WO2007148098A2 - Procédés de production et de propagation de cellules précurseurs neurales - Google Patents

Procédés de production et de propagation de cellules précurseurs neurales Download PDF

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WO2007148098A2
WO2007148098A2 PCT/GB2007/002319 GB2007002319W WO2007148098A2 WO 2007148098 A2 WO2007148098 A2 WO 2007148098A2 GB 2007002319 W GB2007002319 W GB 2007002319W WO 2007148098 A2 WO2007148098 A2 WO 2007148098A2
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colony
cells
npcs
cell
hnm
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WO2007148098A3 (fr
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Siddharthan Chandran
Alexis Joannides
Nicholas Allen
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University College Cardiff Consultants Ltd
Cambridge Enterprise Ltd
Cardiff University
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University College Cardiff Consultants Ltd
Cambridge Enterprise Ltd
Cardiff University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • This invention relates to the production and propagation of mammalian cells, in particular neural precursor cells (NPCs) .
  • NPCs neural precursor cells
  • Parkinson's disease and multiple sclerosis The most widely applied stem cell neuralising systems employ spontaneous differentiation, addition of retinoic acid to stem cell aggregates or co-culture with stromal cells or conditioned media (Hoffman, L.M. & Carpenter, M. K. Nat. Biotechnol. 23, 699-708 (2005)).
  • the present inventors have developed simple and fully defined methods of producing and propagating neural precursor cells using fully humanised and generic culture conditions. These methods are amenable to automation and expansion to bulk culture and may be useful in the generation of clinical grade neural precursors for therapeutic applications, as well as a source of human neural cells and glia for high-throughput drug screening.
  • An aspect of the invention provides a method of production of a population of neural precursor cells (NPCs) comprising: i) slicing a colony of stem cells using an automated tissue sectioning device to produce colony fragments, and; ii) culturing one or more of said colony fragments in human neutralising medium (HNM) to produce said NPCs.
  • NPCs neural precursor cells
  • a neural precursor cell is a daughter or descendant of a undifferentiated stem cell, with a more committed phenotype and/or a more reduced differentiation potential compared to the original stem cell (i.e. multipotent but not totipotent).
  • NPCs are committed to a neural lineage and are capable of giving rise to a plurality of distinct neuronal and glial phenotypes i.e. an NPC is able to differentiate under appropriate conditions into all cell types in the nervous system, including neurons with different neurotransmitter subtypes, astrocytes, oligodendrocytes.
  • An NPC may express one or more NPC associated markers, such as Nestin, Musashil, Pax6 and Soxl .
  • a stem cell is a pluripotent cell which is undifferentiated and is not committed to a neural phenotype.
  • a stem cell may express one or more of the following stem-cell associated markers: Oct-4; Nanog; Sox2; stage specific embryonic antigens (SSEA-3 and 4) , and the tumor rejection antigens TRA-1-60 and -1-81.
  • a stem cell may lack markers associated with specific neuronal fates, such as tyrosine hydroxylase and markers associated with NPCs, such as Nestin, Musashil, Pax ⁇ and Soxl.
  • Suitable stem cells may be derived from any source and may include embryonic, fetal and adult stem cells, e.g. skin derived stem cells.
  • suitable stem cells may be obtained by reprogramming adult somatic cells (Cowan et al, Science 2005 Aug 26; 309 (5739) : 1369-73) .
  • a stem cell may be obtained from an individual in need of therapy, for example for the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue.
  • the stem cell may be obtained directly from the individual or may be obtained by reprogramming a somatic cell obtained from the individual.
  • a population of cells produced from the stem cell as described herein may be administered to the individual to treat the condition.
  • stem cells are human stem cells.
  • Stem cells include embryonic stem cells.
  • An embryonic stem cell An embryonic stem cell
  • ESC is a cell derived from an early stage embryo, which exhibits an undifferentiated phenotype and is potentially totipotent i.e. it is capable of giving rise to any cell type in an individual.
  • An ESC may be a cell obtained from an embryo or may be a descendent of such a cell.
  • the stem cells are human ESCs (hESCs) .
  • hESCs human ESCs
  • Suitable stem cells may be obtained from a cultured hESC cell line, such as H9 or HUES9. Further examples of suitable human embryonic stem cells are described in (Thomson JA et al Science 282: 1145-1147 (1998); Reubinoff et al . Nat Biotechnol 18:399-404 (2000); Cowan, CA. et al. N. Engl. J. Med. 350, 1353-1356(2004), Gage, F. H., et al. Ann. Rev. Neurosci. 18 159-192 (1995); and Gotlieb (2002) Annu. Rev. Neurosci 25 381-407); Carpenter et al .
  • Potentially clinical grade hESCs are described in Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005), Ludwig, T .E . et al. Nat. Biotechnol. 24, 185-187 (2006) .
  • the stem cells are not hESCs.
  • a colony of stem cells may be obtained by culturing cells from an cell line using conventional techniques (Valuer, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, CA. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Joannides, A. et al . Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al . Lancet 365, 1636-1641 (2005), Ludwig, T. E. et al . Nat. Biotechnol.
  • hESCs may be cultured in a culture dish on a layer of feeder cells, such as irradiated mouse embryonic fibroblasts (MEF), at an appropriate density (e.g.lO 5 to 10 6 cells/60mm dish) , or on an appropriate substrate with feeder conditioned or defined medium.
  • hESC colonies may be passaged by enzymatic or mechanical means.
  • Suitable culture media for hESCs include Knockout Dulbecco' s Modified Eagle's Medium (KO-DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, ImM L-Glutamine, 0. ImM ⁇ -mercaptoethanol and 4ng/ml to 10ng/ml human bFGF.
  • a human embryo is used as a source of hESCs, the human embryo is one that would otherwise be destroyed without use, or stored indefinitely, especially a human embryo created for the purpose of IVF treatment for a couple having difficulty conceiving.
  • IVF generally involves creation of human embryos in a number greater than the number used for implantation and ultimately pregnancy. Such spare embryos may commonly be destroyed.
  • an embryo that would otherwise be destroyed can be used in an ethically positive way to the benefit of sufferers of severe neurodegenerative disorders such as Parkinson' s disease.
  • ES cells may be derived from one embryo cell without destroying the whole embryo (Chung Y et al, Nature 2006;439 (7073) :21 ⁇ -9.) .
  • the present invention itself does not concern the use of a human embryo in any stage of its development. As noted, the present invention minimizes the possible need to employ a material derived directly from a human embryo, whilst allowing for development of valuable therapies for common and devastating diseases. Any therapeutic interventions based on the present invention must also be performed according to the relevant national laws and ethical guidelines.
  • a stem cell may be genetically manipulated, for example to reduce or silence expression of one or more genes or to express a heterologous polypeptide .
  • a stem cell may have the genotype of a genetic disease or disorder.
  • the stem cell may be derived from an individual having a genetic disease.
  • a stem cell for use in the present methods may be substantially free from one or more other cell types.
  • Stem cells may, for example, be separated from other cell types, using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (FACS) including the use of antibodies against extracellular regions of molecules found on embryonic stem cells such as SSEA4.
  • FACS fluorescence activated cell sorting
  • a stem cell colony Prior to sectioning, a stem cell colony is preferably detached from feeder cells using standard techniques, for example collagenase treatment, as described herein.
  • a stem cell colony may be sliced or sectioned by making a series of incisions through the colony which divide the colony into a series of separate pieces or fragments.
  • the separate pieces or fragments are uniform in size.
  • colonies are sliced in two different (i.e. non-parallel) directions by making a first series of parallel incisions through the colony in a first direction, and then making a second series of parallel incisions through the colony in a second direction.
  • the first and second series of incisions intersect to divide the colony into separate pieces or fragments.
  • the first and second directions may, for example, be perpendicular to produce substantially square fragments.
  • a colony may be conveniently sliced in two directions by rotating the colony relative to the cutter of the device, for example by mounting the culture dish containing the colony on a rotatable platform or table.
  • the incisions are separated such that each colony fragment which is defined by the incisions contains about 40 to 150 cells, preferably about 70 cells.
  • the incisions in a series of parallel incisions may be at intervals of 50 ⁇ m to 250 ⁇ m, preferably lOO ⁇ m to 200 ⁇ m, and typically 150 ⁇ m.
  • the device may conveniently comprise a blade or cutter mounted on a chopping arm which is positioned to section a subject located on a platform.
  • the platform may be rotatable to allow the cutter to section the subject in different directions.
  • the device may be adapted to section colonies in a culture dish for the passaging of hESCs .
  • the chopping arm may comprise an indentation which is positioned to receive the side of the dish during sectioning, to prevent contact between the dish and the chopping arm.
  • Such adaptation may be unnecessary for passaging cells other than ESCs, including neuralisation fragments, since intact colonies of such cells may be detached from the culture dish, and placed on a suitable stage, and sectioned to the desired fragment size .
  • Suitable automated sectioning devices include automated tissue sectioning devices such as the Mcllwain Tissue Chopper (TC752: Campden Instruments Ltd, Loughborough, LE12 7XT England)
  • the colony fragments may be transferred to one or more secondary culture dishes.
  • the cells are transferred at a cell density which is equivalent to 0.5 to 1.5 x 10 6 cells per 10 cm dish.
  • the fragments of the stem cell colony are cultured in human neutralising medium (HNM) .
  • HNM human neutralising medium
  • the secondary culture dishes are preferably non-adherent, such that colony fragments form small spheres in the HNM medium and the cells therein differentiate into neural precursor cells (NPCs) .
  • Differentiation is characterised by a loss of expression of stem cell markers such as Oct4 and SSEA4 and a gain of expression of neural progenitor markers, such as nestin, Pax6 and mushashil.
  • HNM is a defined medium which is devoid of serum, exogenous mitogens, retinoids, and neural inducers.
  • HNM comprises a basal cell culture medium, such as DMEM, with the addition of human serum albumin (1.80E-02 to 1.20E-01 mM) , human transferrin (30% saturation: 3.80E-05 to 2.85E-03 mM) , glutathione (reduced) (up to 1.00E-02mM), recombinant human insulin (3.51E-04 to 5.26E-03 mM) , superoxide dismutase (up to 1.50E-04mM), L-ascorbic acid (5.00E-02 to 2.00E+00 mM) and N-acetyl-L-cysteine (up to 7.50E+01 mM) .
  • DMEM basal cell culture medium
  • human serum albumin (1.80E-02 to 1.20E-01 mM
  • human transferrin (30% saturation: 3.
  • recombinant human EGF up to 1.29E-05 mM
  • recombinant human FGF2 up to 5.77E-06 mM
  • EGF may also be included in some contexts EGF to maintain long-term cell survival and proliferation.
  • a suitable HNM medium may comprise the ingredients set out in table 1, each ingredient being independently at a concentration between the maximum and minimum concentrations set out for that ingredient. Where no maximum and minimum concentrations are provided, the ingredient may be omitted or may be present at a concentration which is within the range of +/-50% of the stated preferred concentration.
  • the HNM medium may comprise the ingredients set out in table 1, wherein one or more, two or more, three or more or preferably all of the ingredients in the group cons-isting of human serum albumin, human transferring, glutathione (reduced) , recombinant human insulin, superoxide dismutase, L- ascorbic acid and N-acetyl-L-cysteine are at the preferred concentrations stated in table 1.
  • one or more, two or more, three or more or preferably all of the remaining ingredients are at the preferred concentrations stated in table 1.
  • the colony fragments are agitated to prevent re-aggregation of the fragments and maintain small particle size.
  • Agitation may include mechanical stirring or, more preferably, orbital shaking, for example at about 40rpm.
  • Standard mammalian cell culture conditions may be employed, for example 37°C, 21% Oxygen, 5% Carbon Dioxide.
  • Media is preferably changed every two days and cells allowed to settle by gravity. Techniques for mammalian cell culture are well-known in the art (Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) .
  • the cells in the stem cell colony fragment may be cultured in HNM for 12 to 18 days (D12-D18) from the initial seeding of the medium (DO) , preferably about 16 days (D16) , to allow differentiation into NPCs.
  • human recombinant fibroblast growth factor 2 may be added to the culture medium.
  • FGF2 is added to the culture medium (HNM) 4 to 10 days (D4 to DlO) after the colony fragment (DO) , preferably about 8 days (D8) .
  • Human recombinant FGF2 is available from commercial suppliers (e.g. R&D, Minneapolis, MN, USA) .
  • the NPCs produced as described above may be further maintained and propagated in HNM as described herein.
  • the NPCs can be further purified, for example by using a standard enzyme dissociation method (trypsin, accutase, EDTA) to obtain single cells and FACS sorting for one or more NPC markers, such as CD133+, syndecan-l+/ notch-l+/integrin beta-l+, integrin alpha ⁇ or beta 1, dsd-1, nestin, musashil, Pax ⁇ and/or Soxl (Uchida N et al, PNAS 2000 Dec 19; 97 (26) : 14720-5; Nagato M et al, J Neurosci Methods 2005 May 15; 80 (4) : 456-66; Hall PE et al, Stem cells 2006 May 11) .
  • a standard enzyme dissociation method trypsin, accutase, EDTA
  • NPCs neural precursor cells
  • Suitable NPCs may be produced by a method described above or may be NPCs obtained from an individual, for example NPCs from the fetal or adult brain, or cells descended therefrom.
  • a method of propagating NPCs may comprise: (i) slicing a colony of NPCs using an automated tissue sectioning device to produce colony fragments,
  • the fragments are agitated during culturing, for example by orbital shaking, in order to prevent re-aggregation of colony fragments.
  • the cell density is reduced at least 2-fold, at least 3 fold or at least 4 fold for each repetition of steps i) and ii) NPCs propagated using the present methods may be purified, for example by FACS sorting as described above.
  • NPCs produced as described above may be allowed to terminally differentiate into neural cells such as neurons and glia (including astrocytes and oligodendrocytes) .
  • neural cells such as neurons and glia (including astrocytes and oligodendrocytes)
  • glia including astrocytes and oligodendrocytes
  • NPCs may be cultured in the absence of growth factors, for example in DMEM/2% B27 (Invitrogen) or HNM or other suitable media, which may also include combinations of established neurotrophic factors (such as BDNF, GDNF and NT3) .
  • the methods described herein are suitable for the generation of clinical grade neural precursors and terminally differentiated neural cells for therapeutic applications.
  • the present invention extends in various aspects not only to an NPC or terminally differentiated neural cell produced in accordance with any one of the methods disclosed herein or a population of such cells but also a pharmaceutical composition, medicament, drug or other composition comprising such a cell or population, use of such a cell or population or composition in a method of medical treatment, particularly the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue, for example neuronal and/or glial cells, a method comprising administration of such a cell or population or composition to a patient, e.g.
  • a composition for administration e.g. for treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue
  • a method of making a pharmaceutical composition comprising admixing such a cell or population with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients, e.g.
  • a neuroprotective molecule a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, anti-apoptotic factor, or factor that regulates gene expression in NPCs or neural cells or in the host brain.
  • Such optional ingredients may render the cell independent of its environment, i.e. such that its survival is not dependent on the presence of one or more factors or conditions in its environment.
  • a cell or cell population which is administered to an individual may be genetically manipulated to produce a therapeutic molecule, for example a drug or growth factor (Behrstock S et al, Gene Ther 2006 Mar;13(5) :379-88, Klein SM et al, Hum Gene Ther 2005 Apr; 16 (4) : 509- 21)
  • compositions according to the present invention may comprise, in addition to the neuron or cell, a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant or other material well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of the neuron. The precise nature of the carrier or other material will depend on the route of administration.
  • the composition may include one or more of a neuroprotective molecule, a neuroregenerative molecule, a retinoid, growth factor, astrocyte/glial cell, or factor that regulates gene expression in stem, neural stem, precursor or progenitor cells or neuronal cells. Such substances may render the neuron independent of its environment as discussed above.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.
  • a composition may be prepared using artificial cerebrospinal fluid.
  • Cells may be implanted into a patient by any technique known in the art (e.g. Lindvall, 0., (1998) Mov. Disord. 13, Suppl. 1:83-7; Freed, CR. , et al . , (1997) Cell Transplant, 6, 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332, 1118-1124; Freed, C. R., (1992) New England Journal of Medicine, 327, 1549-1555, Le Blanc et al, Lancet 2004 May 1; 363 (9419) : 1439-41) .
  • any technique known in the art e.g. Lindvall, 0., (1998) Mov. Disord. 13, Suppl. 1:83-7; Freed, CR. , et al . , (1997) Cell Transplant, 6, 201-202; Kordower, et al., (1995) New England Journal of Medicine, 332, 1118-1124; Freed, C. R., (1992
  • composition in accordance with the present invention is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a “prophylactically effective amount” or a “therapeutically effective amount” as the case may be, although prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the CNS includes the brain, the spinal cord, and neurons whose cell bodies lie within, or have a primary synapse in, the brain or spinal cord.
  • Examples of such neurons are neurons of origin of the corticospinal tract, ruborospinal tract and retinal ganglion cells and the CNS branch of sensory axons.
  • a condition associated with neuronal cell loss or damage may be a spinal cord injury, for example an injury caused by assault, accident, tumour, intervertebral disc or bone abnormality, or surgery, e.g. surgery for spinal problems and/or surgery to remove tumours .
  • the neuronal cell loss or damage may be CNS damage other than spinal cord injury, particularly CNS damage of the following kinds: hypoxic injury including stroke and perinatal hypoxia; brain injury, including (without limitation) injury caused by assault, accident, tumour (e.g. a brain tumour or a non-brain tumour that affects the brain, such as a bony tumour of the skull that impinges on the brain) or surgery, e.g. surgery to remove tumours or to treat epilepsy; immune-mediated disease including multiple sclerosis; CNS damage following infection, neurodegenerative diseases and genetic diseases affecting the nervous system, including metabolic disorders (e.g. Gaucher's) and leukodystrophies (e.g. Krabbe's).
  • hypoxic injury including stroke and perinatal hypoxia
  • brain injury including (without limitation) injury caused by assault, accident, tumour (e.g. a brain tumour or a non-brain tumour that affects the brain, such as a bony tumour of the skull that impinges on the brain) or surgery
  • Neurodegenerative diseases may include diseases characterised by the intracellular accumulation of protein aggregates. Aggregates may accumulate, for example, in the cytoplasm of a cell. Commonly, aggregates may form in neuronal cells, such as brain cells, for example in disorders such as Alzheimer's disease, Huntington' s disease, Parkinson's disease, and motor neurone disease (amyotrophic lateral sclerosis) .
  • HNM medium for use in culturing mammalian cells which comprises human serum albumin, human transferrin (30% saturation) , glutathione (reduced) , recombinant human insulin, superoxide dismutase, L-ascorbic acid and N-acetyl-L- cysteine .
  • Human serum albumin may be present at 1.80E-02 to 1.20E-01 mM)
  • human transferrin (30% saturation) may be present at 3.80E-05 to 2.85E-03 mM
  • glutathione (reduced) may be present at up to 1.00E- 02mM
  • recombinant human insulin may be present at 3.51E-04 to 5.26E- 03 mM
  • superoxide dismutase may be present at up to 1.50E-04mM
  • L- ascorbic acid may be present at 5.00E-02 to 2.00E+00 mM
  • N- acetyl-L-cysteine may be present at up to 7.50E+01 mM.
  • recombinant human EGF for example, up to 1.29E- 05 mM
  • recombinant human FGF2 for example, up to 5.77E-06 mM
  • EGF may also be included in some contexts EGF to maintain long-term cell survival and proliferation.
  • the medium allows the neuralising of non-neural stem cells and the propagation of NPCs cultured therein.
  • a suitable HNM medium may comprise the ingredients set out in table 1, each ingredient being independently at a concentration which in the range defined by the maximum and minimum concentrations set out for that ingredient in table 1. Where no maximum and minimum concentrations are provided, the ingredient may be omitted or may be present at a concentration which is within the range of +/-50% of the stated preferred concentration.
  • the HNM medium may comprise the ingredients set out in table 1, wherein one or more, two or more, three or more or preferably all of the ingredients in the group consisting of human serum albumin, human transferring, glutathione (reduced) , recombinant human insulin, superoxide dismutase, L- ascorbic acid and N-acetyl-L-cysteine are at the preferred concentrations stated in table 1.
  • one or more, two or more, three or more or preferably all of the remaining ingredients are also at the preferred concentrations that those ingredients set out in table 1.
  • an HNM medium comprises all the ingredients set out in table 1, with each ingredient being at the concentration which is set out as preferred for that ingredient in table 1.
  • Another aspect of the invention provides the use of an HNM medium as described above in the neuralisation of stem cells or the propagation of NPCs, for example in accordance with a method described herein.
  • a method of screening for a compound for use in the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue may include; (i) bringing a cell produced by a method described herein into contact with a test compound;
  • Cells produced by the present methods include NPCs and fully differentiated neural cell types, such as neurons, astrocytes and oligodendrocytes .
  • a cell may be derived from a naturally occurring stem cell disease lines, for example an hESC line with the genotype of a disease such as Down's syndrome and Huntington' s disease (embryos with these genetic disorders are commonly discarded following IVF) .
  • a cell may be derived from a genetically modified stem cell line, for example a cell line which over-expresses an aggregation-prone protein such as alpha-synuclein, which may be useful in determining neuronal/glial dysfunction and possible responses to test compounds as described below, in the study of protein aggregation disorders, such as Parkinson's disease.
  • the growth, proliferation, function, maintenance, viability or neurotransmitter release of the cell in the presence of test compound may be compared with the growth, function, maintenance, viability or neurotransmitter release of the cell in the absence of test compound.
  • An increase in growth, neurotransmission, maintenance, or viability in the presence relative to the absence of test compound may be indicative that the test compound is useful in the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue.
  • the growth, neurotransmission, or maintenance, or viability of the cell may be determined by any method known to those skilled in the art, for example by cell counting, e.g. by the TUNEL technique or active caspase-3 immunostaining, by BrdU or 3 H-Thymidine incorporation, by monitoring morphology, (e.g. sprouting, axonal elongation and/or branching) , and/or by monitoring biochemistry, (e.g. TH activity, e.g. neurotransmitter uptake/release/content), and/or by gene or protein expression (e.g. immunohistochemistry, PCR, genechip, proteome) and/or by electrophysiological screening using multi-electrode arrays.
  • cell counting e.g. by the TUNEL technique or active caspase-3 immunostaining, by BrdU or 3 H-Thymidine incorporation
  • monitoring morphology e.g. sprouting, axonal elongation and/or branching
  • biochemistry e
  • a screening method may employ any known method for analyzing a phenotypic difference between treated and untreated cells and may be at the DNA, mRNA, cDNA or polypeptide level. Differential screening and gene screening are two such techniques. A substance identified by any of the methods of screening described herein may be used as a test substance in any of the other screening methods described herein.
  • a screening method may employ a nucleic expression array, e.g. a cDNA expression array. In this approach, an array of different nucleic acid molecules is arranged on a filter, quartz or another surface, e.g. by cross-linking the nucleic acid to the filter. A test solution or extract is obtained and the nucleic acid within it is labelled, e.g. by fluorescence.
  • the solution or extract is then applied to the filter or genechip.
  • Hybridisation of the test nucleic acid to nucleic acid on the filter or genechip is determined and compared to the hybridisation achieved with a control solution. A difference between the hybridisation obtained with the test and control samples is indicative of different nucleic acid content.
  • Clontech website e.g. www.clontech.com
  • Affymetrix website e.g. www.affymetrix.com
  • Test compounds may be natural or synthetic chemical compounds used in drug screening programmes and, in particular, may include established compounds used to treat unrelated conditions, whose pharmacological properties have been well-characterised. Extracts of plants which contain several characterised or uncharacterised components may also be used.
  • Combinatorial library technology (Schultz, JS (1996) Biotechnol. Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.
  • test substance or compound that may be added to an assay will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.1 to 100 ⁇ M concentrations of putative inhibitor compound may be used, for example from 1 to 10 ⁇ M.
  • the test substance or compound may be membrane permeable in order to access intracellular targets.
  • the compound may be identified as a compound useful in the treatment of a medical condition associated with degeneration, damage to, the loss of, or a disorder in nervous tissue.
  • a compound identified by any one of the methods provided by the invention may be isolated and/or purified and/or further investigated. It may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug.
  • a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof.
  • the modification of a 'lead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be used to avoid randomly screening large number of molecules for a target property.
  • the optimisation commonly comprises several steps. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the autophagy inducing activity of the lead compound.
  • the modified compounds found by this approach can then be screened to see whether they promote or increase the growth and/or proliferation of the NPCs, or the survival of their differentiated derivatives, or to what extent.
  • test compound may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for any of the purposes discussed elsewhere herein.
  • a method may comprise formulating the test compound into a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier as discussed further above.
  • a method of propagating or expanding a population of hESCs may comprise:
  • Steps (iii) and (iii) may be repeated every 6-10 days.
  • the cell density is reduced at least 2-fold, at least 3 fold or at least 4 fold for each repetition of steps il) and iii) .
  • Suitable differentiation-inhibiting culture media include Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM) or DMEM/F12 supplemented with 20% Serum Replacement or fetal bovine serum, 1% Non-Essential Amino Acids, ImM L-Glutamine, 0. ImM ⁇ -mercaptoethanol and 4ng/ml and 10ng/ml human bFGF.
  • the hESCs may conveniently be cultured on a layer of feeder cells, for example, irradiated mouse embryonic fibroblasts (MEF) at a density of 10 5 -10 6 cells/ 60mm dish.
  • MEF mouse embryonic fibroblasts
  • the stem cell colonies are sliced or sectioned at lOO ⁇ m to 300 ⁇ m intervals in two directions.
  • the mechanical sectioning of stem cell colonies using an automatic sectioning device is described in more detail above.
  • Figure 1 shows a schematic of the culture system with corresponding representative phase micrographs (Scale bar, lOO ⁇ m) .
  • Figure 2 shows representative immune micrographs and graphs showing quantification of the temporal profile of Oct4, SSEA4, Nestin and
  • Figure 3 shows representative immune micrographs and graphs showing quantification of the temporal profile of Oct4, SSEA4, Nestin and Pax ⁇ in HUES-9 cultured in HNM at DO, D8 and D16 demonstrating efficient neuralisation in defined conditions (Scale bar, lOO ⁇ m) .
  • Figure 4 shows the overall expansion of hESC-derived neural precursors over 100 days from one representative hESC plate.
  • Figure 5 shows FACS characterisation and quantification with corresponding immune micrographs of D50 NPCs for the neural progenitor markers Nestin and Musashil.
  • Figure 6 shows an exemplar family of superimposed whole-cell currents from NPC-derived neurons after 30 days of terminal differentiation obtained under voltage-clamp conditions showing voltage-activated, rapidly inactivating inward currents (marked as •) followed by sustained outward currents (marked as ⁇ ) .
  • Figure 8 shows membrane potential recording from the same cell as in figure 6 under fast current-clamp conditions, demonstrating action potential generation.
  • Figure 9 shows a family of superimposed current-clamp recordings of membrane potential from the same cell as in figures 6 and 8.
  • Figure 10 shows a scatter representation of hESC colony size before and after mechanical chopping (bars represent mean values) .
  • Figure 11 shows a graph demonstrating sphere growth following addition of FGF2 at D4, D8 or D12. Addition of FGF2 at D8 is optimal for maximising cell yield without persistence of undifferentiated hESCs (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 12 shows a graph demonstrating cell yield following addition of FGF2 at D4, D8 or D12.
  • Figure 13 shows a graph showing persistence of Oct4 at D16, following addition of FGF2 at D4, D8 or D12.
  • Figure 14 shows a schematic of the automated mechanical passaging technique .
  • Figure 15 shows a scatter representation of total hESC number per fragment analyzed after mechanical and enzymatic methods of passaging. Bars represent median values. •
  • Figure 16 shows a frequency distribution of colony fragment sizes analyzed immediately after passaging, x-axis values represent the average value for each frequency interval
  • Figure 17 shows a graph showing colony sizes of passaged human embryonic stem cell populations at 1, 2, and 3 days after passaging.
  • Figure 18 shows a growth curve showing net expansion of individual colonies in culture after passaging at days 2 and 3 (expressed relative to day 1 after passaging) . Examples Methods hESC culture
  • hESC lines H9 WiCeIl Research Institute, Madison; WI, USA
  • HUES-9 hES facility; Harvard University, Cambridge, MA, USA
  • Cells were cultured as previously described (Valuer, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, CA. et al. N. Engl. J. Med.
  • O.lmM ⁇ -mercaptoethanol (all from Invitrogen; Carlsbad, CA, USA) and 4ng/ml and lOng/ml human bFGF (R&D, Minneapolis, MN, USA) for H9 and HUES-9 respectively.
  • Human ESCs were washed in phosphate buffered saline (PBS) and incubated in lmg/ml collagenase IV (Invitrogen) for approximately 90min until colonies could be easily detached from the underlying feeder layer by gentle pipetting. Detached colonies were placed in a 15ml conical tube and allowed to settle by gravity for 5min, before being transferred with a sterile Pasteur pipette onto the lid of a 60mm culture dish.
  • PBS phosphate buffered saline
  • Invitrogen lmg/ml collagenase IV
  • HNM human neuralising medium
  • hESC colonies and coverslip plate-downs were fixed with 4% fresh paraformaldehyde (PFA) for 20min at room temperature.
  • NPC spheres were fixed with 4% fresh PFA for 30min at 4 0 C, and cryoprotected with 30% sucrose before embedding in OCT compound (BDH; Dorset, UK) .
  • Blocks were sectioned at 12 ⁇ m intervals using a Leica cryostat. Fixed cells and sections were blocked for Ih at room temperature with PBS / 5% goat serum / 0.1% Triton X-100 and then incubated overnight with primary antibody at 4 0 C.
  • NPCs were prepared for FACS analysis as previously described?. NPCs were dissociated with accutase, washed in FACS buffer (PBS / 2% serum / 1OmM sodium azide - PFN) and fixed in ice cold 4 % PFA for 30 min. Cells were incubated with primary antibodies (at similar concentrations to immunocytochemistry) in PFN with 0.14% saponin (Sigma) for 30 min at 4 0 C. Secondary antibodies were applied for 30 min at 4 0 C. Cells were then washed in PFN and analysed using a CyAn flow cytometer (Dako) . Data analysis was performed using Summit v4.2 software (Dako).
  • Colony population growth was calculated by determining the number of cells per colony in 15 randomly selected fields at each time point for every condition. Colony growth curves were constructed by determining the size of 10 randomly selected colonies from each condition (on two separate occasions) 24 hours after passaging (day 1) and at two subsequent 24-hour intervals (days 2 and 3) . Net expansion was calculated by dividing total cell number for a given colony at day 2 or 3 by the total cell number for the same colony at day 1. All figures and error bars are represented as mean _ standard error from the mean unless stated otherwise. For parametric analysis, a twotailed Student's t-test to compare population means and a twotailed F-test to compare population variances were used. A Mann-Whitney rank-sum test to compare median values was performed for nonparametric analysis.
  • Terminally differentiated NPCs on glass coverslips were transferred into a perfusion chamber mounted on the stage of an Olympus CK40 inverted microscope equipped with phase- contrast optics.
  • Cells were continuously perfused at room temperature using a gravity fed system at a rate of 2-4 ml/min with a bath solution containing: 135mM NaCl, 5mM KCl, 1.2mM MgCl2, 2.5mM CaC12, 5mM HEPES and 1OmM glucose with the pH adjusted to 7.4 with NaOH.
  • Somae of cells which displayed a neuronal phenotype were voltage- or current-clamped following establishment of the whole-cell configuration of the patch-clamp technique.
  • Whole cell pipettes had measured tip resistances of 6-8 M ⁇ when filled with a solution containing: 117mM KCl, 1OmM NaCl, 2mM MgSO4, ImM CaC12, HmM EGTA, 2mM Na-ATP and HmM HEPES with the pH adjusted to 7.2 with KOH.
  • Whole cell capacitance was measured and compensated whilst series resistance compensation was employed at 80-85%.
  • cells were current-clamped at OpA and two protocols employed: i) continuous recording of resting membrane potential interspersed with current injection to bring membrane potential about threshold for action potential generation [typically 50-10OpA) and; ii) stepped current injection of 100ms duration from OpA to that which elicited action potential generation and beyond (typically from between 10 to 20OpA) . All recordings were obtained using an Axopatch 700A amplifier (Axon Instruments; Forster City, CA, USA) and a DigiData 1320 A/D interface. Current and voltage protocols were generated by PClamp 9 software and recordings were obtained at a digitisation rate of 10kHz through a 4-pole Bessel low pass filter set at 2kHz.
  • hESC colonies were selectively detached, seeded in HNM and cultured as free-floating embryoid bodies (EBs) in suspension culture.
  • EBs free-floating embryoid bodies
  • Preliminary studies showed that neural specification occurred over approximately 16 days.
  • EBs were heterogeneous in nature. Analysis revealed a correlation between EB size and degree of neurogenesis; small aggregates had a uniform, compact structure and were highly neurogenic, while larger aggregates had a non-uniform internal organisation, with limited neurogenic regions dispersed as rosette islands (Supplemen. This finding is consistent with previous mESC observations showing that low cell plating density in suspension culture is critical for efficient neurogenesis (Bouhon,I.A. et al Brain Res Bull.
  • HNM Whilst HNM efficiently supported the neuralisation of hESCs, culture in HNM alone did not support the longer-term propagation of the hESC-derived neural precursor cells (NPCs) much beyond D16. Therefore, to improve cell yield, we supplemented HNM with human recombinant FGF2, an established mitogen for human fetal-derived neural ⁇ recursorsl3. Since FGF2 contributes to the maintenance of hESC pluripotency (Xu, R. H. et al . Nat. Methods 2, 185-190 (2005)), we first determined the optimal time point for adding FGF2 to HNM to support NPC proliferation. Addition of FGF2 at DO increased total cell yield, but as expected maintained a significant persistence of Oct4 expression ( Figures 11 to 13) . Instead, supplementing cultures with FGF2 from D8 onwards was optimal for maximising NPC yield without contaminant undifferentiated hESCs.
  • NPCs at D16 were dissociated and subsequently propagated in HNM under similar conditions to those used for human fetal-derived neural precursors (Svendsen,C.N. et al . J. Neurosci. Methods 85, 141-152 (1998)); automated mechanical passaging and addition of FGF2 together with EGF resulted in long- term NPC propagation with complete loss of Oct4 / SSEA4 at all subsequent timepoints analysed.
  • NPCs had an overall expansion in excess of 250, 000-fold by DlOO ( Figure 4) and maintained a stable karyotype.
  • the behaviour of hESC-derived NPCs was comparable across H9 and HUES9.
  • NPCs were multipotent and could be differentiated into mature neurons (expressing ⁇ -tubulin, MAP2ab and Synapsinl) with different neurotransmitter subtypes, astrocytes and oligodendrocytes.
  • NPC- derived neurons were electrically active, demonstrating fast, voltage-activated and rapidly inactivating inward currents followed by slowly activated but sustained outward currents (Fig. 6) indicative of voltage-activated sodium currents and delayed- rectifier potassium currents (Fig. 7) , and were able to generate action potentials upon current injection (Figs 8 and 9) .
  • Mcllwain tissue chopper was adapted.
  • the Mcllwain tissue chopper originally developed for sectioning fresh brain tissue, has been successfully used for passaging of human fetal neural precursor cultures (Svendsen CN et al . J Neurosci Methods 1998; 85 : 141-152) .
  • the tissue chopper was modified by removing a small square on the chopper arm to avoid contact with the side of the culture dish.
  • Colony plates were fixed on the chopper stage and automatically sectioned.
  • the Mcllwain tissue chopper allows the sectioning interval to be calibrated between 50 and 950 ⁇ m. Pilot studies identified 200- ⁇ m intervals as optimal for AMP, dividing colonies into approximately 15 fragments, depending on the starting colony size.
  • AMP is rapid, with a single 6-cm dish being passaged in less than 60 seconds.
  • the chopped fragments were readily detached from the culture dish and replated at a ratio of 1:3.
  • a striking observation after AMP was the consistency of fragment size after chopping in contrast to the variability of size produced by conventional collagenase passaging.
  • RT-PCR Semi-quantitative reverse transcription (RT)-PCR did not reveal any significant differences in the expression of POU5F1, NANOG, SOX2, or TERT between the two methods. Immunocytochemistry confirmed that mechanically passaged colonies remained positive for Oct-4 and SSEA-4.
  • the methods described herein therefore provide a method for bulk expansion of hESCs by automated mechanical chopping that is both rapid and maintains greater uniformity of colony size than conventional methods.
  • PAXS AACAGACACAGCCCTCACAAACA CGGGAACTTGAACTeGAACTGAC 25 1.5 0 eo

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Abstract

La présente invention concerne la production et la propagation de cellules précurseurs neurales (NPC) par une combinaison de passage mécanique automatisé et de culture dans un milieu neuralisant humain entièrement humanisé et de culture définie (NNM). Les procédés décrits ici sont susceptibles d'automatisation et d'expansion à une culture en vrac et peuvent être utiles dans la génération de NPC de qualité clinique pour des applications thérapeutiques, ainsi qu'une source de cellules neurales humaines et de glies pour un criblage de médicament haut débit.
PCT/GB2007/002319 2006-06-21 2007-06-21 Procédés de production et de propagation de cellules précurseurs neurales Ceased WO2007148098A2 (fr)

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WO2008056166A3 (fr) * 2006-11-09 2008-07-03 Cambridge Entpr Ltd Différenciation de cellules pluripotentes en cellules progénitrices de couches germinales primaires

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WO2000018414A1 (fr) * 1998-09-29 2000-04-06 Diacrin, Inc. Transplantation de cellules neuronales pour le traitement des lesions ischemiques dues a une attaque

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WO2008056166A3 (fr) * 2006-11-09 2008-07-03 Cambridge Entpr Ltd Différenciation de cellules pluripotentes en cellules progénitrices de couches germinales primaires
US8323971B2 (en) 2006-11-09 2012-12-04 Cambridge Enterprise Limited Differentiation of pluripotent cells into primary germ layer progenitors

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