.
  • neurological inhibitors or agents such as neuroblockers or neurotransmitters and receptors therefor, growth inhibitors of growth factors and receptors therefore and enzymes used in their synthesis, more preferably growth factors and neurotrophic factors and the like; more preferably selected from neurotrophic factor such as glial derived neurotrophic factor (GDNF), brain derived neurotrophic factor; neurotrophin such as neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) and the like.
  • GDNF
  • the effect on proliferation of the 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.
  • compounds are additionally screened in parallel against compositions of diseased cells to determine differences in effect on healthy and diseased cells.
  • the compound is provided in solution and compositions are contacted with solution in given concentration(s) and volume(s) in a single dose or at intervals providing a sustained concentration or variations in concentration with time, as compound is consumed.
  • Concentration is suitably in the range of 1 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 mo ⁇ hology, monitoring changes in expressed phenotypes, amount or type of proteins expressed, and the like, monitoring changes in neuronal characteristics including electrophysiological properties such as resting membrane potential, evoked potential, direction and ionic nature of current flow, and dynamics of ion channels.
  • Monitoring may be by techniques such as immunohistochemistry; or biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis, HPLC analysis, western blots, radioimmune assays, nucleic acid analysis such as northern blots and PCR and the like; or extracellular or intracellular voltage recording, voltage clamping and patch clamping, or using voltage sensitive dyes or ion sensitive electrodes.
  • biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis, HPLC analysis, western blots, radioimmune assays, nucleic acid analysis such as northern blots and PCR and the like; or extracellular or intracellular voltage recording, voltage clamping and patch clamping, or using voltage sensitive dyes or ion sensitive electrodes.
  • biochemical analysis including protein assay, enzyme assay, receptor binding assay, enzyme-linked immunosorbant assay (ELISA) electrophoretic analysis
  • the method is for monitoring changes in neural cell development from neural progenitor cells and comprises differentiating cells comprised within a composition of the invention to obtain a composition of neural progenitor cells of purity in excess of 90%, conducting the method for sceening as hereinbefore defined, culturing in the presence of differentiation agent and monitoring changes in neural development such as for example, to assess the effect of compound(s) on neural process outgrowth (formation of neurites, known in the art as neuritogenesis or neurite outgrowth) and the like.
  • measuring neuritogenesis is by measuring levels of expression of proteins which are typically upregulated during normal outgrowth of nerve processes, in particular measuring expression of MAP2 protein.
  • a method for therapy comprising introducing a composition 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.
  • transplantation of tissue into the CNS is a potential route to treatment of neurodegenerative disorders and CNS damage due to injury.
  • Transplantation of new cells into the damaged CNS has the potential to repair damaged circuitries and provide neurotransmitters thereby restoring neurological function.
  • the absence of suitable cells prevents the full potential of this procedure being met.
  • selecting stem cells for transplantation offers the highest success rate, allowing cells to be obtained in large numbers, capable of surviving indefinitely but stop growing after transplantation to the brain for example, cells can be obtained from patients normal tissue lessening the chances of rejection, and are capable of differentiating to form normal neural connections and respond to neurological signals.
  • Stem cell therapy is well recognised and has yet to reach its full potential in terms of successful transplantation for which an improved source of cells having the required diversity or homogeneity and viability are required.
  • the method of the invention provides a pure source of stem cells which is ideally suited for therapy, for transplantation by introduction into a host as hereinbefore defined.
  • Suitably introduction is by grafting or injection 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
  • 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.
  • a graft can be monitored in known manner using non-invasive scan such as computerised axial tomography (CAT) or the like, nuclear magnetic resonance (nmr) or magnetic resonance imaging (MRI).
  • CAT computerised axial tomography
  • nmr nuclear magnetic resonance
  • MRI magnetic resonance imaging
  • Functional integration of a graft into a hosts neural tissue can be assessed by examining restoration of various functions including tests for endocrine, motor, cognitive and sensory functions.
  • 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
  • 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;
  • 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 lO ⁇ M retinoic acid (Sigma- Aldrich Company Ltd, Poole, UK) as previously reported [Przyborski SA Eur J Neurosci 2000 above].
  • 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].
  • 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 .
  • 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 10 7 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.
  • SSEA-3 stage specific embryonic antigen-3
  • BioMag ® magnetic particles are approximately l ⁇ m 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.
  • FIG. 1 shows the isolation and cloning of pluripotent stem cells from the human teratoma line TERA2.
  • RNA was isolated from human EC cells and retinoic acid-induced derivatives, and prepared for northern blotting as previously described
  • 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.
  • 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 ⁇ -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).
  • NSE neuron-specific enolase
  • GAP43 growth-associated protein 43
  • GFAP glial fibrillary acidic protein
  • TERA2.cl.SP-12 pluripotent stem cells 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).
  • TERA2.cl.SP12 stem cells were injected subcutaneously into immune-deficient mice. Xenograft tumours resulted consisting of multiple types of differentiated human tissues.
  • 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).
  • GDNF glial derived neurotrophic factor
  • brain derived neurotrophic factor neurotrophin-3
  • NT- 4 neurotrophin-4
  • GDNF GDNF
  • BDNF BDNF
  • NT-3 or NT- 4 were added to neurons derived from human pluripotent stem cells grown in 6 well plates (5-10 ⁇ l per well).
  • the culture medium was removed and replaced with fresh medium and the test compound.
  • 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.
  • DPBS Dulbecco's phosphate-buffered saline
  • MAP2 microtubule-associated protein 2
  • HRP IgG-horse radish peroxidase

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Abstract

L'invention a trait à un procédé permettant de préparer une ou plusieurs compositions de cellules souches pluripotentes clonales, dérivées d'une cellule souche pluripotente de mammifère, isolée d'une population cellulaire. Ledit procédé consiste : à exposer une population cellulaire hétérogène à une quantité d'indicateur reconnaissant et liant des cellules exprimant des marqueurs de cellules souches pluripotentes de mammifère, l'indicateur contenant également un moyen de détection ; à détecter le marqueur et les cellules liées afin d'obtenir une ou plusieurs cellules sélectionnées individuellement, à extraire une ou plusieurs cellules simples, à cloner les cellules simples pour obtenir une ou plusieurs lignées cellulaires, et à transférer ladite lignée cellulaire vers un récipient dans un milieu de culture, ou à la congeler, ce qui permet de générer la ou les compositions. Ladite composition ou population est caractérisée en ce qu'elle contient des cellules souches pluripotentes clonales d'une pureté, d'une homogénéité ou d'une homologie dépassant 90 %. L'invention concerne également la composition obtenue au moyen du procédé, son utilisation dans le domaine du développement pharmaceutique, notamment en matière de dépistage de drogues, d'essais toxicologiques, et de stratégies de remplacement thérapeutique de cellules, y compris la transplantation, et analogues. L'invention concerne aussi des méthodes de criblage ou thérapeutiques faisant appel auxdites compositions.
EP03766487A 2002-08-02 2003-08-02 Procede d'isolation de cellules souches pluripotentes Withdrawn EP1565550A1 (fr)

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GB0217983 2002-08-02
GB0217983A GB0217983D0 (en) 2002-08-02 2002-08-02 Method for isolation of pluripotent stem cells
PCT/GB2003/003396 WO2004013316A1 (fr) 2002-08-02 2003-08-02 Procede d'isolation de cellules souches pluripotentes

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CA2569079A1 (fr) * 2004-06-03 2005-12-15 The Johns Hopkins University Methodes de criblage de proliferation cellulaire ou de troubles neoplasiques
WO2006084314A1 (fr) * 2005-02-09 2006-08-17 Australian Stem Cell Centre Limited Populations de cellules souches et systeme de classification
GB0911060D0 (en) 2009-06-26 2009-08-12 Ge Healthcare Uk Ltd Methods for predicting the toxicity of a chemical

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