EP1254211A1 - Production de cellules - Google Patents

Production de cellules

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Publication number
EP1254211A1
EP1254211A1 EP01901033A EP01901033A EP1254211A1 EP 1254211 A1 EP1254211 A1 EP 1254211A1 EP 01901033 A EP01901033 A EP 01901033A EP 01901033 A EP01901033 A EP 01901033A EP 1254211 A1 EP1254211 A1 EP 1254211A1
Authority
EP
European Patent Office
Prior art keywords
cells
neurectoderm
cell
neural
differentiated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01901033A
Other languages
German (de)
English (en)
Other versions
EP1254211A4 (fr
Inventor
Peter David Rathjen
Joy Rathjen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Viacyte Georgia Inc
Original Assignee
Bresagen Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPQ5098A external-priority patent/AUPQ509800A0/en
Priority claimed from AUPQ7045A external-priority patent/AUPQ704500A0/en
Priority claimed from AUPQ7143A external-priority patent/AUPQ714300A0/en
Application filed by Bresagen Ltd filed Critical Bresagen Ltd
Publication of EP1254211A1 publication Critical patent/EP1254211A1/fr
Publication of EP1254211A4 publication Critical patent/EP1254211A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • 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)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/14—Coculture with; Conditioned medium produced by hepatocytes
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to neurectoderm cells and to differentiated or partially differentiated cells derived therefrom.
  • the present invention also relates to methods of producing, differentiating and culturing the cells of the invention, and to uses thereof.
  • the inner cells of the epiblast undergo apoptosis to form the proamniotic cavity.
  • the outer, surviving cells, or early primitive ectoderm continue to proliferate and by 6.0-6.5 dpc have formed a pseudo-stratified epithelial layer of pluripotent cells, termed the primitive or embryonic ectoderm.
  • Primitive ectoderm cells are pluripotent, and distinct from cells of the ICM in terms of morphology, gene expression and differentiation potential.
  • dpc pluripotent cells exposed to the blastocoelic cavity have differentiated to form primitive endoderm.
  • the primitive endoderm gives rise to two distinct endodermal cell populations, visceral endoderm, which remains in contact with the epiblast, and parietal endoderm, which migrates away from the pluripotent cells to form a layer of endoderm adjacent to the trophectoderm. Formation of these endodermal layers is coincident with formation of primitive ectoderm and creation of an inner cavity. Visceral endoderm is known to express signals that influence pluripotent cell differentiation.
  • At gastrulation pluripotent cells of the primitive ectoderm differentiate to form the three germ layers of the embryo: mesoderm, endoderm and ectoderm. Pluripotent cells from this time are confined to the germline. Differentiation of primitive ectoderm cells in the distal and anterior regions of the embryo is directed along the ectodermal lineage forming definitive ectoderm, a transient embryonic cell type fated to form neurectoderm and surface ectoderm.
  • Neurectoderm cells are found in the mammalian embryo in the neural plate, which folds and closes to form the neural tube. These cells are the precursors to all neural lineages. They have the capacity to differentiate into all neural cell types present in the central nervous system (CNS) and peripheral nervous system (PNS). In the CNS these cells include multiple neuron subtypes and glia (eg; astrocytes and oligodendrocytes). Neural cells of the peripheral nervous system also include many different types of neurons and glial cells. Peripheral neural cells differentiate from transient embryonic precursor cells termed neural crest cells, which arise from the neural tube. Neural crest cells are also precursor cells to non-neural cells, including melanocytes, cartilage and connective tissue of the head and neck, and cells of cardiac outflow septation (Anderson, 1989).
  • blastocyst In the human and in other mammals, formation of the blastocyst, including development of ICM cells and their progression to pluripotent cells of the primitive ectoderm, and subsequent differentiation to form the embryonic germ layers and differentiated cells, follow a similar developmental process.
  • Pluripotent cells can be isolated from the preimplantation mouse embryo as embryonic stem (ES) cells.
  • ES cells can be maintained indefinitely as a pluripotent cell population in vitro, and, when reintroduced into a host blastocyst, can contribute to all adult tissues of the mouse including the germ cells. ES cells, therefore, retain the ability to respond to all the signals that regulate normal mouse development.
  • EPL cells are a separate population of pluripotent cells distinct from ES cells. EPL cells are equivalent to early primitive ectoderm cells of the post- implantation embryo, and can be maintained, proliferated and differentiated in a controlled manner in vitro. EPL cells and their properties are described in International patent application WO99/53021.
  • ES cells and EPL cells represent powerful model systems for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for embryo manipulation and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES and EPL cells can be used to generate an unlimited source of cells suited to transplantation for treatment of diseases which result from cell damage or dysfunction.
  • pluripotent cells and cell lines including in vivo or in vitro derived
  • ICM/epiblast in vivo or in vitro derived primitive ectoderm, primordial germ cells (EG cells), teratocarcinoma cells (EC cells), and pluripotent cells derived by dedifferentiation or by nuclear transfer will share some or all of these properties and applications.
  • EG cells primordial germ cells
  • EC cells teratocarcinoma cells
  • pluripotent cells derived by dedifferentiation or by nuclear transfer will share some or all of these properties and applications.
  • the differentiation of murine ES cells can be regulated in vitro by the cytokine leukaemia inhibitory factor (LIF) and other gp130 agonists or by culture on feeder cells which promote self-renewal and prevent differentiation of the stem cells.
  • LIF cytokine leukaemia inhibitory factor
  • Differentiation in vitro of human ES cells is not inhibited by LIF, but is inhibited by culture on feeder cells.
  • Selection procedures have been used to obtain cell populations enriched in neural cells from embryoid bodies. These include manipulation of culture conditions to select for neural cells (Okabe et al, 1996), and genetic modification of ES cells to allow selection of neural cells by antibiotic resistance (Li et al, 1998).
  • Chemical inducers such as retinoic acid have also been used to form neural lineages from a variety of pluripotent cells including ES cells (Bain et al, 1995). However the route of retinoic acid-induced neural differentiation has not been well characterised, and the repetoire of neural cell types produced appears to be generally restricted to ventral somatic motor, branchiomotor or visceromotor neurons (Renoncourt et al, 1998).
  • Neural stem cells and precursor cells have also been derived from foetal brain and adult primary central nervous system tissue in a number of species, including rodent and human (e.g. see United States patent 5,753,506 (Johe), United States patent 5,766,948 (Gage), United States patent 5,589,376 (Anderson and Stemple), United States patent 5,851 ,832 (Weiss et al), United States patent 5,958,767 (Snyder et al) and United States patent 5,968,829 (Carpenter).
  • rodent and human e.g. see United States patent 5,753,506 (Johe), United States patent 5,766,948 (Gage), United States patent 5,589,376 (Anderson and Stemple), United States patent 5,851 ,832 (Weiss et al), United States patent 5,958,767 (Snyder et al) and United States patent 5,968,829 (Carpenter).
  • each of these disclosures fails to describe a predominantly homogeneous population of neural stem cells able to differentiate into all neural cell types of the central and peripheral nervous systems, and/or essentially homogeneous populations of partially differentiated or terminally differentiated neural cells derived from neural stem cells by controlled differentiation.
  • Applicant has surprisingly found that maintaining contact of EPL cells with a conditioned medium as hereinafter described, preferably in cell aggregates grown in suspension, may be used to produce an at least partially differentiated cell type equivalent to embryonic neurectoderm, which can differentiate further to all neural cell types.
  • a method of producing neurectoderm cells which method includes providing a source of early primitive ectoderm-like (EPL) cells; a conditioned medium as hereinafter described; or an extract therefrom exhibiting neural inducing properties; and contacting the EPL cells with the conditioned medium or extract, for a time sufficient to generate controlled differentiation to neurectoderm cells.
  • EPL early primitive ectoderm-like
  • the neurectoderm cells so formed may be characterised in that they are synchronous, homogeneous and their formation parallels the formation of neurectoderm in vivo.
  • the neurectoderm is formed in response to molecules of biological origin and has apparently unrestricted neurectoderm differentiation capability.
  • neurontoderm refers to undifferentiated neural progenitor cells substantially equivalent to cell populations comprising the neural plate and/or neural tube.
  • Neurectoderm cells referred to herein retain the capacity to differentiate into all neural lineages, including neurons and glia of the central nervous system, and neural crest cells able to form all cell types of the peripheral nervous system.
  • the neurectoderm cells so formed may be characterised as "early", for example the neurectoderm cells exhibit neural plate-like characteristics.
  • the neurectoderm cells may be further cultured in a suitable culture medium while neurectoderm cells are formed that may be characterised as "late”, for example the neurectoderm cells exhibit neural tube-like characteristics.
  • suitable culture medium as used herein we mean a culture medium which is suitable for culturing neurectoderm cells.
  • the culture medium excludes foetal calf serum (FCS).
  • FCS foetal calf serum
  • the culture medium does not include the conditioned medium as hereinbefore described.
  • the method includes further providing a suitable culture medium as hereinbefore defined, and further culturing the early neurectoderm cells in the presence of the suitable culture medium while late neurectoderm cells are formed.
  • the late neurectoderm cells so produced exhibit neural tube-like characteristics.
  • the method may include the preliminary steps of providing a source of pluripotent cells, a source of a biologically active factor including a low molecular weight component selected from the group consisting of proline and peptides including proline and functionally active fragments and analogues thereof; and a large molecular weight component selected from the group consisting of extracellular matrix proteins and functionally active fragments or analogues thereof, or the low or large molecular weight component thereof; and contacting the pluripotent cells with the biologically active factor, or the large or low molecular weight component thereof, or the conditioned medium, or the extracellular matrix and/or the low molecular weight component, to produce early .primitive ectoderm-like (EPL) cells.
  • EPL early .primitive ectoderm-like
  • the source of the biologically active factor includes a partially or substantially purified form of the biologically active factor, a conditioned medium including the low and/or large molecular weight component thereof; or an extracellular matrix including a large molecular weight component thereof and/or the low molecular weight component , as described in WO99/53021 above.
  • the pluripotent cells from which the EPL cells may be derived may be selected from one or more of the group consisting of embryonic stem (ES) cells, in vivo or in vitro derived ICM/epiblast, in vivo or in vitro derived primitive ectoderm, primordial germ cells (EG cells), teratocarcinoma cells (EC cells), and pluripotent cells derived by dedifferentiation or by nuclear transfer.
  • EPL cells may also be derived from differentiated cells by dedifferentiation.
  • the step of contacting the pluripotent cells with the biologically active factor, etc. to produce EPL cells may be conducted in any suitable manner.
  • EPL cells may be generated in adherent culture or as cell aggregates in suspension culture. It is particularly preferred that the EPL cells are produced in suspension culture in a culture medium such as Dulbecco's Modified Eagles Medium (DMEM), supplemented with the biologically active factor etc. It is also preferred that there is little or no disruption of cell to cell contact (i.e. trypsinisation).
  • DMEM Dulbecco's Modified Eagles Medium
  • conditioned medium includes within its scope a fraction thereof including medium components below approximately 5 kDa, and/or a fraction thereof including medium components above approximately 10 kDa
  • the conditioned medium is prepared using a hepatic or hepatoma cell or cell line, more preferably a human hepatocellular carcinoma cell line such as Hep G2 cells (ATCC HB-8065) or Hepa-1c1c-7 cells (ATCC CRL- 2026), primary embryonic mouse liver cells, primary adult mouse liver cells, or primary chicken liver cells, or an extraembryonic endodermal cell or cell line such as the cell lines END-2 and PYS-2.
  • the conditioned factor may be prepared from a medium conditioned by liver or other cells from any appropriate species, preferably mammalian or avian.
  • the conditioned medium MEDII is particularly preferred.
  • a neural inducing extract from the conditioned medium may be used in place of the conditioned medium.
  • the neural inducing extract does not include the biologically active factor conditioned medium or the large or low molecular weight component thereof.
  • the term "neural inducing extract” as used herein includes within its scope a natural or synthetic molecule or molecules which exhibit(s) similar biological activity, e.g. a molecule or molecules which compete with molecules within the conditioned medium that bind to a receptor on EPL cells responsible for neural induction.
  • the further culturing step is conducted in the presence of a growth factor from the FGF family.
  • the growth factor from the FGF family when present, may be of any suitable type. Examples include FGF2 and FGF4.
  • EPL cells refers to cells derived from pluripotent cells that retain pluripotency and are converted to and/or maintained as cells that express Oct4 and Fgf5 by:
  • the step of contacting the EPL cells with the conditioned medium may be conducted in any suitable manner.
  • neurectoderm cells may be generated in adherent culture or as cell aggregates in suspension culture.
  • the neurectoderm cells are produced in suspension culture in a culture medium such as DMEM, supplemented with the conditioned medium or extract.
  • a culture medium such as DMEM
  • the cells are cultured for approximately 1 to 7 days, more preferably approximately 3 to 7 days, most preferably approximately 5 days.
  • a conditioned medium as hereinbefore described may be used to derive and maintain the neurectoderm cells or the conditioned medium may be fractionated to yield an extract therefrom exhibiting neural inducing properties, which may be added alone or in combination to other media to provide the neurectoderm cell deriving medium.
  • the conditioned medium may be used undiluted or diluted (e.g. approx. 10-80%, preferably approximately 40-60%, more preferably approximately 50%). After day 3, the conditioned medium is preferably replaced with a suitable culture medium as hereinbefore described.
  • the suitable culture medium also includes a growth factor from the FGF family.
  • concentration of the growth factor from the FGF family is preferably in the range approximately 1 to 100 ng/ml, more preferably approximately 5 to 50 ng/ml.
  • concentration is preferably approximately 20 ng/ml.
  • the method includes the further step of identifying the neurectoderm cells by procedures including gene expression markers, morphology and differentiation potential.
  • EPL cells to neurectoderm cells is characterised by down regulation of expression of Oct4 relative to embryonic stem (ES) cells;and absence of expression of brachyury, and one or more of up regulation of expression of N-Cam and nestin; up regulation of expression of Sox1 and Sox2; and initial up regulation of expression of Gbx2; followed by down regulation thereof as neurectoderm cells persist.
  • ES embryonic stem
  • the upregulation of expression of Gbx2 is indicative of neurectoderm cells having neural plate-like characteristics.
  • the subsequent downregulation of Gbx2 is indicative of cells having neural tube-like characteristics.
  • the neurectoderm cell exhibits three of the above characteristics, more preferably four.
  • N-Cam expressed by all neural lineages
  • nestin an intermediate filament protein expressed by undifferentiated neural stem cells
  • Sox1 and Sox2 expressed in neurectoderm and all undifferentiated neural cells
  • Gbx2 expressed in neural plate and open neural tube.
  • Marker genes which may be used to assess the conversion of pluripotent cells to neurectoderm cells and neural lineages include known markers such as Gbx2, Sox1, Sox2, nestin, N-Cam, Oct4 and brachyury. Markers down regulated during the transition from pluripotent cells to neurectoderm include Oct4. Markers up regulated during this transition include Gbx2, Sox1, Sox2, nestin and N-Cam. Markers not expressed during this transition include brachyury (a marker of mesoderm). As neurectoderm cells persist in culture Gbx2 may be down regulated.
  • the neurectoderm cells according to the present invention may also express the neural genes Otx1, Mashl, En1 and En2. They may also express Pax3 and Pax6.
  • a neurectoderm cell derived in vitro As stated above, in a further aspect of the present invention there is provided a neurectoderm cell derived in vitro.
  • the neurectoderm cell may exhibit two of more of the following characteristics down regulation of Oct4 expression; expression of N-CAM and nestin; expression of Sox1 and Sox2 expression of Gbx2; expression of neural genes, as described above.
  • the neurectoderm cell exhibits initial upregulation of Gbx2 indicative of early neurectoderm cells having neural plate-like characteristics.
  • the neurectoderm cell exhibits subsequent down regulation of Gbx2 indicative of late neurectoderm cells having neural tube-like characteristics, the late neurectoderm being further characterised by the substantial absence of patterning marker expression; and up regulation of neural genes
  • the neurectoderm cell according to the present invention has the capacity to differentiate into all neural lineages.
  • the neurectoderm cell according to the present invention may disperse and differentiate in vivo following brain implantation.
  • the cell is capable of dispersing widely along the ventricle walls and moving to the sub-ependymal layer.
  • the cell is further able to move into deeper regions of the brain, including into the uninjected side of the brain into sites that include the thalamus, frontal cortex, caudate putamen and colliculus.
  • neurectoderm cells During embryogenesis in vivo, neurectoderm cells respond to positional signals that lead to the formation of specific differentiated neural cell types. As part of the response to positional signals, position-dependent gene expression is initiated in neurectoderm cells in restricted locations along the rostro-caudal and dorso-ventral axes within the developing nervous system. These genes serve as markers of positional responses, indicative of future developmental restriction.
  • neurectoderm cells according to the present invention do not express the patterning markers HoxB1, Hoxa7, Krox20, Nkx2.2 and Shh. This may distinguish neurectoderm cells produced according to the present invention from neurectoderm cells produced from other sources including for example foetal and adult primary tissues including neural stem cells. Accordingly, the potential for neural development of these cells is expected to be unrestricted, and thus retain the ability to differentiate into all neural cell types, including neuronal cells, glial cells and neural crest cells.
  • HoxB1 is expressed with Phombomere 4
  • Hoxa7 is expressed in the posterior ectoderm and trunk
  • Krox20 is expressed in early neural plate
  • Nkx 2.2 expressed in the developing forebrain and Shh expressed initially in the ventral midbrain, and later extending in a strip of ventral tissue from the rostral limit of the forebrain to the caudal regions of the spine.
  • Neurectoderm formation in vivo proceeds via the formation of definitive ectoderm.
  • definitive ectoderm Although no markers exist for definitive ectoderm in vivo, gene expression analysis identified a population of cells in EPL cells programmed to form neurectoderm which expressed low levels of Oct4 and failed to express neurectodermal markers. See International patent application "Ectodermal Cell Production", to Applicants, filed on even date. These cells were present transiently between primitive ectoderm (high Oct4 expression, low Sox1, Gbx2 expression) and neurectoderm (high Sox1, Gbx2 expression, low Oct4 expression) and suggest that neurectoderm formation proceeds via an intermediate population which may represent definitive ectoderm.
  • the definitive ectoderm may exhibit substantially no Sox1 expression.
  • a method for maintaining neurectoderm cells in vitro in cell populations that are predominantly homogeneous which method includes providing neurectoderm cells produced as described above; and a suitable culture medium as hereinbefore described; further culturing the neurectoderm cells in the culture medium to form aggregates of neurectoderm cells.
  • the further culturing step begins at day 3 or later.
  • a further aspect of the invention there is provided methods for producing differentiated or partially differentiated cells from neurectoderm cells, which method includes providing neurectoderm cells as hereinbefore described, and a suitable culture medium; further culturing the cells in the presence or absence of a growth factor from the FGF family, and optionally in the presence of additional growth factors and/or differentiation agents, to produce the differentiated or partially differentiated cells.
  • the cells produced are cells selected from the group consisting of neuronal cell precursors, neural crest cells, glial cell precursors, or differentiated neurons or glial cells.
  • the neurectoderm cells may differentiate to form neuronal cells with high frequency.
  • the step of further culturing the neurectoderm cells in the presence or absence of a growth factor from the FGF family and in the presence of additional growth factors and/or differentiation agents may be conducted in any suitable manner.
  • the cellular aggregates or explants cultured in the presence or absence of a growth factor from the FGF family in the presence of additional growth factors and/or differentiation agents may be generated in adherent culture or as cell aggregates in suspension culture.
  • the cells are cultured for approximately a further 3 hours to 10 days, more preferably approximately 1 to 6 days.
  • the concentration of the growth factor from the FGF family if included is preferably in the range approximately 1 to 100 ng/ml, more preferably approximately 5 to 50 ng/ml. When FGF4 or FGF2 is used, its concentration is preferably approximately 10 ng/ml.
  • the neurectoderm cells may differentiate in a controlled manner to form predominantly homogeneous populations of neural crest cells.
  • the additional neurectoderm cell culture step is accordingly preferably conducted in the presence of a Protein Kinase Inhibitor, for example staurosporine.
  • a Protein Kinase Inhibitor for example staurosporine.
  • Staurosporine is a Protein Kinase C inhibitor known to induce neural crest formation from premigratory neural cells in quail (Newgreen and Minichiello, 1996).
  • the conversion of neurectoderm cells to neural crest cells is characterised by a change in morphology, cell migration and up regulation of expression of SoxW.
  • a suitable culture medium may include Ham's F12 nurient mixture containing, e.g. 3% FCS and 10 nm/ml FGF2. Plating may occur onto plasticware coated with cellular fibronectin (e.g. 1 ⁇ g/cm 2 ).
  • Culture medium may be supplemented with for example 1 ⁇ M to 200 ⁇ M staurosporine, preferably 25 ⁇ M staurosporine in a suitable solvent (eg DMSO).
  • a suitable solvent eg DMSO
  • the neurectoderm cells may differentiate in a controlled manner to form predominantly homogeneous populations of glial cells.
  • the differentiation of neurectoderm to glial cells is accordingly preferably conducted in a first stage, in the presence of laminin, an FGF growth factor and an EGF growth factor; and in a second stage, in the presence of a PDGF growth factor and in the substantial absence of EGF, FGF and laminin.
  • neurectoderm aggregates or explants are preferably grown in adherent culture in medium that includes a member of the FGF family (eg; FGF2 or FGF4 10 ng/ml) and EGF (eg; 20 ng/ml) and laminin (eg; 1 to 3 ⁇ g/ml) for ⁇ 3 d then cultured for further 2 to 3 d in the absence of EGF, FGF and laminin and presence of PDGF (eg PDGF-AA, 10 ng/ml).
  • FGF family eg; FGF2 or FGF4 10 ng/ml
  • EGF eg; 20 ng/ml
  • laminin eg; 1 to 3 ⁇ g/ml
  • PDGF eg PDGF-AA, 10 ng/ml
  • the conversion of neurectoderm cell to glial cells is characterised by a change in morphology and up regulation of expresion of the cell surface marker GFAP.
  • a partially differentiated neuronal cell or a terminally differentiated neuronal cell, a partially differentiated neural crest cell, or a terminally differentiated neural crest cell, a partially differentiated glial cell, or a terminally differentiated glial cell, produced by the method described above or derived from neurectoderm cells as hereinbefore described.
  • the cells are present as a predominantly homogeneous population.
  • a substantially homogeneous neural crest cell population obtained in vitro exhibiting two or more of the following characteristics: neural crest cell morphology; cell migration; and expression of SoxW.
  • a substantially homogeneous glial cell population obtained in vitro exhibiting one or both of the following characteristics: glial cell morphology; and expression of the cell surface marker GFAP.
  • the substantially homogeneous glial cell population includes glial cell progenitors and terminally differentiated glial cells.
  • a method of producing genetically modified neurectoderm cells or their partially or terminally differentiated progeny including providing pluripotent cells, a source of early primitive ectoderm-like (EPL) cells; and a conditioned medium as hereinbefore defined, or an extract therefrom exhibiting neural inducing properties modifying one or more genes in the EPL cells; and contacting the genetically modified EPL cells with the conditioned medium or extract to produce genetically modified early neurectoderm cells.
  • EPL early primitive ectoderm-like
  • the method further includes providing a suitable culture medium as hereinbefore defined, and further culturing the early neurectoderm cells in the presence of the suitable culture medium for a time sufficient to form late neurectoderm cells.
  • the further culturing step is conducted in the presence of a growth factor from the FGF family.
  • ES cells may be genetically modified before conversion to EPL cells and differentiation to neurectoderm, or neurectoderm cells or their partially or terminally differentiated progeny may be produced as hereinbefore described and then genetically modified.
  • a method of producing genetically modified neurectoderm cells which method includes providing a source of genetically modified pluripotent cells; a source of a biologically active factor including a low molecular weight component selected from the group consisting of proline and peptides including proline and functionally active fragments and analogues thereof; and a large molecular weight component selected from the group consisting of extracellular matrix portions and functionally active fragments or analogues thereof, or the low or large molecular weight component thereof; a conditioned medium as hereinbefore defined; or an extract therefrom exhibiting neural inducing properties; contacting the pluripotent cells with the source of the biologically active factor, or the large or low molecular weight component thereof, to produce genetically modified early primitive ectoderm-like (EPL) cells; and contacting the genetically modified EPL cells with the conditioned medium or extract to produce genetically modified early neurectoderm cells.
  • EPL early primitive ectoderm-like
  • the method preferably further includes providing a suitable culture medium as hereinbefore defined, and further culturing the early neurectoderm cells in the presence of the suitable culture medium for a time sufficient to form genetically modified late neurectoderm cells.
  • the further culturing step is conducted in the presence of a growth factor from the FGF family.
  • Modification of the genes of these cells may be conducted by any means known to the skilled person which includes introducing extraneous DNA, removing DNA or causing mutations within the DNA of these cells. Modification of the genes includes any changes to the genetic make-up of the cell thereby resulting in a cell genetically different to the original cell.
  • the genetically modified or unmodified neurectoderm cells of the present invention and the differentiated or partially differentiated cells derived therefrom are well defined, and can be generated in amounts that allow widespread availability for therapeutic and commercial uses.
  • the cells have a number of uses, including the following:
  • neurodegenerative disorders such as Parkinson's disease, Huntington's disease, lysosomal storage diseases, multiple sclerosis, memory and behavioural disorders, Alzheimer's disease and macular degeneration, and other pathological conditions including stroke and spinal chord injury.
  • neurodegenerative disorders such as Parkinson's disease, Huntington's disease, lysosomal storage diseases, multiple sclerosis, memory and behavioural disorders, Alzheimer's disease and macular degeneration, and other pathological conditions including stroke and spinal chord injury.
  • genetically modified or unmodified neurectoderm cells or their differentiated or partially differentiated progeny may be used to replace or assist the normal function of diseased or damaged tissue.
  • Parkinson's disease the dopaminergic cells of the substantia nigra are progressively lost.
  • the dopaminergic cells in Parkinson's patients could be replaced by implantation of neurectodermal neural cells, produced in the manner described in this application.
  • neural crest cells retain the capacity to form non-neural cells, including cartilage and connective tissue of the head and neck, and are potentially useful in providing tissue for craniofacial reconstruction.
  • neurectoderm cells or their differentiated and partially differentiated products may be genetically modified; eg; so that they provide functional biological molecules.
  • the genetically modified cells can be implanted, thus allowing appropriate delivery of therapeutically active molecules.
  • karyoplasts from neurectoderm or their differentiated or partially differentiated progeny may be reprogrammed by nuclear transfer.
  • Cytoplasts from neurectodermal cells may also be used as vehicles for reprogramming so that nuclear material derived from other cell types are directed along neural lineages.
  • neural stem cells may be reprogrammed in response to environmental and biological signals that they are not normally exposed to.
  • haematopoietic cells cells of mesodermal lineage
  • neurectoderm cells described herein are potentially capable of forming differentiated cells of non-neural lineages, including cells of mesodermal lineage, such as haematopoietic cells and muscle.
  • Reprogramming technology using neural cells potentially offers a range of approaches to derive cells for autologous transplant.
  • karyoplasts from differentiated cells are obtained from the patient, and reprogrammed in neurectoderm cytoplasts to generate autologous neurectoderm.
  • the autologous neurectoderm cells or their differentiated or partially differentiated progeny could then be used in cell therapy to treat neurodegenerative diseases. See Australian provisional patent applications PR1348 and PR2126, the entire disclosures of which are incorporated herein by reference.
  • neurectoderm could be further dedifferentiated to a pluripotent state by fusion with pluripotent cytoplasts, and subsequently directed along alternative differentiation pathways to form cells of mesodermal or endodermal lineage.
  • Neurectoderm cells may be particularly appropriate in evaluating the toxicology and teratogenetic properties of pharmaceutically useful drugs, since many birth defects, including spina bifida are caused by failures in neural tube closure.
  • a method for the treatment of neuronal and other diseases includes treating a patient requiring such treatment with genetically modified or unmodified neurectoderm cells as described above, or their partially differentiated or terminally differentiated progeny, through human or animal cell or gene therapy.
  • a method for the preparation of tissue or organs for transplant which method includes providing neural crest cells or neurectoderm produced as described above; and culturing the neural crest cells to produce neural or non-neural cells and the neurectoderm cells to produce neural cells.
  • A-D 7 ⁇ m sections of paraffin embedded EBM 4 (A, B) and EB 4 (C, D) stained with haematoxylin;eosin (A, C) and Hoescht 22358 (B, D).
  • E. 20 ⁇ g RNA from EB 2"4 and EBM 2'4 was analysed for the expression of Fgf5, brachyury, Oct4 and mGAP by Northern blot analysis.
  • Fgf5 transcripts were 2.7 and 1.8 kb (Herbert et al., 1990), brachyury 2.1 kb (Lake et al., 2000), Oct4 1.55 kb (Rosner et al., 1990) and mGAP 1.5 kb.
  • EBM 7 A and EBM 9 (B). C. 7 ⁇ m section of paraffin embedded EBM 9 stained with haematoxylin. D. 20 ⁇ g RNA from EB 4"8 and EBM 4'8 was analysed for the expression of Oct4 and mGAP. Oct4 transcripts were 1.55 kb (Rosner et al., 1990) and mGAP 1.5 kb. E. In situ hybridisation analysis of seeded EBM 7 two days post-seeding with deoxygenin labelled antisense probes for Oct4.
  • EDII induces neuron formation from EPL cells
  • EBM comprise a homogeneous population of neurectoderm
  • A, B. EBM 9 were analysed by wholemount in situ hybridisation for the expression of Sox1 (A) and Sox2 (B). Stained EBM9 were embedded in paraffin wax and 7 ⁇ m sections analysed for homogeneity of gene expression.
  • C. Flow cytometry of dissociated EBM 10 and EB 10 analysed for expression of the cell surface antigen NCam. NCam positive cells were identified by comparison with cell populations that had been stained with secondary antibody only (data not shown). Cells staining with intensities greater than the secondary antibody only control were determined to be expressing NCam, and are indicated by the bar.
  • Gbx2 is temporally regulated during EBM differentiation
  • cDNA was synthesised from 1 ⁇ g of total RNA isolated from EB 9 (EB),
  • EPLEB 9 EPLEB
  • EBM 9 EBM
  • day 10 mouse embryo day 10
  • Actin Actin was used as a positive control. Primer sequences and product sizes can be found in example 3.
  • ES cell-derived neurectoderm can be directed down neural crest and glial lineages
  • EBM 9 explants were seeded onto cellular fibronectin treated tissue culture plasticware in medium supplemented with 25 nM staurosporine/ 0.1% DMSO (A, C, D) or 0.1% DMSO alone (B). Cultures were examined after 3 (A, B) or 48 hours (C, D). In situ hybridisation analysis of EBM 9 explants with deoxygenin labelled antisense probes for Sox10 (D). E-H.
  • EBM 9 explants were seeded onto poly-L-ornithine treated tissue culture plasticware in medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 1 ⁇ g/ml laminin (E, F) followed by culture in medium supplemented with 10 ng/ml PDGF-AA (G, H). Cultures were examined after 2 (A) or 4 (F), and 6 (G, H) days.
  • GFAP glial fibrillary acidic protein
  • the black represents areas where cells were identified up to 4 weeks post injection and the white represents regions of the brain where the cells were located at times up to 16 weeks post injection. Note that, the two dimensional slice through the rat brain does not depict all the labeled regions.
  • ES cell lines E14 Hooper et al., 1987
  • D3 Doetschman et al., 1985
  • Routine culture of ES and EPL cells and production of MEDII and sfMEDII conditioned medium were as described in Rathjen et al. (1999).
  • D3 ES cells expressing enhanced green fluorescent protein (EGFP; Clontech) under the control of the constitutive EF1 promoter were formed by transfection with pFIRES+EGFP (obtained from Dr. S. Dalton, Department of Molecular Biosciences, Sydney University, Australia).
  • pFIRES+EGFP obtained from Dr. S. Dalton, Department of Molecular Biosciences, Sydney University, Australia.
  • 5x10 6 D3 ES cells in 0.5 ml HBS + glucose (20 mM HEPES, 140 mM NaCI, 5 mM KCl, 7 mM Na 2 HP0 4 , 6 mM glucose, 0.1 mM ⁇ -mercaptoethanol ( ⁇ -ME)) were transfected by electroporation with 15 ⁇ g plasmid DNA (960 ⁇ Fd; 210 V) using a BioRad Gene Pulser.
  • IC DMEM + LIF Dulbecco's Modified Eagles Medium (DMEM; Gibco BRL #12800) supplemented with 10% foetal calf serum (FCS; Commonwealth Serum Laboratories), 40 mg/ml gentamycin, 1 mM L-glutamine, 0.1 mM ⁇ -ME and 1000 units of LIF).
  • FCS foetal calf serum
  • FCS foetal calf serum
  • Stable transformants were picked after 8 days of selection. Single colonies were picked, expanded and assessed morphologically for the expression of EGFP in pluripotent cells and differentiated derivatives.
  • EGFP fluorescence was detected on a Nikon TE300 inverted microscope using a FITC filter.
  • EBM cell aggregates formed and maintained in MEDII, were formed from ES cells aggregated in IC:DMEM (DMEM (Gibco BRL #12800) with 10% foetal calf serum (FCS; Commonwealth Serum Laboratories), 40 mg/ml gentamycin, 1 mM L-glutamine and 0.1 mM ⁇ -mercaptoethanol ( ⁇ -ME)) supplemented with 50% MEDII.
  • DMEM DMEM (Gibco BRL #12800) with 10% foetal calf serum (FCS; Commonwealth Serum Laboratories), 40 mg/ml gentamycin, 1 mM L-glutamine and 0.1 mM ⁇ -mercaptoethanol ( ⁇ -ME)
  • EB 7 and EBM 7 were seeded as described above and assessed on days 8, 10, 12 and 14 for the presence of neurons, identified morphologically by the presence of axonal projections (and confirmed by the expression of NF200; data not shown), and beating cardiocytes, identified morphologically by rhythmical contraction of cells within the aggregate.
  • Cytoplasmic RNA was isolated from cellular aggregates using the following method.
  • Cellular aggregates were resuspended in 1 ml extraction buffer (50 mM NaCI, 50 mM Tris.CI pH 7.5, 5mM EDTA pH 8.0 and 0.5% SDS) and acid washed glass beads (40 mesh; BDH) were added to the meniscus.
  • Suspensions were vigorously vortexed before the addition of a further 3 ml of extraction buffer and 200 ⁇ g/ml proteinase K (Merck) and incubation at 37°C for 60 minutes.
  • One tenth volume of 3M sodium acetate was added before the suspension was phenol:chloroform extracted.
  • the aqueous phase was added to an equal volume of iso-propanol, chilled to -80°C for 30 minutes and the nucleic acids pelleted by centrifugation (Jouan bench centrifuge, 3,000 rpm). The pellet was resuspended in DNase I digestion buffer (50 mM Tris.CI pH 8.0, 1 mM EDTA pH 8.0, 10 mM MgCI, 0.1 mM DTT) and 20 units of RNase free DNase I (Boehringer Mannhiem) added. After incubation at 37°C for 60 minutes nucleic acids were phenol:chloroform extracted and ethanol precipitated. Northern blot analysis was performed as described in Thomas et al. (1995). DNA probes were prepared from DNA fragments using a Gigaprime labelling kit (Bresagen). DNA fragments used were as described in Rathjen et al. (1999) and Lake et al. (2000).
  • Sox2 transcripts were generated from a 748 bp Accl/Xbai cDNA fragment cloned into pBluescript SK (obtained from Dr. R. Lovell-Badge, Division of Developmental Genetics, National Institute for Medical Research, Mill Hill, London). Transcripts were generated from Accl and Xba ⁇ linearised plasmid transcribed with T3 (anti-sense) and T7 (sense) RNA polymerases respectively.
  • EB 4 and EBM 4 were fixed with 4% PFA for 30 minutes before embedding in paraffin wax and sectioning as described in Hogan et al., 1994. 7 ⁇ m sections were stained with haematoxylin:eosin as described by Kaufman, 1992 or with Hoescht 22358 (5 ⁇ g/ml in PBS; Sigma) for 5 minutes.
  • FITC conjugated secondary antibodies For FITC conjugated secondary antibodies, cellular aggregates were washed in PBS and mounted in 80% glycerol containing 5 mg/ml propyl gallate (Sigma). Aggregates were examined on a Nikon TE300 microscope using a FITC filter.
  • Nestin Blocking buffer: 10% goat serum, 2% BSA in PBS.
  • Primary antibody Developmental Studies Hybridoma Bank, reference Rat 401, used at a dilution of 1 :100.
  • Secondary antibody alkaline phosphatase conjugated goat anti- mouse IgG (affinity purified, Rockland) used at a concentration of 1:100.
  • N-Cam Blocking buffer 1% FCS, 1 mg/ml BSA, 1% Triton X 100 in PBS.
  • Primary antibody Santa Cruz Biotech, SC-1507 used at a concentration of 1 :20.
  • Secondary antibody FITC conjugated goat anti-mouse IgM ( ⁇ -specific: Sigma) used at a dilution of 1 :700.
  • NF200 Blocking buffer: 10% goat serum, 2% BSA in PBS
  • Primary antibody anti-neurofilament 200 (Sigma Immunochemicals N-4142) used at a dilution of 1 :200.
  • Secondary antibody alkaline phosphatase conjugated goat anti- rabbit IgG (ZyMaxTM grade, Zymed Laboratories Inc.)
  • Embryonic stem (ES) cells cultured in the presence of medium conditioned by the human hepatocellular carcinoma cell line HepG2 (MEDII) have been shown to form a second pluripotent cell population, EPL cells (Rathjen et al., 1999). EPL cells demonstrate morphology, gene expression, differentiation potential and cytokine responsiveness distinct from ES cells but characteristic of the post- implantation pluripotent cell population of the mouse embryo, primitive ectoderm (Rathjen et al., 1999.
  • ES cells can be aggregated in suspension culture.
  • cytokines such as LIF
  • aggregated ES cells form structures termed embryoid bodies (EB), which recapitulate many aspects of cell differentiation during early mammalian embryogenesis (Doetschman et al., 1985; Shen and Leder, 1992.
  • EB embryoid bodies
  • Outer cells form extraembryonic endoderm and derivatives while inner cells undergo processes equivalent to formation of the proamnioatic cavity (Coucouvanis and Martin, 1995) and primitive ectoderm (Shen and Leder, 1992), followed by pluripotent cell differentiation into differentiated tissues derived from all three germ layers.
  • ES cells were aggregated in medium supplemented with 50% MEDII (EBM) and compared to EB development.
  • EBM MEDII
  • EBM 4 After 4 days cellular aggregates formed in the presence of MEDII (EBM 4 ) could be distinguished from EB 4 by morphology. Histological analysis of sectioned EB 4 and EBM 4 showed EBM 4 to comprise a multi-cell layer of uniform thickness surrounding a single, internal area of cell death indicated by the presence of pyknotic nuclei ( Figure 1A, B). No morphologically distinct outer layer of cells reflecting the presence of extra- embryonic endoderm could be detected at this or later stages of EBM development. In contrast, EB 4 were internally disorganised with sporadic, multiple foci of cell death dispersed throughout the aggregates ( Figure 1C, D). An outer layer of extraembryonic endoderm was apparent at low levels in EB 4 and at higher levels in more advanced EB (data not shown).
  • EB 2"4 and EBM 2"4 were analysed by Northern blot ( Figure 1E) for the expression of Oct4, a marker gene for pluripotent cells (Scholer et al., 1990), and Fgf5, a gene up-regulated in pluripotent cells upon primitive ectoderm formation (Haub and Goldfarb, 1991).
  • Oct4 expression was maintained at high levels throughout these stages of EBM development indicating that pluripotent cell differentiation had not commenced within these aggregates.
  • High level Oct4 expression in EBM 4 was accompanied by elevated Fgf5 expression, indicating that the pluripotent cells had formed primitive ectoderm.
  • highest levels of Oct4 and Fgf5 expression in EB were observed at days 2-3 and day 3 respectively. Downregulation of both genes in EB 4 indicated that pluripotent cells within these aggregates had differentiated.
  • brachyury a marker for nascent mesoderm (Herrmann, 1991), was used to confirm the onset of mesodermal differentiation in the aggregates.
  • Brachyury expression was analysed in EBM 2"4 and EB 2"4 by Northern blot ( Figure 1 E) and in EBM 4 and EB 4 by wholemount in situ hybridisation ( Figure 1 H, K).
  • EB brachyury expression was upregulated on day 4 of development, coincident with the loss of pluripotence in the aggregates.
  • brachyury expression could not be detected by either method in EBM 2"4 , consistent with the maintenance of Oct4 expression and suggesting a lack of differentiation within these aggregates.
  • EBM comprise a homogeneous population of pluripotent cells that have acquired primitive ectoderm-like gene expression with no detectable associated differentiation.
  • MEDII has been shown to contain 50-100 units of human LIF (Rathjen et al., 1999). LIF has been shown to retard the developmental progression of EB in vitro (Shen and Leder, 1992). ES cells aggregated and maintained in medium supplemented with 100 units of LIF did not duplicate the morphology or gene expression profile of EBM (data not shown), indicating the importance of additional secreted factors contained within MEDII (Rathjen et al., 1999) for EPL cell induction.
  • EBM 9 As the morphology of EBM 9 was clearly reminiscent of neurectoderm (Figure 2C), the expression of a number of neural markers was analysed. EBM 7 were seeded onto gelatin treated tissue culture grade plasticware in DMEM containing 10% FCS and the medium was changed to 50% DMEM:50% Hams
  • Sox1 has been shown to delineate the neural plate and is expressed by all undifferentiated neural cells, while Sox2 shows a similar expression pattern but is expressed earlier in embryogenesis (Pevney et al., 1998). Seeded aggregates were also analysed by immunohistochemistry using antibodies directed against nestin, a neurofilament protein expressed in neural progenitor cells (Zimmerman et al., 1994) and N-Cam, a cadherin expressed within the neural system by primitive neurectoderm, neurons and glia (Rutihauser, 1992).
  • EBM 7 were seeded as described above and assessed on days 8, 10 and 12 for the presence of beating cardiocytes, a differentiated mesoderm derivative, and neurons, a differentiated ectoderm derivative.
  • the differentiation of EBM was compared to EB ( Figure 3E, F). Consistent with the up-regulation of neural specific markers, and lack of brachyury expression, neurons could be seen in the differentiated products of the majority of EBM (91.33%). However, ⁇ 2% of EBM formed beating cardiocytes. In contrast, EB comprised a mixed population which differentiated into both beating cardiocytes (54.5%) and neurons (24.9%) on day 12 respectively.
  • EBM 4 which comprise an homogeneous population of EPL cells
  • MEDII programs differentiation of the pluripotent cells to an ectodermal and neurectodermal fate.
  • the great majority of differentiated cells at day 9 expressed neural markers such as Sox1, Sox2, nestin and N-Cam, and spontaneous differentiation resulted in efficient formation of neurons.
  • Neuron formation was increased to >80% of aggregates, with an average of 92.13% +/- 1.944.
  • This result suggests that response to MEDII by differentiation to ectodermal lineages is an inherent property of ES cells and not restricted to a subpopulation within the ES cell population.
  • MEDII-directed differentiation of ES cells as EBM overcame the inherent variability associated with EB differentiation and resulted in uniform, high level production of neurectoderm and neurons.
  • EPL cells differentiate to form neurons in response to MEDII
  • EPL cells form neurons poorly, if at all, when differentiated as EB but form elevated levels of nascent and differentiated mesoderm (see International patent application PCT/AU99/00265, above). This has been interpreted as reflecting disrupted signalling from visceral endoderm or visceral endoderm-derived ECM (Lake et al, 2000).
  • EPL cells were formed from ES cells as described, and aggregated and cultured in suspension for 7 days in either IC:DMEM (EPLEB) or IC:DMEM supplemented with 50% MEDII (EPLEBM).
  • EPLEB IC:DMEM
  • EPLEBM 50% MEDII
  • EPL cell embryoid bodies formed beating cardiocytes efficiently (35.25%), consistent with previous reports and gene expression (Lake et al., 2000).
  • EPL cell embryoid bodies cultured in the presence of 50% MEDII exhibited drastically lower levels of beating cardiocyte formation (0.9%).
  • Aggregation and differentiation of EPL cells in the presence of MEDII resulted in an up regulation in neuron formation from very low levels in EPLEB (3.6%), to 83.73% in EPLEBM (Figure 5B).
  • pluripotent cells can be programmed specifically to an ectodermal and neurectodermal fate by factors within MEDII.
  • the ectodermal cells are formed in the absence of mesodermal cell types, and exhibit a temporal pattern of gene expression equivalent to neurectoderm in vivo. In the embryo these cells are precursors for all neural lineages.
  • neurectoderm described here does not rely on the addition of chemical inducers, such as retinoic acid, or genetic manipulation to promote neural formation. Instead, it relies on biologically derived factors found within the conditioned medium MEDII. Neural progenitors formed in this manner are thought to be differentiated from pluripotent cells in a manner analogous to the formation of neural cells during embryogenesis and are therefore ideal for the production of differentiated neural cells useful for commercial, medical and agricultural applications. Further, in contrast to the formation of limited neural lineages by chemical inducers such as retinoic acid, the identity of neural cell types produced using these methodologies is not likely to be developmentally restricted.
  • EBM comprise a homogeneous and synchronous population of ES cell- derived neural progenitors
  • EBM EB and EBM were aggregated and cultured as described in example 1. Gene expression analysis was as described in example 1. EBM which had been analysed by whole mount in situ hybridisation staining were prepared for histochemical analysis as follows. Stained EBM were fixed in 4% PFA overnight, washed several times with PBS, 0.1% Tween-20, treated with 100% methanol for 5 minutes and then isopropanol for 10 minutes. Bodies were then treated and embedded as described in Hogan et al. (1994).
  • DIG labelled Gbx2 riboprobes were generated from pG290 which contains a 290 bp PCR fragment from base 780 to base 1070 of the Gbx2 cDNA
  • EB 10 and EBM 10 were collected and washed in PBS, then disassociated by incubating for 5 minutes in 0.5 mM EDTA/PBS followed by vigorous pipetting and agitation to a single cell suspension. Cells were washed several times in PBS before fixation with 4% PFA for 30 minutes. Cells were washed with 1 % BSA/PBS, resuspended at 1 x 10 6 cells/ml, and incubated with antibody directed against N- Cam (Santa Cruz Biotech, SC-1507) at a dilution of 1 :2 for 1 hour.
  • FITC conjugated goat anti- mouse IgM ( ⁇ -specific: Sigma) used at a concentration of 1 :100.
  • FITC conjugated goat anti-mouse IgM was pre-adsorbed for 1 hour in 1% BSA/PBS before use. Cells were washed in PBS and fixed in 1% PFA for 30 minutes. Data was collected on 1 x 10 4 cells on a Benton Dickonson FACScan and analysis performed using CellQuest 3.1.
  • EBM comprise a homogeneous population of neural progenitor cells
  • EBM 9 were probed by wholemount in situ hybridisation for Sox1 and Sox2 expression and histological sections were examined for homogeneity of expression. Representative sections ( Figure 6A, B) showed that EBM 9 comprised a morphologically uniform population of cells equivalent to the neurectoderm-like monolayer described in example 1 , in which each cell stained positive for expression of Sox1 and Sox2.
  • EBM 10 and EB 10 were disaggregated to a single cell suspension, labelled immunocytochemically with antibodies directed against N-Cam, a cell adhesion molecule expressed strongly in the nervous system (Ronn et al., 1998) and analysed by FACS analysis (Figure 6C). 95.7% of cells from EBM 10 were scored positive for N-Cam expression, demonstrating relatively uniform differentiation of these aggregates to neural lineages. In comparison, only 42.13% of cells from EB 10 expressed N-Cam, consistent with the established heterogeneity of ES cell differentiation within this system.
  • Neural formation within EBM is relatively synchronous and reflects the temporal formation of neural lineages in the embryo
  • the neural plate which contains the earliest neural precursors, is characterised by expression of Sox1 within a group of cells on the anterior midline of the embryo (Pevney et al., 1998). This population of cells also expresses the homeobox gene Gbx2 (Wassarman et al., 1997). With continued development the neural plate folds at the midline and the outer edges close to form the neural tube. Sox1 expression is maintained after tube closure but Gbx2 expression is down regulated in the majority of cells of the neural lineage and persists only in a restricted population of cells at the mid- brain/hind-brain boundary (Wassarman et al., 1997).
  • EBM differentiation indicates that differentiation of ES cells as EBM results in the formation of a homogeneous population of neural progenitors.
  • Temporal regulation of gene expression indicated relative synchrony of differentiation within and between EBM aggregates, and was conserved in many aspects with formation of the ectodermal/neurectodermal lineages during mammalian embryogenesis. EBM differentiation therefore recapitulates progressive formation of neural plate and neural tube, progenitors for the entire nervous system, from pluripotent cells in the mammalian embryo.
  • EB and EBM were formed and cultured as described in example 1.
  • Gene expression analysis was as described in example 1.
  • PCR was performed using Platinum PCR Supermix (Gibco BRL) following the manufacturers instructions. Reactions were performed in a capillary thermocycler (Corbett Research), with cycling parameters as follows; denaturing 94°C, 10 seconds, annealing 55°C, 10 seconds and extension 72°C, 60 seconds. Cycling times were determined for each primer set to be within the exponential phase of amplification.
  • Primers for amplification of actin, En-1, Hoxa7 and Otx1 have been described previously (Okabe et al., 1996). Primer sequences and the length of amplified products were as follows:
  • PCR products were analysed on 2% agarose gels and visualised with ethidium bromide.
  • the neural tube acquires region specific gene expression with respect to both the rostral/caudal and dorsal/ventral axes, indicative of restricted developmental fate.
  • Expression of neural tube markers expressed in the neural tube shortly after closure in restricted anterior, posterior and ventral domains was analysed in EBM 9 , which expresses Sox1 and Sox2 but not Gbx2, equivalent to closed neural tube in vivo.
  • the ectodermal expression patterns of the analysed genes are described in table 1.
  • EBM 9 Gene expression in EBM 9 was analysed by RT- PCR or in situ hybridisation (Gbx2) and compared to EB 9 and EPLEB 9 , which comprise a mixed population of cells containing ectoderm and mesoderm, and a mesoderm-enriched, ectoderm deficient (International patent application PCT/AU99/00265, above) population respectively.
  • RNA from d10 embryos was used as a positive control.
  • En1, En2 and Otx1 are expressed in a broad region of the anterior neural tube around the time of closure and subsequently within defined regions of the midbrain.
  • These genes were expressed in EBM 9 as was Mashl, a gene expressed in domains of the neuroepithelum of the forebrain, midbrain and spinal cord between days 8.5 and 10.5 (Guillemot and Joyner, 1993).
  • Pax3 and Pax ⁇ were expressed in EBM 9 . While these genes are restricted positionally to dorsal and ventral aspects of the neural tube respectively, evidence from chick (Goulding et al., 1993) suggests that both these genes are expressed widely in neural tube before their expression domains become restricted in response to ventral specification.
  • EBM therefore provide a superior system for the generation of neural progenitors from pluripotent cells compared to chemical induction or differentiation within EB in which positionally restricted genes are expressed.
  • Differentiation of ES cell-derived neurectoderm can be directed to neural crest or glial lineages in response to exogenous signalling
  • Sox 10 probes were transcribed from pSox10E.1 (obtained from Dr. Peter Koopman, 1MB, Brisbane, Australia). Anti-sense and sense probes were transcribed from Hindlll or BamHI cut pSox10E.1 with T7 or T3 RNA polymerase respectively.
  • Anti-glial fibrillary acidic protein (GFAP: Sigma #G9269) was used at a dilution of 1/1000 and detected with alkaline phosphatase conjugated goat anti- rabbit IgG (ZyMax grade, Zymed Laboratories Inc.) used at a dilution of 1/1000. Cells were blocked for 30 minutes in 10% goat serum, 2% BSA in PBS.
  • EBM 9 were collected, washed in PBS, treated with 0.5 mM EGTA pH 7.5 for 3 minutes, washed in PBS and disaggregated to small clumps (20-200 cells) by trituration. Cell clumps were allowed to settle and single cells liberated during trituration were removed with the supernatant before plating onto tissue culture grade plasticware which had been coated with cellular fibronectin (1 ⁇ g/cm 2 ; obtained from M. D. Bettess, Department of Biochemistry, Sydney University, Australia) and allowed to dry.
  • Cells were cultured in Hams F12 containing 3% FCS and 10 ng/ml FGF2 and supplemented with either 0.1% 25 ⁇ M staurosporine (Sigma) in DMSO (final concentration, 25 nM) or 0.1% DMSO. Cellular aggregates were allowed to differentiate for 48 hours before fixation in 4% PFA for 30 minutes.
  • EBM 9 were collected, washed in PBS and broken into small clumps as described above.
  • Cell clumps were transferred to tissue culture plastic pretreated with poly-L-Omithine as per manufacturer's instructions (Sigma) and cultured in 50% DMEM, 50% F12, 1 x ITSS, 1 x N2 supplement (Sigma), 10 ng/ml FGF2, 20 ng/ml EGF (R&D Systems Inc.) and 1 ⁇ g/ml Laminin (Sigma).
  • Medium was changed daily. After 5 days medium was changed to 50% DMEM, 50% F12, 1 x ITSS, 1 x N2 supplement (Sigma), 10 ng/ml FGF2 and 10 ng/ml PDGF-AA (R&D Systems Inc.). Cells were fixed for analysis on day 7 or 8 of culture by treatment with 4% PFA for 30 minutes.
  • EBM 9 were dissociated to clumps and cultured in medium supplemented with either 0.1% of 25 M staurosporine in DMSO (final concentration, 25 nM) or 0.1% DMSO. Cell types produced were assessed morphologically and for expression of the mammalian neural crest marker, Sox10 (Southard-Smith et al., 1998).
  • EBM 9 explants were cultured in medium containing FGF2 (10 ng/ml),
  • EGF (20 ng/ml) and laminin (1 ⁇ g/ml). After 5 days EGF and laminin were omitted from the medium and PDGF-AA was added to a concentration of 10 ng/ml for a further 2-3 days. Cells were not trypsinised or triturated during differentiation. Cultures were analysed by immunohistochemistry for the expression of glial fibrillary acidic protein (GFAP), a marker expressed by both glial precursors and differentiated astrocytes (Landry et al., 1990). Differentiation of EBM 9 explants in response to EGF/laminin and PDGF-AA follows a homogeneous morphological progression depicted in Figure 8E-G. >95% of differentiated cells formed from EBM explants using this protocol expressed GFAP ( Figure 8H), indicating homogeneous differentiation to cells of the glial lineage.
  • GFAP glial fibrillary acidic protein
  • MEDII can be directed to neural crest or glial fates by the additional of biologically relevant exogenous signalling molecules. This indicates that neurectoderm derived by differentiation of pluripotent cells in response to MEDII has differentiation properties equivalent to neurectoderm in vivo.
  • Neural crest cells are precursors in vivo to craniofacial bone and cartilage, and several different types of neurons including sensory cranial nerves, parasympathetic, sympathetic and sensory ganglia. Terminally differentiated glial cells are expected to have wide ranging biological and medical applications including the treatment of neuronal diseases using cell and gene therapy.
  • Unpatterned neural progenitors produced by differentiation of pluripotent cells in response to MEDII can therefore be used as a superior substrate for directed formation of ectodermal lineages of medical importance.
  • Neural progenitors derived by differentiation of pluripotent cells in response to MEDII are capable of incorporation and differentiation in the rat brain.
  • D3 ES cells expressing EGFP were formed by the transfection of D3 ES cells with pFIRES+EGFP (Example 1), and used throughout. Routine tissue culture and maintenance of ES cells was as described in Example 1. EBM were formed and cultured as described in Example 1.
  • EBM 7 and EBM 10 were allowed to settle and media was aspirated.
  • Cell aggregates were washed with 10ml PBS, and treated with 0.5mM EGTA pH7.5 for 3 minutes. This was removed and aggregates were treated with 3ml of trypsin (0.05%, Gibco, UK) /EDTA (0.5mM, Gibco, UK) for 1 minute.
  • 1 ml of FCS (Gibco, UK) was then added and cells were dissociated by vigorous pipetting. Cells were collected by centrifugation and re-suspended to 1 x 10 7 and 1 x 10 8 cells/ml in Dulbecco's MEM (DMEM). The cell suspensions were chilled on ice for no more than 2 hours before implantation.
  • DMEM Dulbecco's MEM
  • Sprague Dawley rats no older than 8 hours were chilled on ice for up to 20 minutes to reduce their metabolic rate and reduce movement. 2 ⁇ l of cell suspension or DMEM was injected into the left lateral ventricle.
  • Rats were sacrificed by cervical dislocation at 1, 2, 4, 8 and 16 weeks after implantation of cells.
  • 0.5mm sections were visualised under the fluorescent (Nikon TE300, FITC, excitation 465/95 emission 515/55nm, with the dichroic mirror set at 505nm) or confocal (Bio/Rad MRC 1000uv confocal system attached to a Nikon Diphot 3000 microscope) microscope.
  • fluorescent Nekon TE300, FITC, excitation 465/95 emission 515/55nm, with the dichroic mirror set at 505nm
  • confocal Bio/Rad MRC 1000uv confocal system attached to a Nikon Diphot 3000 microscope
  • excitation and emission bandwidths were 488/8 nm and 522/35 nm respectively and images were taken using a water immersion x 40 lens with a numerical aperture of 1.15.
  • Sections containing green fluorescent regions were embedded in paraffin wax as follows; tissue was dehydrated through 80% ethanol for 1 hour, followed by 95 % ethanol for 1.5 hours, then 100% ethanol for 4 hours. Sections were then immersed in Histoclear (Ajax, Australia)/ethanol (50%:50%) for 1 hour, then 100% Histoclear for 4 hours, followed by paraffin wax (Oxford labware, USA) at 60°C for 2 hours and then paraffin wax under vacuum (15-20KPa) for a further 2 hours. The 0.5mm brain slices were removed from the molten wax bath and placed on a hot plate at 60°C. A pre-warmed mould was half filled with molten wax.
  • the lid of the cassette was removed and tissue was positioned at the bottom of the mould and then transferred to a cold (-20°C) surface.
  • the mould was removed from the cold surface after the wax at the bottom solidified.
  • the bottom of the cassette was placed on top of the base mould and tissue and the mould was filled with wax and allowed to solidify.
  • the wax block containing tissue was separated from the mould before thin sections (7 ⁇ m, Leica RM2135 Microtome) were cut. Using fine paintbrushes, the sections were floated on the surface of a water bath heated at 45-55°C. These sections were floated onto superfrost+ slides (Bayer, Australia) and allowed to dry completely prior to immunohistochemical analysis.
  • Paraffin wax embedded 7 ⁇ m thick brain sections were washed with Histoclear twice for 4 minutes, in order to remove paraffin wax from the tissue.
  • the slices were then treated with graded ethanol (100%, 80% and 70% each for 2 minutes) before washing with PBS twice for 5 minutes.
  • the tissue was then permeablised with 0.1% triton-X100 (Sigma, UK), in PBS for 5 minutes and treated with blocking solution (10% goat serum, Gibco, UK, 3% bovine serum albumin, Roche, Germany) in PBS for 30 minutes at room temperature.
  • Antibodies for Nestin and NF200 were used as previously described in Example 1. Use of other antibodies is outlined below.
  • GFP Blocking buffer containing primary antibody to GFP raised in mouse and used at 1 in 1000, Clonetec, USA. Secondary antibody: Anti-mouse IgG conjugated to alkaline phosphatase, 1 in 4000, Rockland, USA.
  • GFAP Blocking buffer containing primary antibody to glial fibrilliary acidic protein, GFAP raised in rabbit used at 1 in 500, Sigma, UK; Secondary antibody: Anti-rabbit IgG conjugated to alkaline phosphatase, 1 in 500, Zymed, USA.
  • Neural progenitors derived by differentiation of pluripotent cells in response to MEDII persists and disperses in the rat brain.
  • GFP positive cells were still present in the left lateral ventricle wall of all implanted rats, however additional regions of GFP positive cells in other sites such as the 3 rd ventricle wall and the cerebral aqueducts were identified, indicating that the cells had moved within the cerebrospinal fluid.
  • Implanted cells at 2 weeks formed multiple clumps per brain, each clump consisting of approximately 50 to 300 GFP positive cells within the walls of the ventricular system (a typical clump is shown in Fig. 10). No apparent difference between the implantation of EBM 7 or EBM 10 cells in the brain was observed at 1 and 2 weeks.
  • GFP positive cells could no longer be identified around the ventricle walls and were difficult to find. This may be due to dispersal of the cells within the brain or represent a loss of cells due to immuno rejection.
  • GFP positive cells were identified in deeper brain regions such as the thalamus, (Fig. 12), frontal cortex, caudate putamen (Fig. 13) and colliculus, midbrain and in these sites in the un-injected right side of the brain. There were no apparent differences in the distribution of EBM 7 and EBM 10 at either 8 or 16 weeks. The distribution of implanted GFP positive cells within rat brains over 16 weeks is shown in Fig. 14.
  • the black areas represent the location of the GFP positive cells at time points up to 4 weeks and the white at later times up to 16 weeks.
  • the cells were initially located within the CSF, then incorporated into the ependyma and were later found in brain regions between the main ventricles and the ventricular canals.
  • Fig. 13 arrows Using confocal microscopy cellular processes (Fig. 13 arrows), were identified at 8 weeks in rats implanted with EBM 7 , which was indicative of differentiation to neurons or glia.
  • thin serial sections were taken from a GFP positive brain region for immunohistochemical analysis to assess gene expression.
  • Fig. 15 shows serial sections from a GFP positive region located in the caudate putamen of a rat injected with EBM 7 cells after 2 weeks.
  • Serial sections were stained for nestin (Fig. 15A), NF200 (Fig. 15B), GFP (Fig. 15C) and GFAP (Fig. 15D).
  • Fig 15A shows that the GFP positive cells did not express the neural precursor nestin, however nestin was expressed in EBM (Example 1).
  • neural progenitors derived by differentiation of pluripotent cells in response to MEDII can incorporate, differentiate and disperse in the rat brain.
  • the neural progenitors derived by differentiation of pluripotent cells in response to MEDII are therefore potentially of use for the replacement of damaged or dysfunctional brain cells in conditions such as Parkinson's disease, dementia, central ischaemic injury resulting from trauma or stroke, spinal injury and movement disorders.
  • Embryonic stem cell-derived glial precursors a source of myelinating transplants. Science. 285, 754-756.
  • Pax-3 a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10,1135-1147.
  • Rathjen J., Lake, J.-A., Bettess, M. D., Washington, J. M., Chapman, G. and Rathjen, P. D. (1999). Formation of a primitive ectoderm like cell population from ES cells in response to biologically derived factors. J. Cell Sci. 112, 601-612.
  • NCAM neural cell adhesion molecule
  • NCAM and its polysialic acid moiety a mechanism for pull/push regulation of cell interactions during development. Development 1992 Supplement, 99- 104.
  • Leukemia inhibitory factor is expressed by the preimplantation uterus and selectively blocks primitive ectoderm formation in vitro. Proc. Natl. Acad. Sci. U.S.A. 89, 8240-8244.
  • Pax-6 a murine paired box gene, is expressed in the developing CNS. Development 113,1435-1449.

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Abstract

La présente invention concerne un procédé permettant de produire des cellules neurectodermiques, qui consiste à obtenir une source de cellules primitives précoces de type ectodermique (EPL) ; un milieu conditionné tel que défini dans les pièces descriptives de l'invention ; ou un extrait de ce dernier possédant des propriétés d'induction neuronale ; et à mettre les cellules EPL en contact avec le milieu conditionné pendant une durée suffisamment longue pour générer une différenciation contrôlée en cellules neurectodermiques.
EP01901033A 2000-01-14 2001-01-12 Production de cellules Withdrawn EP1254211A4 (fr)

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US7015037B1 (en) 1999-08-05 2006-03-21 Regents Of The University Of Minnesota Multiponent adult stem cells and methods for isolation
US10638734B2 (en) 2004-01-05 2020-05-05 Abt Holding Company Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof
US7250294B2 (en) 2000-05-17 2007-07-31 Geron Corporation Screening small molecule drugs using neural cells differentiated from human embryonic stem cells
JP2004529621A (ja) 2001-02-14 2004-09-30 ティー ファークト，レオ 多能性成体幹細胞、その起源、それを得る方法および維持する方法、それを分化させる方法、その使用法、ならびにそれ由来の細胞
US7799324B2 (en) 2001-12-07 2010-09-21 Geron Corporation Using undifferentiated embryonic stem cells to control the immune system
EP1463803B1 (fr) 2001-12-07 2018-02-14 Asterias Biotherapeutics, Inc. Obtention de cellules hematopoietiques a partir de cellules souches embryonnaires humaines
EP2264146A1 (fr) 2001-12-07 2010-12-22 Geron Corporation Cellules d'îlots de Langerhans à partir de cellules souches embryonnaires humaines
US7285415B2 (en) 2002-07-11 2007-10-23 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US20050101014A1 (en) 2002-07-11 2005-05-12 Keirstead Hans S. Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US7153650B2 (en) 2003-03-13 2006-12-26 Geron Corporation Marker system for preparing and characterizing high-quality human embryonic stem cells
US8426200B2 (en) 2003-07-02 2013-04-23 Regents Of The University Of Minnesota Neuronal differentiation of stem cells
AU2005271723B2 (en) 2004-07-13 2010-12-16 Asterias Biotherapeutics, Inc. Medium for growing human embryonic stem cells
AU2006262369B2 (en) 2005-06-22 2012-07-05 Asterias Biotherapeutics, Inc. Suspension culture of human embryonic stem cells
KR101529317B1 (ko) 2005-06-22 2015-06-16 아스테리아스 바이오세라퓨틱스, 인크. 영장류 다능성 줄기 세포의 심근세포 계통 세포로의 분화
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