EP3370744A1 - Le blocage du cycle cellulaire améliore l'efficacité de production de cellules souches pluripotentes induites - Google Patents

Le blocage du cycle cellulaire améliore l'efficacité de production de cellules souches pluripotentes induites

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Publication number
EP3370744A1
EP3370744A1 EP16862739.6A EP16862739A EP3370744A1 EP 3370744 A1 EP3370744 A1 EP 3370744A1 EP 16862739 A EP16862739 A EP 16862739A EP 3370744 A1 EP3370744 A1 EP 3370744A1
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EP
European Patent Office
Prior art keywords
cell
cells
phase
arresting
cell cycle
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Withdrawn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
EP16862739.6A
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German (de)
English (en)
Other versions
EP3370744A4 (fr
Inventor
Robin Y. SMITH
Marcie A. Glicksman
Nikhat F. ZAIDI
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.)
Seaport Diagnostics Inc
Original Assignee
Orig3n Inc
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Publication date
Application filed by Orig3n Inc filed Critical Orig3n Inc
Publication of EP3370744A1 publication Critical patent/EP3370744A1/fr
Publication of EP3370744A4 publication Critical patent/EP3370744A4/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Withdrawn legal-status Critical Current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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-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
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/604—Klf-4
    • 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
    • C12N2501/606—Transcription factors c-Myc
    • 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
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system 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
    • C12N2510/00—Genetically modified cells

Definitions

  • iPSCs induced pluripotent stem cells
  • the invention relates to a method for generating an induced pluripotent stem cell (iPSC), comprising: providing a cell; arresting the cell cycle of the cell; and transforming the cell, thereby generating the induced pluripotent stem cell.
  • This method may also be used to generate a plurality of induced pluripotent stem cells.
  • the invention relates to a method for generating a plurality of induced pluripotent stem cells, comprising: providing a plurality of cells; selecting a subset of the plurality of cells, wherein the subset of cells is enriched in cells of one or more cell cycle phases; and transforming the subset of cells, thereby generating the plurality of induced pluripotent stem cells.
  • Synchronization may be accomplished, for example, by arresting the cell cycle of a cell, or by selecting cells enriched in one or more cell cycle phases from a plurality of cells.
  • the invention relates to a method for generating an induced pluripotent stem cell, comprising: providing a cell; arresting the cell cycle of the cell; and transforming the cell, thereby generating the induced pluripotent stem cell.
  • the cell is preferably not a stem cell (e.g., the cell may be a differentiated cell). This method may also be used to generate a plurality of induced pluripotent stem cells.
  • the invention relates to a method for generating a plurality of induced pluripotent stem cells, comprising: providing a plurality of cells; selecting a subset of the plurality of cells, wherein the subset of cells is enriched in cells of one or more cell cycle phases; and transforming the subset of cells, thereby generating the plurality of induced pluripotent stem cells.
  • the cells are not stem cells.
  • the plurality of cells may comprise stem cells, and such a plurality would also comprise cells that are not stem cells (e.g., the plurality would also comprise differentiated cells).
  • the cell may be a eukaryotic cell, such as a metazoan cell.
  • the cell may be a mammalian cell.
  • the cell may be from a rodent, lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate.
  • the cell may be a human cell.
  • the cell is a somatic cell.
  • the cell is a diploid cell.
  • the cell may be a differentiated cell.
  • the cell may be derived from the ectoderm, endoderm, or mesoderm.
  • the cell may originate from the epithelium, connective tissue, muscle tissue, or nervous tissue.
  • the cell may be a peripheral blood mononuclear cell (PBMC) or a fibroblast.
  • PBMC peripheral blood mononuclear cell
  • the cell may be a lymphocyte. In some embodiments, the cell is an adipocyte.
  • the invention relates to methods comprising arresting the cell cycle of the cell.
  • Arresting the cell cycle of the cell may comprise any known method that inhibits mitosis (see, e.g. , BANFALVI, GASPAR, CELL CYCLE SYNCHRONIZATION (Humana Press, 201 1); and PCT Patent Application Publication No. WO 2010/1 18709 (hereby incorporated by reference)).
  • Cells that are in the mitosis phase of the cell cycle (M phase) are more receptive to transduction and/or transfection.
  • arresting the cell cycle comprises arresting the cell cycle at M phase ⁇ e.g. , by contacting the cell with an agent such as colchicine, colcemid, razoxane, or noscapine).
  • arresting the cell cycle may comprise arresting the cell cycle at interphase, such as GO phase, Gl phase, S phase, or G2 phase.
  • a cell may be arrested in Gl/S phase ⁇ e.g. , using thymidine, such as 4 mM thymidine for 16-24 hours), and the method may comprise incubating the cell for a period of time after releasing the cell from arrest, prior to transforming the cell ⁇ e.g. , 12 hours after release from a thymidine block, such that the cell is in M phase when it is transformed).
  • a method may comprise arresting the cell cycle of a plurality of cells, and for such embodiments, the phrase "arresting the cell cycle” is synonymous with “synchronizing the cell cycle”.
  • arresting the cell cycle does not halt the cell cycle at a specific phase, and yet the cell cycle is inhibited such that transformation is more efficient than without arresting the cell cycle.
  • aphidicolin and nocodazole may be used to arrest a cell at G2/M phase, which is useful to increase transformation efficiency.
  • Arresting the cell cycle may comprise arresting the cell cycle at interphase, GO phase,
  • G0/G1 phase early Gl phase, Gl phase, late Gl phase, Gl/S phase, S phase, G2/M phase, or M phase.
  • arresting the cell cycle comprises incubating the cell in media comprising a serum concentration and/or amino acid concentration that restricts cell growth.
  • the cell may be incubated in media comprising a serum concentration and/or amino acid concentration that restricts cell growth for about 1 hour to about 10 days, such as about 1 day to about 7 days, such as about 1, 2, 3, 4, 5, 6, or 7 days.
  • the period of time may depend on different factors, e.g., because different cells and different cell culture conditions result in cell cycles of varying duration.
  • the media may have a serum concentration, for example, of about 0% to about 10%, such as about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1.0%.
  • the media may have a serum concentration of less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%.
  • the media may have a serum concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0%.
  • the media may be serum free media (i.e., media comprising no serum).
  • the cell cycle may then be restarted, for example, by contacting the cell with media comprising a second serum concentration (i.e., a higher serum concentration than the serum concentration that restricts cell growth).
  • a second serum concentration i.e., a higher serum concentration than the serum concentration that restricts cell growth.
  • the method may comprise incubating the cell in media comprising a second serum concentration and/or a second amino acid concentration, e.g., wherein the second serum concentration and/or second amino acid concentration is higher than the serum
  • the second serum concentration may be about 2% to about 35%, such as about 5% to about 30%, or about 10%) to about 25%.
  • the second serum concentration may be about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%.
  • the serum may be, for example, fetal bovine serum.
  • Arresting the cell cycle may comprise contacting the cell with an agent. Contacting the cell with an agent may comprise incubating the cell in media comprising the agent.
  • the agent may be a small molecule or a biomolecule.
  • small molecule refers to molecules with a molecular weight less than 5,000 amu, such as about 30 to about 1000 amu, about 40 amu to about 800 amu, or about 50 amu to about 750 amu.
  • biomolecule refers to molecules comprising peptides, proteins, nucleic acids, sugars, and/or carbohydrates.
  • a biomolecule may be, for example, a cytokine (e.g., Transforming Growth Factor ⁇ ).
  • the agent may be an inhibitor of a transcription factor, enzyme (e.g., a kinase or phosphorylase), or cellular pathway.
  • the agent may be a cyclin dependent kinase inhibitor, a cyclin dependent kinase 4 inhibitor, a cyclin dependent kinase 6 inhibitor, a DNA polymerase inhibitor, a FDVIG CoA reductase inhibitor, an inhibitor of nucleotide biosynthesis, or an inhibitor of microtubule polymerization.
  • the agent is a reversible inhibitor of the cell cycle, e.g., the cell can restart its cell cycle after the agent is removed from media comprising the agent.
  • the agent is non-toxic, e.g., a concentration of the agent that is non-toxic to the cell is sufficient to arrest the cell cycle of the cell.
  • incubating the cell in media comprising the agent comprises incubating the cell in media comprising a concentration of the agent that is not toxic to the cell.
  • the agent may be abemaciclib, aminopterin, aphidicolin, blebbistatin, butyrate, cathinone, colcemid, colchicine, compactin, cytochalasin D, cytosine arabinoside, fluorodeoxyuridine, hydroxyurea, lovastatin, methotrexate, mevinolin, MG132, mimosine, nocodazole, noscapine, palbociclib, pantopon, razoxane, reveromycin A, RO-3306, roscovitine, ribociclib, vincristine, or vomciclib.
  • Incubating the cell in media comprising an agent may comprise incubating the cell in media comprising the agent for about 1 hour to about 10 days, such as about 2 hours to about 7 days, such as about 1, 2, 3, 4, 5, 6, or 7 days.
  • the period of time may depend on different factors, e.g., because different cells and different cell culture conditions result in cell cycles of varying duration.
  • the method comprises incubating the cell in media that does not comprise the agent, e.g., after incubating the cell in media comprising the agent, to restart the cell cycle.
  • the method may comprise incubating the cell in media that does not comprise the agent for about 4 hours to about 24 hours following a double thymidine block, e.g., such that the cell is in M phase during transformation.
  • arresting the cell cycle comprises modulating a CEK interacting protein (cip) pathway, kinase inhibitory protein (kip) pathway, inhibitor of kinase 4 (INK4a) pathway, or alternative reading frame (ARF) pathway.
  • cip CEK interacting protein
  • kip kinase inhibitory protein
  • INK4a inhibitor of kinase 4
  • ARF alternative reading frame pathway
  • the cip/kip family members p21, p27, and p57 arrest the cell cycle at Gl phase.
  • Transforming Growth Factor ⁇ TGFP
  • the INK4a/ARF family includes pl6 INK4a , which binds to cyclin dependent kinase 4 (CDK4) to arrest the cell cycle at Gl phase.
  • CDK4 cyclin dependent kinase 4
  • arresting the cell cycle comprises activating pl4 ARF , pl6 INK4a , pl8, pl9, p21, p27, p53, or p57 (see, e.g., U.S. Patent No. 6,033,847; hereby incorporated by reference).
  • arresting the cell cycle comprises modulating a cyclin D pathway.
  • Arresting the cell cycle may comprise inhibiting cyclin dependent kinase 4 (CDK4) or cyclin dependent kinase 6 (CDK6).
  • Arresting the cell cycle may comprise contacting the cell with a cyclin dependent kinase inhibitor.
  • the cyclin dependent kinase inhibitor may be, for example, a cyclin dependent kinase 4 inhibitor or a cyclin dependent kinase 6 inhibitor.
  • Palbociclib is a selective inhibitor of both CDK4 and CDK6.
  • the cyclin dependent kinase inhibitor is palbociclib, ribociclib, vomciclib, or abemaciclib (see also PCT Patent Application Publication No. WO 2014/109858; hereby incorporated by reference).
  • arresting the cell cycle comprises inhibiting nucleotide biosynthesis in the cell.
  • Inhibiting nucleotide biosynthesis in the cell comprises contacting the cell with an inhibitor of nucleotide biosynthesis.
  • arresting the cell cycle comprises incubating the cell in media comprising hydroxyurea or thymidine.
  • Hydroxyurea and thymidine cause the decrease of free deoxyribonucleotide triphosphates (dNTPs), the main structural units of DNA.
  • dNTPs free deoxyribonucleotide triphosphates
  • the inhibition of DNA replication arrests cells on the transition between the Gl phase and S phase.
  • arresting the cell cycle comprises inhibiting microtubule polymerization in the cell.
  • Inhibiting microtubule polymerization may comprise contacting the cell with an inhibitor of microtubule polymerization, such as nocodazole.
  • arresting the cell cycle comprises inhibiting HMG CoA reductase in the cell.
  • Inhibiting HMG CoA reductase may comprise contacting the cell with an HMG CoA reductase inhibitor, such as lovastatin.
  • arresting the cell cycle comprises inhibiting DNA
  • Inhibiting DNA polymerase may comprises contacting the cell with a DNA polymerase inhibitor, such as aphidicolin.
  • arresting the cell cycle comprises contacting the cell with abemaciclib, aminopterin, aphidicolin, blebbistatin, butyrate, cathinone, colcemid, colchicine, compactin, cytochalasin D, cytosine arabinoside, fluorodeoxyuridine, hydroxyurea, lovastatin, methotrexate, mevinolin, MG132, mimosine, nocodazole, noscapine, palbociclib, pantopon, razoxane, reveromycin A, RO-3306, roscovitine, ribociclib, vincristine, or voruciclib.
  • arresting the cell cycle comprises incubating the cell at low temperature.
  • the low temperature may be, for example, less than about 35°C, less than 34°C, less than 33°C, less than 32°C, less than 31°C, or less than 30°C.
  • the temperature may be about 20°C to about 35°C, such as about 27°C to about 33°C.
  • the low temperature may be about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, or about 33°C.
  • the cell may be incubated in at low temperature for about 1 hour to about 10 days, such as about 1 day to about 7 days, such as about 1, 2, 3, 4, 5, 6, or 7 days.
  • the period of time may depend on different factors, e.g., because different cells and different cell culture conditions result in cell cycles of varying duration.
  • the method comprises arresting the cell cycle using more than one of the methods described herein, including serum starvation or nutrient starvation, contacting the cell with an agent, contacting the cell with more than one agent, modulating a cellular pathway, and incubating the cell at low temperature.
  • the invention relates to methods comprising selecting a subset of the plurality of cells, wherein the subset of cells is enriched in cells of one or more cell cycle phases.
  • the methods may further comprise arresting the cell cycle for the cells of the plurality, e.g., prior to selecting the subset, using any method described herein.
  • the methods may further comprise arresting the cell cycle for the subset of cells, e.g., after selecting the subset, using any method described herein.
  • the method may comprise selecting a subset of the plurality of cells that is enriched in GO phase, G0/G1 phase, early Gl phase, Gl phase, late Gl phase, Gl/S phase, S phase, G2/M phase, or M phase cells.
  • the method comprises selecting a subset of cells that is enriched in M phase cells.
  • the method may comprise contacting a population of cells with a dye, wherein selecting the subset of cells comprises selecting cells that comprise a similar amount of the dye.
  • a dye refers to a molecule or particle that can absorb or emit light at an infrared, visible, or ultraviolet wavelength, such as a chromophore or fluorophore.
  • the dye may comprise fluorescein, Alexa Fluor® 488, phycoerythrin, R-phycoerythrin, Texas Red®, cyanine 5 (Cy5), bisbenzimidazole (Hoechst 33342), 4',6-diamidino-2-phenylindole (DAPI), actinomycin, mithramycin, anthraquinone, TO-PRO-3, or propidium iodide.
  • the dye may bind (e.g., specifically bind) to a cellular component, such as nucleic acids, double stranded nucleic acids, DNA, chromatin, or microtubules.
  • the dye may bind to a molecule on the surface of a cell.
  • the dye may be an intercalating agent, such as propidium iodide.
  • cells at one stage of the cell cycle comprise more of a molecule that specifically binds to the dye (e.g., DNA or microtubules) than cells at a different stage of the cell cycle, so that the dye allows for the differentiation of cells at different stages.
  • the dye e.g., DNA or microtubules
  • dyes that bind to DNA allow for the differentiation of cells at different stages of the cell cycle because G2 and M phase cells contain twice as much DNA as GO or Gl phase cells, and S phase cells contain an intermediate amount of DNA.
  • the method comprise fluorescence activated cell sorting (FACS) (see, e.g., PCT Patent Application Publication Nos. WO 2014/109713 and WO 2010/118709, each of which is hereby incorporated by reference).
  • FACS fluorescence activated cell sorting
  • the method does not comprise contacting the population of cells with a dye.
  • the forward scatter and side scatter channels of a FACS system may be used to differentiate cells at different stages of a cell cycle (e.g.,
  • fluorescence activated cell sorting may rely on forward scatter and side scatter rather than fluorescence). Similarly, methods such as centrifugation elutriation and mitotic shake-off do not rely on dyes.
  • Selecting a subset of the plurality of cells may comprise centrifugation elutriation (see, e.g., PCT Patent Application Publication Nos. WO 2013/067038 and WO
  • a centrifugation elutriation system consists of a specialized centrifuge rotor in which the centrifugal force and opposing bulk medium flow create a gradient, with smaller cells at the top and larger cells at the bottom.
  • the rotor speed or medium flow is manipulated such that the gradient of size-separated cells is pushed toward the top and the small cells at the top of the gradient are eventually pushed out of the elutriation chamber and into a collection vessel.
  • With further manipulation of the rotor speed and medium flow progressively larger cells are pushed out of the elutriation chamber. Since Gl cells are roughly half the size of mitotic or late G2 cells, centrifugal elutriation can be used to select cells according to their position in the cell cycle.
  • Selecting a subset of the plurality of cells may comprise mitotic shake-off (see, e.g.,
  • Mitotic shake-off allows for the selection of spherical, mitotic (M) phase cells, which adhere less firmly to surfaces than GO phase, Gl phase, S phase, and G2 phase cells.
  • M mitotic
  • Gl phase GO phase
  • S phase S phase
  • G2 phase cells GO phase
  • G2 phase cells GO phase
  • shaking cultures of adherent cells allows for the separation of M phase cells from cells at other phases.
  • the method preferentially comprises transforming the cell or cells to generate the induced pluripotent stem cell(s).
  • Transforming the cell or cells may comprise transforming the cell(s) with one or more proteins, one or more nucleic acids, one or more vectors, and/or one or more small molecules.
  • Generating an induced pluripotent stem cell may comprise transducing the cell with at least one gene selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene (see, e.g., U.S. Patent Application Publication No. 2009/0227032; hereby incorporated by reference).
  • generating an induced pluripotent stem cell may comprise transducing the cell with an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene.
  • the method may comprise transducing the cell with a gene for Kruppel-like factor 4 (Klf4), octamer-binding transcription factor 3/4 (Oct-3/4), octamer-binding transcription factor 4 (Oct-4), SRY (sex determining region Y)-box 2 (Sox2), and/or c-Myc.
  • Generating a pluripotent stem cell may comprise transducing the cell with a gene for Oct3/4, Oct4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Glisl, Esrrb, and/or Esrrg.
  • generating a pluripotent stem cell comprises transducing the cell with a gene for Klf4, Oct-3/4, Oct-4, Sox2, and c-Myc.
  • Transducing the cell may comprise transducing the cell with at least one vector, e.g., wherein the at least one vector comprises a gene for Klf4, Oct-3/4, Oct-4, Sox2, and/or c- Myc.
  • the vector may comprise a plasmid, virus, transposable element, or nanoparticle.
  • the vector may be, for example, a plasmid vector or a viral vector, such as a Sendai virus vector or an adenovirus vector.
  • Transducing the cell may comprise transducing the cell with at least one Sendai virus vector, e.g., wherein the at least one Sendai virus vector comprises a gene for Klf4, Oct-3/4, Oct-4, Sox2, and/or c-Myc.
  • the method comprises transforming the cell with at least one of a glycogen synthase kinase inhibitor, TGFP receptor inhibitor, cyclic AMP agonist, S- adenosyl homocysteine hydrolase inhibitor, and agent that promotes histone acetylation.
  • the method comprises transforming the cell with a small molecule, such as valproic acid, BIX-01294, SB431412, or PD0325901.
  • the method may comprise transforming the cell with valproic acid, BIX-01294, SB431412, and PD0325901.
  • the method comprises transforming the cell with one or more microRNAs (see, e.g., U.S. Patent No. 8,852,941; hereby incorporated by reference).
  • the method comprises differentiating the induced pluripotent stem cell(s).
  • the method may comprise differentiating the iPSC(s) into fibroblast s), B cell(s), T cell(s), hematopoietic cell(s), macrophage(s), monocyte(s), mononuclear cell(s), dendritic cell(s), myocyte(s), ketatinocyte(s), melanocyte(s), adipocyte(s), epithelial cell(s), epidermal cell(s), chondrocyte(s), neural cell(s), glial cell(s), astrocyte(s), cardiac cell(s), cardiomyocyte(s), esophageal cell(s), gastric cell(s), pancreatic cell(s), hepatocyte(s), cumulus cell(s), or gametocyte(s).
  • Methods for differentiating stem cells, such as iPSCs are well known in the art (see, e.g., U.S. Patent Application Public
  • Example 1 Harvesting peripheral blood mononuclear cells.
  • a 12 mL LeucoSepTM tube is filled with 3 mL LeucoSepTM separation medium (Greiner Bio One). The tube is centrifuged for 30 seconds at 1000 rcf at room temperature to position the separation medium in the tube below the porous barrier.
  • PBS phosphate buffered saline
  • Human blood in a 4 mL vacutainer is inverted 10 times to mix the blood.
  • the blood is then added to the conical tube containing the PBS, and the blood and PBS is mixed.
  • the blood and PBS mixture is then poured into the LeucoSepTM tube.
  • the LeucoSepTM tube is centrifuged at room temperature for 30 minutes at 1250 rcf in a Labnet Centrifuge (or 2100 rpm in a Beckman swinging bucket centrifuge).
  • the enriched cell fraction containing lymphocytes and peripheral blood mononuclear cells, is collected by pouring off both the plasma supernatant and enriched cell fraction above the porous barrier into a new 15 mL centrifuge tube.
  • the cells are pelleted at 500 rcf for 10 minutes in a Labnet centrifuge (or for 10 minutes at 1 100 rpm in a Beckman centrifuge), and the supernatant is discarded.
  • the pellet is resuspended in 1 mL of freezing media (10% DMSO in heat- inactivated Fetal Bovine Serum).
  • the 1 mL sample is divided into two 0.5 mL aliquots and frozen in a -80°C freezer. Each 0.5 mL aliquot contains approximately 1,000,000 peripheral blood mononuclear cells.
  • Example 2 Transducing peripheral blood mononuclear cells.
  • peripheral blood mononuclear cells A 0.5 mL aliquot of peripheral blood mononuclear cells is washed with 0.5 mL of expansion media and placed in a 15 mL conical vial. The cells are pelleted at 250 rcf for 7 minutes, and the supernatant is decanted, leaving approximately 100 uL of media in the tube.
  • Transduction media is prepared, containing 0.4 mL StemPro-34 Lance Media; 5 iL hKOS; 5 ⁇ hc-Myc; 3 ⁇ h-Klf4; 2 ⁇ Polybrene in water (1 mg/mL dilution); and Polybrene reagent (10 mg/mL).
  • Frozen CytoTune virus vials are placed in a 37°C bath for 8 seconds, causing the reagent to melt, and then placed in a 4°C cold block. The virus is mixed into the PBMC expansion media.
  • the transduction media is then placed in the 15 mL conical vial to resuspend the cell pellet.
  • the transduction media and cells are placed in one well of a 24-well plate and incubated overnight at 37°C in a humidified atmosphere of 5% C0 2 .

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Abstract

L'invention concerne des méthodes permettant de produire des cellules souches pluripotentes induites (CSPi) en arrêtant le cycle cellulaire d'une cellule, puis en transformant la cellule en CSPi.
EP16862739.6A 2015-11-02 2016-10-27 Le blocage du cycle cellulaire améliore l'efficacité de production de cellules souches pluripotentes induites Withdrawn EP3370744A4 (fr)

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