WO2025085598A2 - Cellules régénératives personnalisées - Google Patents

Cellules régénératives personnalisées Download PDF

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Publication number
WO2025085598A2
WO2025085598A2 PCT/US2024/051719 US2024051719W WO2025085598A2 WO 2025085598 A2 WO2025085598 A2 WO 2025085598A2 US 2024051719 W US2024051719 W US 2024051719W WO 2025085598 A2 WO2025085598 A2 WO 2025085598A2
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cells
mesenchymal stem
cell
stem cell
interleukin
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WO2025085598A3 (fr
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Thomas E. Ichim
Boris Reznik
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Immorta Bio Inc
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Immorta Bio Inc
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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
    • 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
    • C12N2501/065—Modulators of histone acetylation
    • 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
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal 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
    • 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
    • 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
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages

Definitions

  • the invention herein relates to generation of dedifferentiated cells possessing pluripotent or multipotent ability.
  • a goal of the invention herein is to use dedifferentiation to generate pluripotent stem cells.
  • Preferred embodiments include methods of generating personalized regenerative cells, said method comprising the steps of a) extracting an aliquot of blood; b) separating mononuclear cells from said blood; c) isolating cells with proclivity towards retrodifferentiation; d) optionally expanding in vitro said cells with potential for retrodifferentiation; e) contacting said cells with potential for retrodifferentiation with one or more inducers of retrodifferentiation; f) assessing extent of retrodifferentiation; g) optionally performing another contact with said one or more retrodifferentiating agents; h) assessing pluripotency status of said cells; and i) optionally differentiating said cells into mesenchymal stem cells.
  • Preferred methods include embodiments wherein personalized regenerative cells are pluripotent.
  • Preferred methods include embodiments wherein said pluripotent stem cells are capable of proliferation for more than 100 passages without exhibiting characteristics of senescence.
  • Preferred methods include embodiments wherein said pluripotent stem cells are capable of proliferation for more than 100 passages without exhibiting karyotypic abnormalities. [0008] Preferred methods include embodiments wherein said pluripotent stem cells are capable of proliferation for more than 100 passages without alteration of rate of proliferation.
  • Preferred methods include embodiments wherein said mononuclear cells isolated from blood are purified by use of a density gradient.
  • Preferred methods include embodiments wherein said density gradient is ficoll.
  • Preferred methods include embodiments wherein said density gradient is percoll.
  • Preferred methods include embodiments wherein said mononuclear cell is further purified for expression of CD 105.
  • Preferred methods include embodiments wherein said expression of CD 105 is used to magnetically isolate cells from a heterogenous population of cells.
  • Preferred methods include embodiments wherein said mononuclear cell is further purified for expression of CD34.
  • Preferred methods include embodiments wherein said expression of CD34 is used to magnetically isolate cells from a heterogenous population of cells.
  • Preferred methods include embodiments wherein said mononuclear cell is further purified for expression of VASA.
  • Preferred methods include embodiments wherein said expression of VASA is used to magnetically isolate cells from a heterogenous population of cells.
  • Preferred methods include embodiments wherein said CD 105 expressing cells are expanded in vitro.
  • Preferred methods include embodiments wherein said CD 105 expressing cells are placed on mesenchymal stem cell feeder cells.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are plastic adherent.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells express CD73.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells express CD39.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells express IL- 10 receptor. [0025] Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells express TSGP.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells express c-met.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from bone marrow.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from Whatron’s Jelly.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from umbilical cord.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from umbilical cord blood.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from adipose tissue.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are derived from amniotic membrane tissue.
  • Preferred methods include embodiments wherein said mesenchymal stem cell feeder cells are treated with a histone deacetylase inhibitor prior to contact with said CD 105 expressing cells.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is valproic acid.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 10 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 20 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of HGF from 1 million mesenchymal stem cells. [0039] Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 1 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 2 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said valproic acid is added to said feeder cells at a concentration and duration sufficient to induce production of more than 5 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is trichostatin A.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 10 pg of HGF from 1 million mesenchymal stem cells. [0050] Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 20 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 1 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 2 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said trichostatin A is added to said feeder cells at a concentration and duration sufficient to induce production of more than 5 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is phenylbutyrate.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 10 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 20 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of HGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of VEGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 50 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 100 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 500 pg of IGF from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 1 ng of stem cell factor from 1 million mesenchymal stem cells. [0072] Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 2 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein said phenylbutyrate is added to said feeder cells at a concentration and duration sufficient to induce production of more than 5 ng of stem cell factor from 1 million mesenchymal stem cells.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing CD105.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing c-kit.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing CD105.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing c-met.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing leukemia inhibitory factor receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing IL-3 receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing G- CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing GM- CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing M- CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing erythropoietin receptor. [0084] Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and GM-CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and stem cell factor receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and M-CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and oxytocin receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and IL-3 receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and CD200.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and c-kit.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA and DAZL receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA, DAZL and GM-CSF receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA, DAZL and IL-3 receptor. [0095] Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA, DAZL and IL-6 receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA, DAZL and IL-11 receptor.
  • Preferred methods include embodiments wherein isolating cells with proclivity towards retrodifferentiation is performed by isolation of cells expressing VASA, DAZL and NANOG.
  • Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with cytoplasmic extract from a pluripotent stem cell.
  • Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing microRNAs. [0100] Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing small molecules. [0101] Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing RNA interference stimulating molecules.
  • Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing genes.
  • Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing mRNAs. [0104] Preferred methods include embodiments wherein cells with proclivity towards retrodifferentiation are treated with retrodifferentiation inducing proteins.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of superoxide dismutase as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of manganese dependent superoxide dismutase as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of bone morphogenic protein 2 as compared to a nonengineered mesenchymal stem cell. [0108] Preferred methods include embodiments wherein said cell is engineered to possess increased expression of bone morphogenic protein 4 as compared to a nonengineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of VEGF as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of VEGF-C as compared to a non-engineered mesenchymal stem cell.
  • [OHl] Preferred methods include embodiments wherein said cell is engineered to possess increased expression of CXCL12 as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of TIMP-1 as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of TIMP-3 as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of TIMP-5 as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of IL- 10 as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of Fas ligand as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of HLA-G as compared to a non-engineered mesenchymal stem cell.
  • Preferred methods include embodiments wherein said cell is engineered to possess increased expression of TIMP-5 as compared to a non-engineered mesenchymal stem cell. [0119] Preferred methods include embodiments wherein said cell is utilized to enhance engraftment of a chondrocytic progenitor.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is autologous to the recipient.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is allogeneic to the recipient.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is xenogeneic to the recipient.
  • Preferred methods include embodiments wherein said mesenchymal stem cell is cultured with said chondrocytic progenitor before administration of said chondrocytic progenitor.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is utilized to treat a defect of hyalin cartilage.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is utilized to treat rheumatoid arthritis.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is utilized to treat osteoarthritis.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is utilized to treat disc degenerative disease.
  • Preferred methods include embodiments wherein said chondrocytic progenitor is administered together with FGF-5.
  • Preferred methods include embodiments wherein said cell is utilized to treat a non-union bone fracture.
  • Preferred methods include embodiments wherein said mesenchymal stem cell is administered together with an anti-inflammatory agent.
  • Preferred methods include embodiments wherein said anti-inflammatory agent is capable of inhibiting activation of NF-kappa B.
  • Preferred methods include embodiments wherein said anti-inflammatory agent is capable of inhibiting degradation of i-kappa B.
  • Preferred methods include embodiments wherein said anti-inflammatory agent is n-acetylcysteine.
  • Preferred methods include embodiments wherein said n-acetylcysteine is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 25% as compared to baseline.
  • Preferred methods include embodiments wherein said n-acetylcysteine is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 50% as compared to baseline.
  • Preferred methods include embodiments wherein said n-acetylcysteine is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 100% as compared to baseline.
  • Preferred methods include embodiments wherein said anti-inflammatory agent is quercetin.
  • Preferred methods include embodiments wherein said quercetin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 25% as compared to baseline. [0139] Preferred methods include embodiments wherein said quercetin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 50% as compared to baseline. [0140] Preferred methods include embodiments wherein said quercetin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 100% as compared to baseline. [0141] Preferred methods include embodiments wherein said anti-inflammatory agent is indomethacin.
  • Preferred methods include embodiments wherein said indomethacin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 25% as compared to baseline.
  • Preferred methods include embodiments wherein said indomethacin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 50% as compared to baseline.
  • Preferred methods include embodiments wherein said indomethacin is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 100% as compared to baseline.
  • Preferred methods include embodiments wherein said anti-inflammatory agent is ampiroxicam [0146]
  • Preferred methods include embodiments wherein said ampiroxicam is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 25% as compared to baseline.
  • Preferred methods include embodiments wherein said ampiroxicam is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 50% as compared to baseline.
  • Preferred methods include embodiments wherein said ampiroxicam is administered at a concentration sufficient to increase production of IL- 10 from mesenchymal stem cells stimulated with HMGB1 by over 100% as compared to baseline.
  • Preferred methods include embodiments wherein said cell is maintained in an undifferentiated state.
  • Preferred methods include embodiments wherein said maintenance in said undifferentiated state is accomplished by culture in a media conditioned by dedifferentiated fibroblasts.
  • Preferred methods include embodiments wherein said dedifferentiated fibroblasts are fibroblasts transfected with OCT4.
  • Preferred methods include embodiments wherein said dedifferentiated fibroblasts are fibroblasts transfected with NANOG.
  • Preferred methods include embodiments wherein said dedifferentiated fibroblasts are fibroblasts transfected with lin28.
  • Preferred methods include embodiments wherein said dedifferentiated fibroblasts are fibroblasts transfected with one or more proteins selected from a group comprising of: a) PIM1; b) c-myc; c) k-ras; d) bcr-abl; e) KLF4; f) c-met; g) OCT4; h) NANOG; and i) AIRE.
  • Preferred methods include embodiments wherein said dedifferentiated fibroblasts are obtained from a tissue possessing immature properties.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from testicular tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from ovarian tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from amniotic fluid. [0162] Preferred methods include embodiments wherein said fibroblasts are obtained from bone marrow.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with G-CSF.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with GM-CSF.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with M-CSF.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with an agonist of CXCR4.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with mozibil.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with flt3 ligand.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with beta glucan.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from peripheral blood of a patient treated with Poly IC.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from fallopian tube tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from adipose tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from deciduous tooth tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from Wharton’s Jelly.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from omental tissue.
  • Preferred methods include embodiments wherein said fibroblasts are obtained from adherent pluripotent stem cells.
  • Preferred methods include embodiments wherein said pluripotent stem cells are induced pluripotent stem cells. [0179] Preferred methods include embodiments wherein said pluripotent stem cells are parthenogenic derived pluripotent stem cells.
  • Preferred methods include embodiments wherein said pluripotent stem cells are stress derived pluripotent stem cells.
  • Preferred methods include embodiments wherein said pluripotent stem cells are somatic nuclear transfer derived pluripotent stem cells.
  • Preferred methods include embodiments wherein said fibroblasts are dedifferentiated by treatment with a “dedifferentiating agent”.
  • Preferred methods include embodiments wherein said dedifferentiating agent is a histone deacetylase inhibitor.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is valproic acid.
  • Preferred methods include embodiments wherein said valproic acid is added together with lithium chloride.
  • Preferred methods include embodiments wherein said valproic acid is added together with a ROCK inhibitor.
  • Preferred methods include embodiments wherein said valproic acid is added together with GDF -15.
  • Preferred methods include embodiments wherein said valproic acid is added together with a cyclooxygenase 2 inhibitor.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is trichostatin A.
  • Preferred methods include embodiments wherein said trichostatin A is added together with lithium chloride.
  • Preferred methods include embodiments wherein said trichostatin A is added together with a ROCK inhibitor.
  • Preferred methods include embodiments wherein said trichostatin A is added together with GDF -15.
  • Preferred methods include embodiments wherein said trichostatin A is added together with a cyclooxygenase 2 inhibitor.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is phenylbutyrate.
  • Preferred methods include embodiments wherein said phenylbutyrate is added together with lithium chloride. [0196] Preferred methods include embodiments wherein said phenylbutyrate is added together with a ROCK inhibitor.
  • Preferred methods include embodiments wherein said phenylbutyrate is added together with GDF-15.
  • Preferred methods include embodiments wherein said phenylbutyrate is added together with a cyclooxygenase 2 inhibitor.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is vorinostat.
  • Preferred methods include embodiments wherein said vorinostat is added together with lithium chloride.
  • Preferred methods include embodiments wherein said vorinostat is added together with a ROCK inhibitor.
  • Preferred methods include embodiments wherein said vorinostat is added together with a cyclooxygenase 2 inhibitor.
  • a method of creating an autologous pluripotent stem cell derived from peripheral blood sources comprising of: a) selecting a population of blood mononuclear cell; b) isolating from said blood mononuclear cells a population of cells expressing the marker DAZL; c) expanding said DAZL expressing cell population in vitro; and d) contacting said DAZL expressing cells with a dedifferentiation means.
  • Preferred methods include embodiments wherein said dedifferentiating means are proteins, nucleic acids, or small molecules capable of endowing multipotency.
  • Preferred methods include embodiments wherein said dedifferentiating means are proteins, nucleic acids, or small molecules capable of endowing pluripotency.
  • Preferred methods include embodiments wherein said dedifferentiating means are proteins, nucleic acids, or small molecules capable of endowing ability to form a teratoma when said cells are administered into an immunodeficient animal.
  • Preferred methods include embodiments wherein said dedifferentiating means are proteins, nucleic acids, or small molecules capable of endowing immortality in treated cells.
  • Preferred methods include embodiments wherein said dedifferentiation means involves transfer of cytoplasm from an immature cell into a cell which is meant to be dedifferentiated.
  • Preferred methods include embodiments wherein said cytoplasm transfer is facilitated by the use of cell fusion.
  • Preferred methods include embodiments wherein said fusogenic agent is polyethylene glycol.
  • Preferred methods include embodiments wherein said promoting agent is GDF-11.
  • Preferred methods include embodiments wherein said promoting agent is amniotic fluid.
  • Preferred methods include embodiments wherein said promoting agent is BMP2.
  • Preferred methods include embodiments wherein said promoting agent suppresses NF-kappa B activation.
  • Preferred methods include embodiments wherein said promoting agent increases NRF2 activation.
  • Preferred methods include embodiments wherein said promoting agent increases heme-oxygenase-1 activation.
  • Preferred methods include embodiments wherein said promoting agent increases bcl-2 activation.
  • Preferred methods include embodiments wherein said promoting agent increases bcl-2XL activation. [0225] Preferred methods include embodiments wherein said promoting agent increases survivin activation.
  • Preferred methods include embodiments wherein said promoting agent increases livin activation.
  • Preferred methods include embodiments wherein said promoting agent is an HD AC inhibitor.
  • Preferred methods include embodiments wherein said HD AC inhibitor is valproic acid.
  • Preferred methods include embodiments wherein said HD AC inhibitor is trichostatin A.
  • Preferred methods include embodiments wherein said HD AC inhibitor is sodium phenylbutyrate.
  • Preferred methods include embodiments wherein said HD AC inhibitor is butyrate.
  • Preferred methods include embodiments wherein said promoting agent is a GSK-3 inhibitor.
  • Preferred methods include embodiments wherein said GSK-3 inhibitor is lithium.
  • Preferred methods include embodiments wherein said promoting agent causes a change in the genotype of the cell population.
  • Preferred methods include embodiments wherein at least one promoting agent comprises or consists of an immortalizing oncogene.
  • Preferred methods include embodiments wherein said promoting agent is PIM I .
  • Preferred methods include embodiments wherein said promoting agent is SV40 large T antigen.
  • Preferred methods include embodiments wherein said promoting agent is abll.
  • Preferred methods include embodiments wherein said promoting agent is AFF4.
  • Preferred methods include embodiments wherein said promoting agent is AKT2.
  • Preferred methods include embodiments wherein said promoting agent is AKL. [0242] Preferred methods include embodiments wherein said promoting agent is AML I .
  • Preferred methods include embodiments wherein said promoting agent is MTG8.
  • Preferred methods include embodiments wherein said promoting agent is BCL6.
  • Preferred methods include embodiments wherein said promoting agent is MCF2.
  • Preferred methods include embodiments wherein said promoting agent is DCF3.
  • Preferred methods include embodiments wherein said promoting agent is EGFR.
  • Preferred methods include embodiments wherein said promoting agent is MLLT11.
  • Preferred methods include embodiments wherein said promoting agent is ERBB2.
  • Preferred methods include embodiments wherein said promoting agent is ETS1.
  • Preferred methods include embodiments wherein said promoting agent is CSF1R.
  • Preferred methods include embodiments wherein said promoting agent is FOS.
  • Preferred methods include embodiments wherein said promoting agent is FES.
  • Preferred methods include embodiments wherein said promoting agent is GNAS.
  • Preferred methods include embodiments wherein said promoting agent is HER2.
  • Preferred methods include embodiments wherein said promoting agent is FGF3.
  • Preferred methods include embodiments wherein said promoting agent is FGF4.
  • Preferred methods include embodiments wherein said promoting agent is JUN. [0259] Preferred methods include embodiments wherein said promoting agent is c- kit.
  • Preferred methods include embodiments wherein said promoting agent is K- SAM.
  • Preferred methods include embodiments wherein said promoting agent is AKAP13.
  • Preferred methods include embodiments wherein said promoting agent is LCK.
  • Preferred methods include embodiments wherein said promoting agent is LM01.
  • Preferred methods include embodiments wherein said promoting agent is LYL I .
  • Preferred methods include embodiments wherein said promoting agent is MASI.
  • Preferred methods include embodiments wherein said promoting agent is MDM2.
  • Preferred methods include embodiments wherein said promoting agent is MOS.
  • Preferred methods include embodiments wherein said promoting agent is MYH11.
  • Preferred methods include embodiments wherein said promoting agent is MYB.
  • Preferred methods include embodiments wherein said promoting agent is MYCN.
  • Preferred methods include embodiments wherein said promoting agent is PAX5.
  • Preferred methods include embodiments wherein said promoting agent is RAF.
  • Preferred methods include embodiments wherein said promoting agent is RAS.
  • Preferred methods include embodiments wherein said promoting agent is REL.
  • Preferred methods include embodiments wherein said promoting agent is ROS1.
  • Preferred methods include embodiments wherein said promoting agent is SKI (PDGF-BB).
  • Preferred methods include embodiments wherein said promoting agent is SET.
  • Preferred methods include embodiments wherein said promoting agent is SRC.
  • Preferred methods include embodiments wherein said promoting agent is TALI.
  • Preferred methods include embodiments wherein said promoting agent is TAN1.
  • Preferred methods include embodiments wherein said promoting agent is TIAN.
  • Preferred methods include embodiments wherein said promoting agent is TSC2.
  • Preferred methods include embodiments wherein said promoting agent is TRK.
  • Preferred methods include embodiments wherein said promoting agent can be conditionally inactivated.
  • Preferred methods include embodiments wherein said tissue specific progenitors are immortalized.
  • Preferred methods include embodiments wherein said immortalization is performed by transfection with an immortalizing factor.
  • said immortalizing factor is one or more oncogenes selected from a group comprising of: ABCB1, ABCG2, ABI1, ABL1, ABL2, ACKR3, ACSL3, ACSL6, ACVR1B, ACVR2A, AFF1, AFF3, AFF4, AKAP9, AKT1, AKT2, AKT3, ALDH1A1, ALDH2, ALK, AMER1, ANGPT1, ANGPT2, ANKRD23, APC, AR, ARAF, AREG, ARFRP1, ARHGAP26, ARHGEF12, ARID1A, ARID1B, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATP1A1, ATP2B3, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BBC3, BCL10, BCL11 A, BCL1 IB, BCL2, BCL2L1, BCL2L11, BCL2L2,
  • Preferred methods include embodiments wherein said retroviral expression vector is a pMXs vector.
  • Preferred methods include embodiments wherein said retroviral expression vector is a lentiviral vector.
  • Preferred methods include embodiments wherein said retroviral expression vector is an adenoviral vector.
  • Preferred methods include embodiments wherein said retroviral expression vector is a pMXs adeno-associated vector.
  • Preferred methods include embodiments wherein said retroviral expression vector is a herpesvirus vector.
  • Preferred methods include embodiments wherein said dedifferentiation genes are delivered by means of a plasmid based vector.
  • Preferred methods include embodiments wherein said dedifferentiation genes are delivered by means of an RNA based vector.
  • Preferred methods include embodiments wherein said dedifferentiation genes are delivered by means of an mRNA based vector.
  • Preferred methods include embodiments wherein said dedifferentiation genes are delivered by means of an microRNA based vector.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with ascorbic acid.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with ascorbic acid.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with a stimulator of the RAS pathway.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with a stimulator of the myc pathway.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with a stimulator of the PIM-1 pathway.
  • Preferred methods include embodiments wherein said dedifferentiation factors are administered together with a stimulator of the j anus activated kinase pathway.
  • a method of reducing age associated fibrosis in a patient comprising administration of T regulatory cells, and/or T regulatory cell derived cell-free means together with one or more senolytic agents.
  • Preferred methods include embodiments wherein said T regulatory cells generated subsequent to administration of mesenchymal stem cells or cell free mesenchymal stem cell derived means.
  • Preferred methods include embodiments wherein said mesenchymal stem cells are selected for release of brain derived neurotrophic factor upon stimulation with molecular signals associated with tissue injury.
  • Preferred methods include embodiments wherein mesenchymal stem cells are selected for expression of CD56.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 10 pg of brain derived neurotrophic factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 25 pg of brain derived neurotrophic factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 100 pg of brain derived neurotrophic factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein said mesenchymal stem cells are selected for release of ciliary neurotrophic growth factor upon stimulation with molecular signals associated with tissue injury.
  • Preferred methods include embodiments wherein mesenchymal stem cells are selected for expression of CD73.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 4 pg of ciliary neurotrophic growth factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 8 pg of ciliary neurotrophic growth factor per milliliter subsequent to stimulation with Poly IC. [0315] Preferred methods include embodiments wherein mesenchymal stem cells produce at least 12 pg of ciliary neurotrophic growth factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein said mesenchymal stem cells are selected for release of basic fibroblast growth factor upon stimulation with molecular signals associated with tissue injury.
  • Preferred methods include embodiments wherein mesenchymal stem cells are selected for expression of CD 105.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 50 pg of basic fibroblast growth factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 100 pg of basic fibroblast growth factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein mesenchymal stem cells produce at least 100 pg of basic fibroblast growth factor per milliliter subsequent to stimulation with Poly IC.
  • Preferred methods include embodiments wherein said T regulatory cells and/or cell free means secreted by said T regulatory cells are administered subsequent to administration of a fibrinolytic enzyme.
  • Preferred methods include embodiments wherein said enzyme is a matrix metalloprotease.
  • Preferred methods include embodiments wherein said matrix metalloprotease is MMP-3.
  • Preferred methods include embodiments wherein said matrix metalloprotease is MMP-5.
  • Preferred methods include embodiments wherein said matrix metalloprotease is MMP-6.
  • Preferred methods include embodiments wherein said matrix metalloprotease is MMP-9.
  • Preferred methods include embodiments wherein said matrix metalloprotease is MMP-12.
  • Preferred methods include embodiments wherein said T regulatory cells and/or cell free means generated by said T regulatory cells are administered subsequent to administration of an inhibitor of NOGO.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is an antisense oligonucleotide.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a one or more molecules capable of inducing RNA interference.
  • Preferred methods include embodiments wherein said molecule capable of inducing RNA interference is a short interfering RNA.
  • Preferred methods include embodiments wherein said molecule capable of inducing RNA interference is a short hairpin RNA.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a small molecule inhibitor.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is an antibody.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is an aptamer.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a somamer.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is bispecific antibody.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a ribozyme.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is an antibody.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a microantibody.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a hammerhead ribozyme.
  • Preferred methods include embodiments wherein said inhibitor of NOGO is a soluble receptor.
  • Preferred methods include embodiments wherein said T regulatory cells are administered subsequent to administration of a myeloid lineage cell.
  • Preferred methods include embodiments wherein said myeloid lineage cell is a myeloid derived dendritic cell.
  • Preferred methods include embodiments wherein said myeloid dendritic cell is immature.
  • Preferred methods include embodiments wherein said myeloid dendritic cell generated from a monocytic progenitor.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with G-CSF.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with GM-CSF.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with M-CSF.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with interleukin-3.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with interleukin-10.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatment with beta glucan.
  • Preferred methods include embodiments wherein said monocyte is isolated from peripheral blood that is mobilized by treatm6ent with interferon gamma.
  • Preferred methods include embodiments wherein said monocyte is isolated from menstrual blood.
  • Preferred methods include embodiments wherein said monocyte is isolated from umbilical cord blood.
  • Preferred methods include embodiments wherein said monocyte is isolated from Wharton’s Jelly.
  • Preferred methods include embodiments wherein said monocyte is isolated from bone marrow.
  • Preferred methods include embodiments wherein said monocyte is isolated from adipose tissue.
  • Preferred methods include embodiments wherein said monocyte is isolated from omental tissue.
  • Preferred methods include embodiments wherein said monocyte is pretreated with one or more agents to induce a tolerogenic phenotype prior to differentiation into a myeloid dendritic cells with immature phenotype and exosomes are derived from said myeloid dendritic cells.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PGE2 at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PGE2 at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PGE2 at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with genistein at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with genistein at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with genistein at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with quercetin at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with quercetin at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with quercetin at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with hypertonic saline at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with hypertonic saline at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with hypertonic saline at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with VEGF at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with VEGF at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with VEGF at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PDGF-BB at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PDGF-BB at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with PDGF-BB at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with IGF-1 at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with IGF-1 at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with IGF-1 at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with salinomycin at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with salinomycin at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with salinomycin at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with erythropoietin at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with erythropoietin at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with erythropoietin at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with NF-kappa B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with NF-kappa B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with NF-kappa B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with Ikk-B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with Ikk-B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with Ikk-B decoy oligonucleotides at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with interleukin-35 at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with interleukin-35 at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with interleukin-35 at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with TGF-beta at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with TGF-beta at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with TGF-beta at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with ascorbic acid at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with ascorbic acid at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with ascorbic acid at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with n-acetylcysteine at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with n-acetylcysteine at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with alpha lipoic acid at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with alpha lipoic acid at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with alpha lipoic acid at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with methylene blue at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with methylene blue at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with methylene blue at a sufficient concentration and for a time period to stimulate production of at least 40 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with valproic acid at a sufficient concentration and for a time period to stimulate production of at least 10 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said monocyte is pretreated with valproic acid at a sufficient concentration and for a time period to stimulate production of at least 20 pg/ml of interleukin- 10 from a culture of 1 million monocytes.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin-4.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin-20.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin-22.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin-35.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin-37.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses HLA-G.
  • Preferred methods include embodiments wherein said immature dendritic cell expresses interleukin- 12 p40 homodimer.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses increased phagocytic activity as compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses increased migratory activity towards chemotactic gradients as compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine IL-8.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine MIP-1 alpha.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine MIP-1 beta.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine CXCL12.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine TNF-alpha.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine lymphotoxin.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine hyaluronic acid degradation products.
  • Preferred methods include embodiments wherein said chemotactic gradient is comprised of the chemokine HMGB1.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased HL A II compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased CD40 compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased interleukin- 15 receptor compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased interferon gamma compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased interleukin- 18 receptor compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased progesterone receptor compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell possesses decreased c-kit compared to a mature dendritic cell.
  • Preferred methods include embodiments wherein said immature dendritic cell is capable of inducing generation of T regulatory cells upon culture with allogeneic naive T cells.
  • Preferred methods include embodiments wherein said immature dendritic cells upregulate expression of AIRE upon contact with said allogeneic naive T cells.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in an interleukin- 10 dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in a TGF-beta dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in a soluble HLA-G dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in an endoglin dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in an FGF-1 dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in an FGF-2 dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in an FGF-5 dependent manner.
  • Preferred methods include embodiments wherein said dendritic cells upregulate expression of AIRE in a NOTCH dependent manner.
  • Preferred methods include embodiments wherein said myeloid lineage cell is an immature neutrophil.
  • Preferred methods include embodiments wherein said immature neutrophil is a neutrophil progenitor.
  • Preferred methods include embodiments wherein said immature neutrophil is capable of differentiating along the neutrophilic lineage or the monocytic lineage.
  • Preferred methods include embodiments wherein said immature neutrophil expresses PU1.1.
  • Preferred methods include embodiments wherein said immature neutrophil expresses G-CSF receptor.
  • Preferred methods include embodiments wherein said immature neutrophil expresses stem cell factor receptor.
  • Preferred methods include embodiments wherein said immature neutrophil expresses c-met.
  • Preferred methods include embodiments wherein said immature neutrophil expresses M-CSF receptor.
  • Preferred methods include embodiments wherein said immature neutrophil expresses GM-CSF receptor.
  • Preferred methods include embodiments wherein said immature neutrophil produces interleukin- 10 upon stimulation with a TLR-4 agonist.
  • Preferred methods include embodiments wherein said TLR-4 agonist is beta glucan.
  • Preferred methods include embodiments wherein said TLR-4 agonist is hyaluronic acid degradation products.
  • Preferred methods include embodiments wherein said TLR-4 agonist is HMGB1.
  • Preferred methods include embodiments wherein said TLR-4 agonist is histones. [0477] Preferred methods include embodiments wherein said myeloid lineage cell is a myeloid suppressor cell.
  • Preferred methods include embodiments wherein said myeloid suppressor cell is capable of producing Reptimed.
  • Preferred methods include embodiments wherein said myeloid suppressor cell is capable of differentiating into monocytes when treated with All Trans Retinoic Acid.
  • Preferred methods include embodiments wherein said myeloid suppressor cell is capable of differentiating into monocytes when treated with vitamin D3.
  • Preferred methods include embodiments wherein said myeloid suppressor cell is capable of differentiating into monocytes when treated with M-CSF.
  • Preferred methods include embodiments wherein said myeloid suppressor cell is capable of differentiating into monocytes when treated with GM-CSF.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of nitric oxide.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of reactive oxygen species.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of superoxide.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of hydrogen peroxide.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of arginase.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of interleukin- 10.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of soluble PD-L1.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of soluble VISTA.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of LAG-3.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of TIM3.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of PGE2.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of secreted vimentin.
  • Preferred methods include embodiments wherein said myeloid suppressor cell inhibit T cell proliferation by production of secreted calreticulin.
  • Preferred methods include embodiments wherein said T regulatory cell is administered together with a mesenchymal cell differentiated to the oligodendrocyte linage.
  • Preferred methods include embodiments wherein said T regulatory cell is administered together with a pluripotent stem cell differentiated to the oligodendrocyte linage.
  • Preferred methods include embodiments wherein said pluripotent stem cell is generated by transfecting a somatic cell with cytoplasm from an oocyte.
  • Preferred methods include embodiments wherein said cytoplasm is transfected by use of electroporation.
  • Preferred methods include embodiments wherein said cytoplasm is transfected by use of cell fusion.
  • Preferred methods include embodiments wherein said cytoplasm is transfected by use of streptolysin O to generate transient holes in the cytoplasm of said recipient cell.
  • Preferred methods include embodiments wherein said cytoplasm is transfected by use of cell penetrating peptides to generate transient holes in the cytoplasm of said recipient cell.
  • Preferred methods include embodiments wherein said pluripotent stem cell is an induced pluripotent stem cell.
  • Preferred methods include embodiments wherein said induced pluripotent stem cell is created by transfection of cells with pluripotency inducing factors.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is OCT4.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is PIM-1.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is NANOG. [0508] Preferred methods include embodiments wherein said pluripotency inducing factor is c-met.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is hTERT.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is OCT4.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is KLF4.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is RAS.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is NOTCH.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is BMP2.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is BMP4.
  • Preferred methods include embodiments wherein said pluripotency inducing factor is AIRE.
  • a method of regenerating aged tissue comprising administration of T regulatory cell derived cell-free means, wherein said T regulatory cells are optionally treated with one or more histone deactylase inhibitors.
  • Preferred methods include embodiments wherein said cell free means are exosomes.
  • Preferred methods include embodiments wherein said cell free means are exosomes expressing CD8.
  • Preferred methods include embodiments wherein said cell free means are exosomes expressing phosphatidylserine.
  • Preferred methods include embodiments wherein said cell free means are apoptotic bodies
  • Preferred methods include embodiments wherein said cell free means are ferroptotic bodies.
  • Preferred methods include embodiments wherein said cell free means is conditioned media.
  • Preferred methods include embodiments wherein said cell free means is conditioned media from stressed cells.
  • Preferred methods include embodiments wherein said cellular stress is exposure to inflammatory stimuli.
  • Preferred methods include embodiments wherein said inflammatory stimuli is activation of one or more danger associated receptors.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with beta glucan.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with poly IC.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with poly LC.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with CpG DNA.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with BCG.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with lipopolysaccharide.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with low molecular weight hyaluronic acid.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with free histone.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with imiquimod.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with Poly A:U.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with MPL.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with Poly G3.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with Poly GIO.
  • Preferred methods include embodiments wherein said toll like receptor is activated by treatment with hyperthermia exposed cells.
  • Preferred methods include embodiments wherein said hyperthermia exposed cells possess an upregulation of hsp90 of more than 50% as compared to untreated cells of the same type.
  • Preferred methods include embodiments wherein a histone deacetylase inhibitor is administered locally and/or systemically to enhance reparative activity of said T regulatory cell.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor i s CXD 101.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor i s HD AC 10-IN - 1.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Tubastatin A TFA.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is ACY-775.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Panobinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Trichostatin A.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Vorinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Entinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is BML-210.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Abexinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Dacinostat. [0557] Preferred methods include embodiments wherein said histone deacetylase inhibitor is Quisinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Mocetinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Valproic Acid.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is CUDC-101
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is GSK3117391.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Droxinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is MCI 568.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Pracinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Divalproex Sodium.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Diferuloylmethane.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Sodium butyrate.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is PCI-34051.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is SR-4370.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Givinostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Tubacin.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is AR-42.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is (-)-Parthenolide. [0574] Preferred methods include embodiments wherein said histone deacetylase inhibitor is Tubastatin A HC1.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Resminostat.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is CUDC-907.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is M344.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is 4-Phenylbutyric acid.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Sulforaphane.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is UFO 10.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Suberohydroxamic acid.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is NKL 22.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is ITSA-1.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is KA2507.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Isoguanosine.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Raddeanin A.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is BRD3308. [0591] Preferred methods include embodiments wherein said histone deacetylase inhibitor is TH34.
  • Preferred methods include embodiments wherein said histone deacetylase inhibitor is Tinostamustine.
  • Preferred methods include embodiments wherein exosomes from a pluripotent stem cell derived mesenchymal stem cell population are administered systemically from either a culture vessel or through the use of a bioreactor.
  • Preferred methods include embodiments wherein said mesenchymal stem cell is transfected with a growth factor.
  • Preferred methods include embodiments wherein said growth factor is HGF- 1.
  • Preferred methods include embodiments wherein said growth factor is FGF- 1.
  • Preferred methods include embodiments wherein said growth factor is EGF- 1.
  • Preferred methods include embodiments wherein said growth factor is angiopoietin.
  • Preferred methods include embodiments wherein said growth factor is placental growth factor.
  • Preferred methods include embodiments wherein said growth factor is vasoactive intestinal peptide precursor.
  • Preferred methods include embodiments wherein said growth factor is endoglin.
  • Preferred methods include embodiments wherein said growth factor is myostatin.
  • Preferred methods include embodiments wherein said growth factor is TGF- beta.
  • Preferred methods include embodiments wherein said growth factor is vascular endothelial growth factor.
  • Preferred methods include embodiments wherein said growth factor is GDF- 11.
  • Preferred methods include embodiments wherein said growth factor is GDF- 15.
  • Preferred methods include embodiments wherein said growth factor is hyaluronic acid synthease.
  • Preferred methods include embodiments wherein said growth factor is interleukin-33.
  • Preferred methods include embodiments wherein said growth factor is osteosarcoma-derived growth factor.
  • Preferred methods include embodiments wherein said growth factor is midkine.
  • Preferred methods include embodiments wherein said growth factor is PDGF-BB.
  • Preferred methods include embodiments wherein said growth factor is IGF- 1.
  • Figure l is a bar graph showing the results of generating pluripotent stem cells from VASA expressing peripheral blood cells.
  • Figure 2A is a bar graph showing the results of generating pluripotent stem cells from VASA expressing peripheral blood mononuclear cells.
  • Figure 2B is a bar graph showing the results of generating pluripotent stem cells from VASA expressing peripheral blood mononuclear cells.
  • Figure 3 A is a bar graph showing the results of generating pluripotent stem cells from peripheral blood mononuclear cell derived T cells.
  • Figure 3B is a bar graph showing the results of generating pluripotent stem cells from peripheral blood mononuclear cell derived T cells.
  • Figure 4 is a bar graph showing the results of generating pluripotent stem cells from monocytes using cytoplasmic extract.
  • Figure 5 is a bar graph showing the superior activity of combined senolytic immunotherapy with mesenchymal stem cells and hepatic progenitor cells.
  • Peripheral blood derived germline stem cells are any multipotent cells obtained from peripheral blood that include a population of male or female germline stem cells.
  • “Expansion” refers to the propagation of a cell or cells without terminal differentiation. “Isolation phenotype” refers to the structural and functional characteristics of the peripheral blood derived germline stem cells upon isolation. “Expansion phenotype” refers to the structural and functional characteristics of the peripheral blood derived germline stem cells during expansion. The expansion phenotype can be identical to the isolation phenotype, or alternatively, the expansion phenotype can be more differentiated than the isolation phenotype.
  • “Differentiation” refers to the developmental process of lineage commitment.
  • a “lineage” refers to a pathway of cellular development, in which precursor or “progenitor” cells undergo progressive physiological changes to become a specified cell type having a characteristic function (e.g., nerve cell, muscle cell or endothelial cell). Differentiation occurs in stages, whereby cells gradually become more specified until they reach full maturity, which is also referred to as “terminal differentiation.”
  • a “terminally differentiated cell” is a cell that has committed to a specific lineage, and has reached the end stage of differentiation (i.e., a cell that has fully matured). Oocytes are an example of a terminally differentiated cell type.
  • isolated refers to a peripheral blood derived germline stem cell or its progenitor cell, in a non-naturally occurring state (e.g., isolated from the body or a biological sample, such as peripheral blood, from the body).
  • Progenitor cells as used herein are germ lineage cells that are 1) derived from germline stem cells of the invention as the progeny thereof which contain a set of common marker genes; 2) are in an early stage of differentiation; and 3) retain mitotic capacity.
  • Progeny as used herein are all cells derived from peripheral blood derived germline stem cells of the invention, including progenitor cells, differentiated cells, and terminally differentiated cells.
  • Endgraft refers to the process of cellular contact and incorporation into an existing tissue of interest (e.g., ovary) in vivo.
  • tissue of interest e.g., ovary
  • Agents refer to cellular (e.g., biologic) and pharmaceutical factors, preferably growth factors, cytokines, hormones or small molecules, or to genetically- encoded products that modulate cell function (e.g., induce lineage commitment, increase expansion, inhibit or promote cell growth and survival).
  • expansion agents are agents that increase proliferation and/or survival of peripheral blood derived germline stem cells.
  • “Differentiation agents” are agents that induce peripheral blood derived germline stem cells to differentiate into committed cell lineages, such as oocytes or sperm cells.
  • a “follicle” refers to an ovarian structure consisting of a single oocyte surrounded by somatic (granulosa without or with theca-interstitial) cells. Somatic cells of the gonad enclose individual oocytes to form follicles. Each fully formed follicle is enveloped in a complete basement membrane. Although some of these newly formed follicles start to grow almost immediately, most of them remain in the resting stage until they either degenerate or some signal(s) activate(s) them to enter the growth phase.
  • ovarian structure, function and physiology see Gougeon, A., (1996) Endocr Rev. 17: 121-55; Anderson, L. D., and Hirshfield, A. N. (1992) Md Med J. 41 : 614-20; and Hirshfield, A. N. (1991) hit Rev Cytol. 124: 43-101.
  • a “sperm cell” refers to a male germ cell, in either a pre-meiotic (i.e., mitotically competent) or post-meiotic state of development, including a fully mature spermatozoan. “Spermatogenesis” is the developmental process by which a sperm cell is formed.
  • Mitotically competent refers to a cell that is capable of mitosis, the process by which a cell divides and produces two daughter cells from a single parent cell.
  • a “non-embryonic” cell refers to a cell that is obtained from a post-natal source (e.g., infant, child or adult tissue).
  • a “subject” is a vertebrate, preferably a mammal, more preferably a primate and still more preferably a human. Mammals include, but are not limited to, primates, humans, farm animals, sport animals, and pets.
  • obtaining as in “obtaining the agent” is intended to include purchasing, synthesizing or otherwise acquiring the agent (or indicated substance or material).
  • polypeptides are administered, in one embodiment of the invention, to cells which already possess a dedifferentiated phenotype.
  • stem cells are utilized to generated pluripotent stem cells.
  • Said stem cells may be mesenchymal stem cells, or in some embodiments hematopoietic stem cells.
  • the iPSCs can be cultured using suitable culturing conditions.
  • iPSCs can be maintained using protocols such as those disclosed in Gao Y, Guo X, Santostefano K et al. Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for Development of Autologous Stem Cell Therapy. Mol Ther. 2016; 24: 1378-1387; Xia G, Gao Y, Jin S et al. Genome modification leads to phenotype reversal in human myotonic dystrophy type 1 induced pluripotent stem cell-derived neural stem cells. Stem Cells.
  • these protocols may be modified to meet the criteria of clinically-clean iPSCs, including the use of feeder-free, xeno-free culture and coating media. While common cultures call for the use of an extracellular matrix such as, for example, the Coming Matrigel matrix (Corning, New York, N. Y.), it should be noted that the Corning Matrigel matrix contains a mixture of matrix proteins and growth factors of non-human origin. Accordingly, for applications wherein the cells are ultimately to be implanted in a human subject, it may be desirable to use cultures conditions that do not utilize non-human origin additives.
  • cultured cells may be coated with laminin and collagen IV from human cell culture (for example, Sigma-Aldrich C6745, Sigma-Aldrich Co.) and adapted to Laminin 521 coating culture conditions.
  • Laminin 521 (LaminStemTM 521,05-753-lF, Biological Industries) is a chemically defined, animal component-free, xeno-free matrix. Those of skill in the art will be familiar with other suitable culturing conditions as well as the adaptation of those conditions for the specific uses of the presently described genome corrected cells.
  • the genetically altered iPSC colonies can then be cultured for harvest as needed to obtain the genetically altered cells.
  • iPSC colonies are detached and resuspended in embryoid body (EB) culture medium containing BMP -4 (50 ng/ml), VEGF (50ng/ml), FGF (10 ng/ml) and Y-27632 (10 pM) at a concentration of 1.2x 105. 100 pl is then seeded to into 96-well ultra-low adherence plate for EB formation.
  • EB embryoid body
  • EBs are transferred into six-well tissueculture plate (8 EBs per well) and cultured in differentiation medium (containing IL-3 (25-50 ng/ml) and M-CSF (50-100 ng/ml)). After four days, 4 ml of the differentiation medium will be added, and cells can be harvested at day 8.
  • differentiation medium containing IL-3 (25-50 ng/ml) and M-CSF (50-100 ng/ml)
  • 4 ml of the differentiation medium will be added, and cells can be harvested at day 8.
  • Differentiation of the genetically altered iPSCs into monocyte cells can also be achieved using methods described in, for example: Lachmann N., et al., Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Reports. 2015; 4:282-296; Yanagimachi M D., et al. Robust and highly-efficient differentiation of functional monocy
  • Cells to be dedifferentiated are subsequently made to express proteins/polypeptides associated with dedifferentiation. This can be accomplished through the administration of nucleic acid vectors designed to express AIRE, Pim-1, Oct3/4, Sox2, Klf4, and c-Myc.
  • AIRE, Pim-1, Oct3/4, Sox2, Klf4, and c-Myc polypeptides can be directly delivered into target cells to obtain induced pluripotent stem cells using a polypeptide transfection method (e.g., liposome or electroporation).
  • nucleic acid vectors designed to express Oct3/4, Sox2, and Klf4 polypeptides, and not a c-Myc polypeptide can be used to obtain induced pluripotent stem cells.
  • Oct3/4, Sox2, and Klf4 polypeptides can be directly delivered into target cells to obtain induced pluripotent stem cells using a polypeptide transfection method.
  • any appropriate cell type can be used to obtain induced pluripotent stem cells.
  • skin, lung, heart, liver, blood, kidney, or muscle cells can be used to obtain induced pluripotent stem cells.
  • Such cells can be obtained from any type of mammal including, without limitation, humans, mice, rats, dogs, cats, cows, pigs, or monkeys.
  • any stage of the mammal can be used, including mammals at the embryo, neonate, newborn, or adult stage.
  • fibroblasts obtained from an adult human patient can be used to obtain induced pluripotent stem cells.
  • Such induced pluripotent stem cells can be used to treat that same human patient (or to treat a different human) or can be used to create differentiated cells that can be used to treat that same human patient (or a different human).
  • somatic cells from a human patient can be treated as described herein to obtain induced pluripotent stem cells.
  • the obtained induced pluripotent stem cells can be differentiated into cardiomyocytes that can be implanted into that same human patient.
  • the obtained induced pluripotent stem cells can be directly administered to that same human patient.
  • Any appropriate method can be used to introduce nucleic acid (e.g., nucleic acid encoding polypeptides designed to induce pluripotent stem cells from cells) into a cell.
  • nucleic acid encoding polypeptides designed to induce pluripotent stem cells from other cells (e.g., non-embryonic stem cells) can be transferred to the cells using recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, transposons, phage integrases, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells.
  • the exogenous nucleic acid that is delivered typically is part of a vector in which a regulatory element such as a promoter is operably linked to the nucleic acid of interest.
  • the promoter can be constitutive or inducible.
  • constitutive promoters include cytomegalovirus (CMV) promoter and the Rous sarcoma virus promoter.
  • CMV cytomegalovirus
  • inducible refers to both up-regulation and down regulation.
  • An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed.
  • the inducer can be a chemical agent such as a protein, metabolite, growth regulator, phenolic compound, or a physiological stress imposed directly by, for example heat, or indirectly through the action of a pathogen or disease agent such as a virus.
  • Additional regulatory elements that may be useful in vectors, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, or introns. Such elements may not be necessary, although they can increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such elements can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cells. Sufficient expression, however, can sometimes be obtained without such additional elements. Vectors also can include other elements.
  • viral vectors can be used to introduce sternness-related factors, such as Oct3/4, Klf4, Sox2 and c-Myc.
  • viral vectors include, without limitation, vectors based on DNA or RNA viruses, such as adenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses, vaccinia virus, measles viruses, herpes viruses, baculoviruses, and papilloma virus vectors. See, Kay et al., Proc. Natl. Acad. Sci.
  • cells obtained from a human can be provided nucleic acid encoding human Oct3/4, Sox2, Klf4, and c-Myc polypeptides using viral vectors that do not integrate the exogenous nucleic acid into the cells.
  • the polypeptides are expressed and induced pluripotent stem cells are obtained, the induced pluripotent stem cells can be maintained in culture such that the induced pluripotent stem cells are devoid of the exogenous nucleic acid.
  • Any appropriate non-viral vectors can be used to introduce sternness-related factors, such as Oct3/4, Klf4, Sox2, and c-Myc.
  • induced pluripotent stem cells can be obtained using culture conditions that do not involve the use of serum or feeder cells.
  • cells obtained from a human can be provided nucleic acid encoding human Oct3/4, Sox2, Klf4, and c-Myc polypeptides and cultured using media lacking serum (e.g., human or non-human serum) and lacking feeder cells (e.g., human or non-human feeder cells).
  • the present disclosure provides a population of iPSCs, wherein the genetically modified somatic cells comprise a Cbx family gene sequence and one or more reprogramming factor sequences other than a cMyc family gene sequence and a Klf4 family gene sequence.
  • the invention suppression of p53 gene expression is used to increase efficacy of iPSC generation.
  • the invention teaches the use of dedifferentiated cells such as adult stem cells, however in some embodiments, somatic cells may be used. Somatic cells are cells that have differentiated sufficiently that they will not naturally generate cells of all three germ layers of the body, i.e. ectoderm, mesoderm and endoderm.
  • somatic cells include the cells from ectodermal (e.g., keratinocytes), mesodermal (e.g., fibroblast), endodermal (e.g., pancreatic cells), or neural crest lineages (e.g. melanocytes).
  • Certain embodiments include fibroblasts, keratinocytes, pancreatic beta cells, neurons, oligodendrocytes, astrocytes, hepatocytes, hepatic stem cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hematopoietic cells, osteoclasts, osteoblasts, pericytes, vascular endothelial cells, Schwann cells, and the like.
  • Somatic cells are reprogrammed using a Cbx family gene sequence and one or more reprogramming factor sequences (other than a cMyc family gene sequence and a Klf4 family gene sequence).
  • the Cbx family gene sequence is a nucleic acid sequence having at least 70% identical to the sequence of Cbx7.
  • the Cbx family gene sequence is Cbx7.
  • the one or more reprogramming factor sequences are preferably Oct family gene sequence, a Sox family gene sequence, a Nanog family gene sequence, a Lin28 family gene sequence.
  • the one or more reprogramming factor sequences include Oct family gene sequence and a Sox family gene sequence.
  • the Oct family gene sequence is a nucleic acid sequence having at least 70% identical to the amino acid sequence of Oct 3/4.
  • the Sox family gene sequence is a nucleic acid sequence having at least 70% identical to the amino acid sequence of Sox2.
  • the Nanog family gene sequence is a nucleic acid sequence having at least 70% identical to the amino acid sequence of Nanog.
  • the Lin28 family gene sequence is a nucleic acid sequence having at least 70% identical to the amino acid sequence of Lin28.
  • the iPSC cells of the present disclosure are generated by a method comprising a step of (a) introducing somatic cells with a vector expressing a Cbx family gene and one or more vectors expressing one or more reprogramming factor genes rather than a cMyc family gene and a Klf4 family gene; and (b) culturing the resulting somatic cells of (a) under conditions which reprogram the resulting somatic cells of (a) to produce the iPSCs.
  • Any appropriate vector expressing the reprogramming factors described herein may be used to introduce transgenes into somatic cells.
  • Suitable vectors notably include plasmid vectors and viral vectors.
  • Viral vectors can be replication-competent or -selective (e.g. engineered to replicate better or selectively in specific host cells), or can be genetically disabled so as to be replication-defective or replication-impaired.
  • such vectors are commercially available (e.g. in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depository institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of production, allowing the artisan to apply them.
  • Representative examples of suitable viral vectors are generated from a variety of different viruses (e.g.
  • retrovirus encompasses vector DNA, genomic DNA as well as viral particles generated therefrom, and especially infectious viral particles.
  • a retrovirus vector or lentivirus vector In a preferred embodiment, a lentivirus is used to introduce transgenes into differentiated cells.
  • suitable plasmid vectors include, without limitation, pREP4, pCEP4 (Invitrogen), pCI (Promega), pVAX (Invitrogen) and pGWiz (Gene Therapy System Inc.).
  • Vectors used for providing reprogramming factors to the subject cells as nucleic acids will typically comprise suitable promoters for driving the expression, that is, transcriptional activation, of the reprogramming factor nucleic acids.
  • suitable promoters for driving the expression that is, transcriptional activation, of the reprogramming factor nucleic acids.
  • This may include ubiquitously acting promoters, for example, the CMV-b-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline.
  • the genetically modified somatic cells harboring the Cbx family gene sequence and reprogramming factor sequences as described herein can transform to iPSCs by culturing and expanding the resulting somatic cells of under conditions which reprogram the resulting somatic cells to produce the iPSCs in the presence of feeder cells.
  • the iPSCs are substantially isolated if it is mixed with carriers or diluents, such as culture medium, which will not interfere with its intended use.
  • the iPSC of the invention may be present in a growth matrix or immobilized on a surface.
  • the invention calls for generation of iPSC form peripheral blood or mobilized peripheral blood.
  • Stem cell mobilization is a common procedure and usually involves administration of a mobilizing agent such as G-CSF.
  • the invention generates iPSC by reprogramming blood cells (BCs) including peripheral mononuclear blood cells (PBMCs) into iPSCs (BC-iPSCs) and show that these iPSC lines are superior in terms of cytogenetic stability in comparison to their fibroblast- derived iPSC (Fib-iPSCs) lines obtained from public repositories or local clinics.
  • BCs peripheral mononuclear blood cells
  • PBMCs peripheral mononuclear blood cells
  • Fib-iPSCs fibroblast- derived iPSC
  • the alternative source of blood progenitors contain an intact genome.
  • they can be expanded in culture conditions that favor the proliferation of myeloid cells or erythroid cells.
  • Blood stem/progenitor cells express surface marker CD34 and reside in the stem cell niche. However, only about 1% stem/progenitor cells enter circulation each day and as a result, only 0.01-0.1% cells in PB are CD34+ cells. This population can be enriched by magnetic-activated cell sorting (MACS) or culture of MNCs for several days can be relied upon to expand CD34+ cells to a 5-20% purity, which can be used for reprogramming without further purification.
  • Other nucleated peripheral blood cells include granulocytes (mostly neutrophils), monocytes, T lymphocytes, B lymphocytes and a few progenitor cells.
  • PBMCs peripheral blood mononuclear cells
  • lymphocytes T cells, B cells, NK cells
  • monocytes dendritic cells
  • Lymphocytes are Small (5-10 pm) and Medium (10-18 pm) and constitute 70-90% of PBMCs.
  • CD3+ T cells 40-70% of PBMCs
  • CD4 Helper T cells 25-60% of PBMCs
  • CD8 ratio of 2 typically with CD4 to CD8 ratio of 2: 1
  • CD8 “Cytotoxic” compartment T cells 5-30% of PBMCs.
  • the remaining compartment includes 5-20% B Cells (up to 15% of PBMCs) and 5-20% NK Cells (up to 15% of PBMCs).
  • Monocytes are 16-25 pm and 10-30% of PBMCs (macrophages).
  • Dendritic cells 1-2% of PBMCs.
  • episomal vectors allow for generation of iPSCs substantially free of the vectors used in their production, as episomal or similar vectors do not encode sufficient viral genome sufficient to give rise to infection or a replication-competent virus.
  • these vectors do possess a limited degree of self-replication capacity in the beginning somatic host cells. This self-replication capacity provides a degree of persistent expression understood to be beneficial in allowing the dedifferentiation process to initiate take hold in a target host cell.
  • a plasmid vector satisfying these criteria includes the Epstein Barr oriP/Nuclear Antigen-1 (“EBNA1”) combination, which is capable of limited self-replication and known to function in mammalian cells.
  • EBNA1 Epstein Barr oriP/Nuclear Antigen-1
  • binding of the EBNA1 protein to the virus replicon region oriP maintains a relatively long-term episomal presence of plasmids in mammalian cells.
  • This particular feature of the oriP/EBNAl vector makes it ideal for generation of integration-free iPSCs. More specifically, persistent expression of reprogramming factor encoded in an oriP/EBNAl vector occurs across multiple cell divisional cycles.
  • reprogramming factors Sufficiently high levels of reprogramming factors across several cell divisions allows for successful reprogramming even after only one infection. While sustained expression of reprogramming factors is understood to be beneficial during initial programming stages, otherwise unlimited constitutive expression would hamper subsequent stages of the reprogramming process. For example, unabated expression of reprogramming factors would interfere with subsequent growth, development, and fate specification of the host cells.
  • oriP/EBNAl The persistent, yet eventual abrogation of reprogramming factor expression on oriP/EBNAl is highly coincident with the needs for different stages of the reprogramming process and eliminates the need for further manipulation steps for excision of the reprogramming factors, as has been attempted through use of transposons and excisable polycistronic lentiviral vector elements.
  • oriP/EBNAl has been applied by others in reprogramming studies, the reported efficiencies are extremely low (as few as 3 to 6 colonies per million cells nucleofected), which may be due, in-part, to reliance on large plasmids encoding multiple reprogramming factors (e.g., more than 12 kb), negatively impacting transfection efficiency.
  • reprogramming factors include pluripotency-related genes Oct-4, Sox-2, Lin-28, Nanog, Sal 14, Fbx-15 and Utf-1. These factors, traditionally are understood be normally expressed early during development and are involved in the maintenance of the pluripotent potential of a subset of cells that will constituting the inner cell mass of the pre-implantation embryo and post-implantation embryo proper. Their ectopic expression of is believed to allow the establishment of an embryonic-like transcriptional cascade that initiates and propagates an otherwise dormant endogenous core pluripotency program within a host cell.
  • reprogramming determinants such as Tert, Klf-4, c-Myc, SV40 Large T Antigen (“SV40LT”) and short hairpin RNAs targeting p53 (“shRNA-p53”) have been applied.
  • SV40LT Large T Antigen
  • shRNA-p53 short hairpin RNAs targeting p53
  • TERT and SV40LT are understood to enhance cell proliferation to promote survival during reprogramming, while others such as short hairpin targeting of p53 inhibit or eliminate reprogramming barriers, such as senescence and apoptosis mechanisms. In each case, an increase in both the speed and efficiency of reprogramming is observed.
  • miRNAs are also known to influence pluripotency and reprogramming, and some miRNAs from the miR-290 cluster have been applied in reprogramming studies. For example, the introduction of miR-291- 3p, miR-294 or miR-295 into fibroblasts, along with pluripotency-related genes, has also been reported to increase reprogramming efficiency.
  • IVS internal ribosome entry site
  • a further advantage of the techniques described herein is the use of defined media conditions for the reprogramming process, including the use of ESC media and/or E7 media. While certain additives may be present to spur the reprogramming process (e.g., L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF), no serum or animal components are used. In some instances, there may be further benefits in altering the chemical and/or atmospheric conditions under which reprogramming will take place.
  • additives e.g., L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF
  • hypoxic similar to bone marrow stem-cell niches
  • reprogramming under hypoxic conditions of 5% 02 instead of the atmospheric 21% 02, may further provide an opportunity to increase the reprogramming efficiency.
  • chemical induction techniques have been used in combination with reprogramming, particularly histone deacetylase (HD AC) inhibitor molecule, valproic acid (VP A), which has been found wide use in different reprogramming studies.
  • HD AC histone deacetylase
  • VP A valproic acid
  • MAPK kinase (MEK)-ERK (“MEK”) inhibitor PD0325901 MAPK kinase (MEK)-ERK (“MEK”) inhibitor PD0325901
  • TGF- transforming growth factor beta
  • ALK5 and ALK7 inhibitor SB431542 transforming growth factor beta
  • GSK3 glycogen synthase kinase-3
  • CHIR99021 have been applied for activation of differentiation-inducing pathways (e.g. BMP signaling), coupled with the modulation of other pathways (e.g. inhibition of the MAPK kinase (MEK)-ERK pathway) in order to sustain self-renewal.
  • differentiation-inducing pathways e.g. BMP signaling
  • other pathways e.g. inhibition of the MAPK kinase (MEK)-ERK pathway
  • Rho-associated coiled-coil-containing protein kinase (“ROCK”) inhibitors such as Y-27632 and thiazovivin (“Tzv”) have been applied in order to promote survival and reduce vulnerability of pSCs to cell death, particularly upon single-cell dissociation.
  • ROCK Rho-associated coiled-coil-containing protein kinase
  • Tzv thiazovivin
  • further variations must consider the nature of the host target cell for reprogramming. To date, a wide variety of cells have served as sources for reprogramming including fibroblasts, stomach and liver cell cultures, human keratinocytes, adipose cells, and frozen human monocyte.
  • PB cells peripheral blood
  • MNCs mononuclear cells
  • PB cells are relatively easy to isolate (e.g., blood draw) compared to isolation from other sources such as fibroblasts (e.g., skin biopsy). These cells do not require laborious culturing and propagation prior to reprogramming, thereby reducing the overall time from which reprogramming iPSCs can be obtained.
  • clonal selection allows for generation of pluripotent stem cell lines.
  • pluripotent stem cell lines possess requisite morphology (i.e., compact colony, high nucleus to cytoplasm ratio and prominent nucleolus), self-renewal capacity for unlimited propagation in culture (i.e., immortal), and with the capability to differentiate into all three germ layers (e.g., endoderm, mesoderm and ectoderm).
  • Further techniques to characterize the pluripotency of a given population of cells include injection into an immunocompromised animal, such as a severe combined immunodeficient (“SCID”) mouse, for formation of teratomas containing cells or tissues characteristic of all three germ layers.
  • SCID severe combined immunodeficient
  • the composition of blood cell derived induced pluripotent stem cells includes cells generated by providing a quantity of blood cells, delivering a quantity of reprogramming factors into the blood cells, culturing the blood cells in a reprogramming media for at least 4 days, wherein delivering the reprogramming factors, and culturing generates the blood cells derived induced pluripotent stem cells.
  • the blood cells are T-cells. In other embodiments, the blood cells are non-T-cells.
  • the blood cells are mononuclear cells (MNCs), including for example peripheral blood mononuclear cells (PBMCs).
  • MNCs mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • the cells are primary granulocytes, monocytes and B lymphocytes.
  • the reprogramming factors are Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”).
  • these reprogramming factors are encoded in a combination of vectors including pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNAl.
  • the reprogramming media includes PD0325901, CHIR99021, HA-100, and A-83-01.
  • the culturing the blood cells in a reprogramming media is for 4-30 days.
  • the blood cells are plated on a treated cell culture surface after delivering a quantity of reprogramming factors.
  • treatment includes plating of feeder cells, such as mouse embryonic fibroblasts.
  • treatment includes coating with extracellular matrix proteins.
  • extracellular matrix proteins include laminin.
  • the BC-iPSCs are capable of serial passaging as a cell line.
  • the BC-iPSCs possess genomic stability.
  • Genomic stability can be ascertained by various techniques known in the art. For example, G-band karyotyping can identify abnormal cells lacking genomic stability, wherein abnormal cells possess about 10% or more mosaicism, or one or more balanced translocations of greater than about 5, 6, 7, 8, 9, 10 or more Mb.
  • genomic stability can be measured using comparative genomic hybridization (aCGH) microarray, comparing for example, BC-iPSCs against iPSCs from a non-blood cell source such as fibroblasts.
  • aCGH comparative genomic hybridization
  • Genomic stability can include copy number variants (CNVs), duplications/deletions, and unbalanced translocations.
  • BC-iPSCs exhibit no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mb average size of amplification and deletion.
  • BC-iPSCs exhibit no more than about 20-30 Mb average size of amplification and deletion.
  • BC- iPSCs exhibit no more than about 30-40 Mb average size of amplification and deletion.
  • BC-iPSCs exhibit no more than about 40-50 Mb average size of amplification and deletion.
  • the average number of acquired de novo amplification and deletions in BC-iPSCs is less than about 5, 4, 3, 2, or 1.
  • de novo amplification and deletions in fib-iPSCs are at least two-fold greater than in PBMC-iPSCs.
  • reprogramming factors can also include one or more of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sal 14, Fbx-15, Utf-1, Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR- 294 or miR-295.
  • the reprogramming factors are encoded by a vector.
  • the vector can be, for example, a non-integrating episomal vector, minicircle vector, plasmid, retrovirus (integrating and non-integrating) and/or other genetic elements known to one of ordinary skill.
  • the reprogramming factors are encoded by one or more oriP/EBNAl derived vectors.
  • the vector encodes one or more reprogramming factors, and combinations of vectors can be used together to deliver one or more of Oct-4, Sox-2, Klf- 4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sal 14, Fbx-15, Utf-1, Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295.
  • oriP/EBNAl is an episomal vector that can encode a vector combination of multiple reprogramming factors, such as pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and pCXWB-EBNAl.
  • the reprogramming factors are delivered by techniques known in the art, such as nuclefection, transfection, transduction, electrofusion, electroporation, microinjection, cell fusion, among others.
  • the reprogramming factors are provided as RNA, linear DNA, peptides or proteins, or a cellular extract of a pluripotent stem cell.
  • the reprogramming media is embryonic stem cell (ESC) media. In various embodiments, the reprogramming media includes bFGF. In various embodiments, the reprogramming media is E7 media. In various embodiments, the reprogramming E7 media includes L- Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF In different embodiments, the reprogramming media comprises at least one small chemical induction molecule. In different embodiments, the at least one small chemical induction molecule comprises PD0325901, CHIR99021, HA-100, A-83-01, valproic acid (VP A), SB431542, Y-27632 or thiazovivin (“Tzv”).
  • culturing the BCs in a reprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • the BC-iPSCs are derived from blood cells previously isolated from a subject, by for, example, drawing a blood sample from the subject.
  • the blood cells are isolated from a subject possessing a disease mutation.
  • subjects possessing any number of mutations, such as autosomal dominant, recessive, sex-linked can serve as a source of blood cells to generate BC-iPSCs possessing said mutation.
  • the disease mutation is associated with a neurodegenerative disease, disorder and/or condition.
  • the disease mutation is associated with an inflammatory bowel disease, disorder, and/or condition.
  • the BC-iPSCs possess features of pluripotent stem cells. Some exemplary features of pluripotent stem cells including differentiation into cells of all three germ layers (ectoderm, endoderm, mesoderm), either in vitro or in vivo when injected into an immunodeficient animal, expression of pluripotency markers such as Oct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, SSEA4, high levels of alkaline phosphatase (“AP”) expression, indefinite propagation in culture, among other features recognized and appreciated by one of ordinary skill.
  • pluripotency markers such as Oct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, SSEA4, high levels of alkaline phosphatase (“AP”) expression, indefinite propagation in culture, among other features recognized and appreciated by one of ordinary skill.
  • the cells are primary culture cells.
  • the cells are blood cells (BCs).
  • the blood cells are T-cells.
  • the blood cells are non- T-cells.
  • the cells are mononuclear cells (MNCs), including for example peripheral blood mononuclear cells (PBMCs).
  • the cells are primary granulocytes, monocytes and B lymphocytes.
  • the reprogramming factors are Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”).
  • these reprogramming factors are encoded in a combination of vectors including pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNAl.
  • the reprogramming media is embryonic stem cell (ESC) media.
  • the reprogramming media includes bFGF. In various embodiments, the reprogramming media is E7 media. In various embodiments, the reprogramming E7 media includes L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF. In different embodiments, the reprogramming media comprises at least one small chemical induction molecule. In certain other embodiments, the reprogramming media includes PD0325901, CHIR99021, HA-100, and A-83-01. In other embodiments, the culturing the blood cells in a reprogramming media is for 4-30 days. In various embodiments, the BC-iPSCs are capable of serial passaging as a cell line.
  • the BC-iPSCs possess genomic stability.
  • Genomic stability can be ascertained by various techniques known in the art. For example, G-band karyotyping can identify abnormal cells lacking genomic stability, wherein abnormal cells possess about 10% or more mosaicism, or one or more balanced translocations of greater than about 5, 6, 7, 8, 9, 10 or more Mb.
  • genomic stability can be measured using comparative genomic hybridization (aCGH) microarray, comparing for example, BC-iPSCs against iPSCs from a non-blood cell source such as fibroblasts.
  • Genomic stability can include copy number variants (CNVs), duplications/deletions, and unbalanced translocations.
  • BC-iPSCs exhibit no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mb average size of amplification and deletion. In various embodiments, BC-iPSCs exhibit no more than about 20-30 Mb average size of amplification and deletion. In various embodiments, BC-iPSCs exhibit no more than about 30-40 Mb average size of amplification and deletion. In various embodiments, BC-iPSCs exhibit no more than about 40-50 Mb average size of amplification and deletion. In various embodiments, the average number of acquired de novo amplification and deletions in BC-iPSCs is less than about 5, 4, 3, 2, or 1.
  • de novo amplification and deletions in fib-iPSCs are at least two-fold greater than in PBMC-iPSCs.
  • the methods produces iPSC cell lines collectively exhibiting about 20%, 15%, 10%, 5% or less abnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or more passages when serially passaged as a cell line.
  • reprogramming factors can also include one or more of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sal 14, Fbx-15, Utf-1, Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295.
  • the reprogramming factors are encoded by a vector.
  • the vector can be, for example, a nonintegrating episomal vector, minicircle vector, plasmid, retrovirus (integrating and nonintegrating) and/or other genetic elements known to one of ordinary skill.
  • the reprogramming factors are encoded by one or more oriP/EBNAl derived vectors.
  • the vector encodes one or more reprogramming factors, and combinations of vectors can be used together to deliver one or more of Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sal 14, Fbx-15, Utf-1, Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295.
  • oriP/EBNAl is an episomal vector that can encode a vector combination of multiple reprogramming factors, such as pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and pCXWB-EBNAl.
  • the reprogramming factors are delivered by techniques known in the art, such as nuclefection, transfection, transduction, electrofusion, electroporation, microinjection, cell fusion, among others.
  • the reprogramming factors are provided as RNA, linear DNA, peptides or proteins, or a cellular extract of a pluripotent stem cell.
  • the cells are treated with sodium butyrate prior to delivery of the reprogramming factors.
  • the cells are incubated or 1, 2, 3, 4, or more days on a tissue culture surface before further culturing. This can include, for example, incubation on a Matrigel coated tissue culture surface.
  • the reprogramming conditions include application of norm-oxygen conditions, such as 5% 02, which is less than atmospheric 21% 02.
  • the reprogramming media is embryonic stem cell (ESC) media.
  • the reprogramming media includes bFGF.
  • the reprogramming media is E7 media.
  • the reprogramming E7 media includes L-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF.
  • the reprogramming media comprises at least one small chemical induction molecule.
  • the at least one small chemical induction molecule comprises PD0325901, CHIR99021, HA-100, A-83- 01, valproic acid (VP A), SB431542, Y-27632 or thiazovivin (“Tzv”).
  • culturing the BCs in a reprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • Efficiency of reprogramming is readily ascertained by one of many techniques readily understood by one of ordinary skill. For example, efficiency can be described by the ratio between the number of donor cells receiving the full set of reprogramming factors and the number of reprogrammed colonies generated. Measuring the number donor cells receiving reprogramming factors can be measured directly, when a reporter gene such as GFP is included in a vector encoding a reprogramming factor. Alternatively, indirect measurement of delivery efficiency can be provided by transfecting a vector encoding a reporter gene as a proxy to gauge delivery efficiency in paired samples delivering reprogramming factor vectors.
  • the number of reprogrammed colonies generated can be measured by, for example, observing the appearance of one or more embryonic stem cell-like pluripotency characteristics such as alkaline phosphatase (AP)-positive clones, colonies with endogenous expression of transcription factors Oct or Nanog, or antibody staining of surface markers such as Tra-1-60.
  • efficiency can be described by the kinetics of induced pluripotent stem cell generation.
  • efficiency can include producing cell lines of normal karyotype, including the method producing iPSC cell lines collectively exhibiting about 20%, 15%, 10%, 5% or less abnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or more passages when serially passaged as a cell line.
  • the pluripotency of a cell is tested in vivo by examining its capability of growing into teratoma containing all three germ cells.
  • the pluripotency is tested by the expression of certain markers in cultured cells ex vivo.
  • the pluripotency of a cell is tested by its contribution to the development of an embryo into a living organism.
  • the pluripotent stem cells are injected into the inner cell mass (ICM) of an embryonic blastocyst, which is then implanted into the uterus of a female organism and developing into a fetus.
  • ICM inner cell mass
  • chimerism refers to the contribution of the stem cells and their progenies to all three germ layers that give rise to various tissues in a living organism.
  • the differentiation occurs ex vivo; in some cases the differentiation occurs in vivo.
  • neural stem cells may be generated by culturing the induced cells as floating aggregates in the presence of noggin, or other bone morphogenetic protein antagonist
  • the composition of the present invention may be formulated with an excipient, carrier or vehicle including, but not limited to, a solvent.
  • the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent.
  • the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
  • Suitable pharmaceutically acceptable carriers for the compositions of the present invention include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like.
  • Such carrier solutions also can contain buffers, diluents and other suitable additives.
  • buffer refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
  • buffers include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiologic saline (0.9% NaCl).
  • PBS Dulbecco's phosphate buffered saline
  • D5W 5% dextrose in water
  • normal/physiologic saline (0.9% NaCl).
  • the infusion solution is isotonic to subject tissues.
  • the infusion solution is hypertonic to subject tissues.
  • compositions of the present invention that are for parenteral administration can include pharmaceutically acceptable carriers such as sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in a liquid oil base.
  • the compositions of the present invention may be administered parenterally in the form of a sterile injectable aqueous or oleaginous suspension.
  • parenteral or “parenterally” as used herein refers to introduction into the body by way of an injection (i.e., administration by injection), including, but not limited to, infusion techniques.
  • parenteral administration includes but is not limited to intravascular delivery (meaning into a blood vessel), intravenous delivery (meaning into a vein), intra-arterial delivery (meaning into an artery), intraosseous delivery (meaning into the bone marrow), intramuscular delivery (meaning into a muscle), subcutaneous delivery (meaning under the skin), cardiac delivery (meaning into the heart, myocardium), etc.
  • the delivery route may vary and depend on the origin of degenerative diseases.
  • the delivery route for treating degenerative conditions in central nervous system is intracranial injection.
  • the generation of pluripotent stem cells is provided in a manner which is scalable to production of commercial-grade cellular products. Although several methodologies have been reported for generation of pluripotent stem cells, these are characterized by low level of cellular dedifferentiation, as well as problems with large-scale expansion.
  • the invention provides conditions for enhancing the generation of pluripotent stem cells in part by creating environments that resemble embryonic stem cell development. Accordingly target cells to be dedifferentiated are grown in conditions that possess the multiple cells associated with the three dimensional embryonic environment. Specifically, in some embodiments amniotic membrane stem cells are utilized as feeder layers or as a cellular composite to support the cells which are to be dedifferentiated.
  • Cells included in culture to support dedifferentiation include mesenchymal stem cells, monocytes, B cells and NKT cells.
  • bone marrow endothelial cells are utilized to expand various cells to be dedifferentiated. It is occasionally desired to utilized defined liquid media in order to avoid reproducibility issues associated with fetal calf serum lot to lot variability.
  • the invention provides the utilization of liquid media containing various histone deacetylase inhibitors to allow for expansion of stem cells without differentiation.
  • useful histone deacetylase inhibitors include valproic acid, trichostatin A, sodium phenylbutyrate and sulforaphane.
  • the invention discloses culture medium being serum- free and devoid of non-human contaminants comprising ascorbic acid at a concentration range of about 200-6000 pg/ml, basic fibroblast growth factor (bFGF) at a concentration range of about 5-2000 ng/ml, serum replacement and a lipid mixture, wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state and with a stable karyotype for at least 40 passages in the absence of feeder cell support.
  • the invention provides for feeder layers as well.
  • the expansion of stem cells may be performed utilizing various types of liquid culture media as a base. For example, the GIBCOTM KNOCKOUTTM Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, N.Y.
  • GIBCOTM KnockoutTM Serum Replacement includes Albumax (Bovine serum albumin enriched with lipids) which is from an animal source (International Patent Publication No. WO 98/30679 to Price, P. J.
  • the concentration of GIBCOTM KNOCKOUTTM Serum Replacement in the culture medium is in the range of from about 1% [volume/volume (v/v)] to about 50% (v/v), e.g., from about 5% (v/v) to about 40% (v/v), e.g., from about 5% (v/v) to about 30% (v/v), e.g., from about 10% (v/v) to about 30% (v/v), e.g., from about 10% (v/v) to about 25% (v/v), e.g., from about 10% (v/v) to about 20% (v/v), e.g., about 10% (v/v), e.g., about 15% (v/v), e.g., about 20% (v/v), e.g., about 30% (v/v).
  • cytokine For expansion of iPSC there may be various cytokines added to said media.
  • cytokine is leukemia inhibitory factor, which may be added at concentrations of 1 pg/ml to 500 ng/ml, more preferably 10 pg/ml to 200 ng/ml, more preferably 100 pg/ml to 100 ng/ml.
  • leukemia inhibitory factor There exist other commercially available serum replacement is the B27 supplement without vitamin A which is available from Gibco-Invitrogen, Corporation, Grand Island, N.Y. USA, Catalogue No. 12587-010.
  • the B27 supplement is a serum-free formulation which includes d-biotin, fatty acid free fraction V bovine serum albumin (BSA), catalase, L-camitine HC1, corticosterone, ethanolamine HC1, D-galactose (Anhyd.), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCl, sodium selenite, superoxide dismutase, T-3/albumin complex, DL alpha-tocopherol and DL alpha tocopherol acetate.
  • BSA bovine serum albumin
  • catalase L-camitine HC1, corticosterone
  • ethanolamine HC1 D-galactose
  • glutathione glutathione
  • recombinant human insulin linoleic acid, linolenic acid, progesterone
  • leukemia inhibitory factor is added to said B27 supplement.
  • animal-free media such as in cases in which generation of autoantibodies, alloantibodies or xenoantibodies are to be avoided, it may be preferably to utilizing animal free media, which is sometimes referred to as “xeno-free”.
  • xeno-free serum replacement compositions include the premix of ITS (Insulin, Transferrin and Selenium) available from Invitrogen corporation (ITS, Invitrogen, Catalogue No. 51500-056); Serum replacement 3 (Sigma, Catalogue No.
  • the xeno-free serum replacement formulations ITS (Invitrogen corporation) and SR3 (Sigma) are diluted in a 1 to 100 ratio in order to reach a xl working concentration.
  • the culture medium is capable of maintaining pluripotent stem cell in a proliferative, pluripotent and undifferentiated state for at least about 5 passages, at least about 10 passages, at least about 15 passages, at least about 20 passages, at least about 22 passages, at least about 25 passages, at least about 30 passages, at least about 35 passages, at least about 40 passages, at least about 45 passages, at least about 50 passages and more.
  • the culture medium is capable of expanding the pluripotent stem cells in an undifferentiated state.
  • One of the important aspects of the current invention is the proliferation or expansion of pluripotent stem cells without their differentiation.
  • the number of pluripotent stem cells over the culturing period (by at least about 5%, 10%, 15%, 20%, 30%, 50%, 100%, 200%, 500%, 1000%, and more). It will be appreciated that the number of pluripotent stem cells which can be obtained from a single pluripotent stem cell depends on the proliferation capacity of the pluripotent stem cell.
  • the proliferation capacity of a pluripotent stem cell can be calculated by the doubling time of the cell (i.e., the time needed for a cell to undergo a mitotic division in the culture) and the period the pluripotent stem cell culture can be maintained in the undifferentiated state (which is equivalent to the number of passages multiplied by the days between each passage).
  • the culture medium of some embodiments of the invention is capable of supporting expansion of a single pluripotent stem cell (e.g., hESC or human iPS cell) or a population of pluripotent stem cells by at least 223 (i.e., 8 ⁇ 106) within about one month, e.g., at least 224 (i.e., 16.7x 106) within about one month.
  • a single pluripotent stem cell e.g., hESC or human iPS cell
  • a population of pluripotent stem cells e.g., 8 ⁇ 106
  • at least 224 i.e., 16.7x 106
  • the serum-free and xeno-free culture medium comprises basic fibroblast growth factor (bFGF), transforming growth factor beta-3 (TGFP3) and ascorbic acid, wherein a concentration of the ascorbic acid in the culture medium is at least 50 pg/ml and wherein the culture medium is capable of maintaining pluripotent stem cells in an undifferentiated state in the absence of feeder cell support.
  • Ascorbic acid also known as vitamin C
  • Ascorbic acid is a sugar acid (C6H8O6; molecular weight 176.12 grams/mole) with antioxidant properties.
  • the ascorbic acid used by the culture medium of some embodiments of the invention can be a natural ascorbic acid, a synthetic ascorbic acid, an ascorbic acid salt (e.g., sodium ascorbate, calcium ascorbate, potassium ascorbate), an ester form of ascorbic acid (e.g., ascorbyl palmitate, ascorbyl stearate), a functional derivative thereof (a molecule derived from ascorbic acid which exhibits the same activity/function when used in the culture medium of the invention), or an analogue thereof (e.g., a functional equivalent of ascorbic acid which exhibits an activity analogous to that observed for ascorbic acid when used in the culture medium of the invention).
  • an ascorbic acid salt e.g., sodium ascorbate, calcium ascorbate, potassium ascorbate
  • an ester form of ascorbic acid e.g., ascorbyl palmitate, ascorbyl stearate
  • a functional derivative thereof a molecule derived from as
  • Non-limiting examples of ascorbic acid formulations which can be used in the culture medium of some embodiments of the invention include L-ascorbic acid and ascorbic acid 3 -phosphate.
  • Ascorbic acid can be obtained from various manufacturers such as Sigma, St Louis, Mo., USA (e.g., Catalogue numbers: A2218, A5960, A7506, A0278, A4403, A4544, A2174, A2343, 95209, 33034, 05878, 95210, 95212, 47863, 01-6730, 01-6739, 255564, A92902, W210901).
  • the concentration of ascorbic acid in the culture medium is at least about 50 pg/ml.
  • the ascorbic acid can be used in a range of concentrations such as from about 50 pg/ml to about 50 mg/ml, e.g., from about 50 pg/ml to about 5 mg/ml, e.g., from about 50 pg/ml to about 1 mg/ml, e.g., from about 100 pg/ml to about 800 pg/ml, e.g., from about 200 pg/ml to about 800 pg/ml, e.g., from about 300 pg/ml to about 700 pg/ml, e.g., from about 400 pg/ml to about 600 pg/ml, e.g., from about 450 pg/ml to about 550 pg/ml.
  • concentrations such as from about 50 pg/ml to about 50 mg/ml, e.g., from about 50 pg/ml to about 5 mg/ml,
  • the concentration of ascorbic acid in the culture medium is at least about 75 pg/ml, e.g., at least about 100 pg/ml, e.g., at least about 150 pg/ml, e.g., at least about 200 pg/ml, e.g., at least about 250 pg/ml, e.g., at least about 300 pg/ml, e.g., at least about 350 pg/ml, e.g., at least about 400 pg/ml, e.g., at least about 450 pg/ml, e.g., about 500 pg/ml.
  • Basic fibroblast growth factor (also known as bFGF, FGF2 or FGF-P) is a member of the fibroblast growth factor family.
  • the bFGF used in the culture medium of some embodiments of the invention can be a purified, a synthetic or a recominantly expressed bFGF protein [(e.g., human bFGF polypeptide GenBank Accession No. NP — 001997.5 (SEQ ID N0:31); human bFGF polynucleotide GenBank Accession No. NM — 002006.4 (SEQ ID NO:32).
  • bFGF is preferably purified from a human source or is recombinantly expressed as is further described hereinbelow.
  • bFGF can be obtained from various commercial sources such as Cell Sciences®, Canton, Mass., USA (e.g., Catalogue numbers CRF001 A and CRF001B), Invitrogen Corporation products, Grand Island N.Y., USA (e.g., Catalogue numbers: PHG0261, PHG0263, PHG0266 and PHG0264), ProSpec-Tany TechnoGene Ltd.
  • the concentration of bFGF in culture medium is in the range from about 1 ng/ml to about 10 pg/ml, e.g., from about 2 ng/ml to about 1 pg/ml, e.g., from about 1 ng/ml to about 500 ng/ml, e.g., from about 2 ng/ml to about 500 ng/ml, e.g., from about 5 ng/ml to about 250 ng/ml, e.g., from about 5 ng/ml to about 200 ng/ml, e.g., from about 5 ng/ml to about 150 ng/ml, e.g., about 10 ng/ml, e.g., about 20 ng/ml, e.g.
  • the concentration of bFGF in the culture medium is at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 55 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, at least about 95 ng/ml,
  • Transforming growth factor beta-3 (TGFP3) is involved in the control of proliferation, differentiation, and other functions in many cell types, acts in inducing transformation and as a negative autocrine growth factor.
  • TGFP3 can be obtained from various commercial sources such as R&D Systems Minneapolis Minn., USA.
  • the concentration of TGFP3 in the culture medium is in the range of about 0.05 ng/ml to about 1 pg/ml, e.g., from 0.1 ng/ml to about 1 pg/ml, e.g., from about of about 0.5 ng/ml to about 100 ng/ml.
  • the concentration of TGFP3 in the culture medium is at least about 0.5 ng/ml, e.g., at least about 0.6 ng/ml, e.g., at least about 0.8 ng/ml, e.g., at least about 0.9 ng/ml, e.g., at least about 1 ng/ml, e.g., at least about 1.2 ng/ml, e.g., at least about 1.4 ng/ml, e.g., at least about 1.6 ng/ml, e.g., at least about 1.8 ng/ml, e.g., about 2 ng/ml.
  • the culture medium comprises bFGF at a concentration range of about 0.1 ng/ml to about 500 ng/ml, TGFP3 at a concentration range of about 0.1 ng/ml to about 20 ng/ml, and ascorbic acid at a concentration range of about 50 pg/ml to about 5000 pg/ml.
  • the culture medium of some embodiments of the invention comprises bFGF at a concentration range of about 5 ng/ml to about 150 ng/ml, TGFP3 at a concentration range of about 0.5 ng/ml to about 5 ng/ml, and ascorbic acid at a concentration range of about 400 pg/ml to about 600 pg/ml.
  • the concentration of the lipid mixture in the culture medium is from about 0.5% [volume/volume (v/v)] to about 3% v/v, e.g., from about 0.5% v/v to about 2% v/v, e.g., from about 0.5% v/v to about 1% v/v, e.g., about 1% v/v.
  • the culture medium of some embodiments of the invention comprises bFGF at a concentration range of about 0.1 ng/ml to about 500 ng/ml, TGFP3 at a concentration range of about 0.1 ng/ml to about 20 ng/ml, ascorbic acid at a concentration range of about 50 pg/ml to about 5000 pg/ml, xeno-free serum replacement and a lipid mixture.
  • Non-limiting examples of xeno-free and serum-free culture media which comprise TGFP3, bFGF and ascorbic acid at a concentration of at least 50 pg/ml and which can be used to maintain pluripotent stem cells in a proliferative and undifferentiated states include the HA75 and HA78 culture media.
  • the culture medium further comprises sodium bicarbonate.
  • Sodium bicarbonate can be obtained from Biological Industries, Beit HaEmek, Israel.
  • the concentration of sodium bicarbonate in the culture medium is from about 5% to about 10%, e.g., from about 6% to about 9%, e.g., from about 7% to about 8%, e.g., about 7.5%.
  • the present inventors uncovered that pluripotent stem cells can be maintained in a proliferative, pluripotent and undifferentiated state for at least 15 passages when cultured in a serum-free and xeno-free culture medium which comprises bFGF and ascorbic acid but does not comprise a TGFP isoform.
  • the culture medium comprises no more than 1 ng/ml of the TGFP isoform, e.g., no more than 0.5 ng/ml, e.g., no more than 0.1 ng/ml, e.g., no more than 0.05 ng/ml, e.g., no more than 0.01 ng/ml of the TGFP isoform.
  • the culture medium comprises ascorbic acid at a concentration range of about 400-600 pg/ml and basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml.
  • bFGF basic fibroblast growth factor
  • the culture medium which comprises ascorbic acid at a concentration range of about 400-600 pg/ml and basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml is capable of maintaining pluripotent stem cells in an undifferentiated state in the absence of feeder cell support.
  • bFGF basic fibroblast growth factor
  • the concentration of ascorbic acid in the culture medium is between about 410 pg/ml to about 590 pg/ml, between about 420 pg/ml to about 580 pg/ml, between about 450 pg/ml to about 550 pg/ml, between about 460 pg/ml to about 540 pg/ml, between about 470 pg/ml to about 530 pg/ml, between about 490 pg/ml to about 520 pg/ml, e.g., between about 490 pg/ml to about 510 pg/ml, e.g., about 500 pg/ml.
  • the concentration of bFGF in the culture medium is between about 50 ng/ml to about 200 ng/ml, between about 60 ng/ml to about 190 ng/ml, between about 70 ng/ml to about 180 ng/ml, between about 80 ng/ml to about 170 ng/ml, between about 90 ng/ml to about 160 ng/ml, between about 90 ng/ml to about 150 ng/ml, between about 90 ng/ml to about 130 ng/ml, between about 90 ng/ml to about 120 ng/ml, e.g., about 100 ng/ml.
  • the concentration of bFGF in the culture medium is about 50, about 55, about 60, about 65, about 70, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200 ng/ml.
  • the culture medium which comprises ascorbic acid at a concentration range of about 400-600 pg/ml and basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml further comprises xeno-free serum replacement.
  • the culture medium which comprises ascorbic acid at a concentration range of about 400-600 pg/ml and basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml further comprises a lipid mixture.
  • the culture medium comprises bFGF at a concentration of about 50-200 ng/ml and ascorbic acid at a concentration of about 400-600 pg/ml is devoid of sodium-bicarbonate. According to some embodiments of the invention, the culture medium comprises bFGF at a concentration of about 50-200 ng/ml and ascorbic acid at a concentration of about 400- 600 pg/ml, xeno-free serum replacement at a concentration of about 1% and lipid mixture at a concentration of about 1%.
  • a non-limiting example of a xeno-free, serum-free, and TGFP isoform-free culture medium which comprises ascorbic acid at a concentration range of about 400-600 pg/ml, bFGF at a concentration range of about 50-200 ng/ml, xeno-free serum replacement and a lipid mixture and which is capable of maintaining pluripotent stem cells such as hESCs and human iPS cells in a proliferative and undifferentiated state for at least 21 passages in the absence of feeder cell support is the HA77 culture medium or a culture medium similar to the HA77 medium but which is devoid of sodium bi-carbonate such as a culture medium which consists of DMEM/F12 (94%) (Biological Industries, Israel, Sigma Israel), L-glutamine 2 mM (Invitrogen corporation, Sigma, Israel), ascorbic acid 500 pg/ml (Sigma, Israel), bFGF — 100 ng (Invitrogen corporation), SR3
  • the present inventors have uncovered novel serum-free and highly defined culture media, which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three-dimensional (i.e., a suspension culture) systems in the absence of feeder cell support.
  • the phrase “suspension culture” refers to a culture in which the pluripotent stem cells are suspended in a medium rather than adhering to a surface.
  • the serum-free culture medium which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three-dimensional culture systems in the absence of feeder cell support comprises basic fibroblast growth factor (bFGF) at a concentration range of about 50-200 ng/ml.
  • bFGF basic fibroblast growth factor
  • the culture medium comprises between about 55-190 ng/ml, e.g., between about 60-190 ng/ml, e.g., between about 70-180 ng/ml, e.g., between about 80-160 ng/ml, e.g., between about 90-150 ng/ml, e.g., between about 90-140 ng/ml, e.g., between about 90- 130 ng/ml, e.g., between about 90-120 ng/ml, e.g., between about 90-110 ng/ml, e.g., between about 95-105 ng/ml, e.g., about 100 ng/ml.
  • the culture medium which comprises bFGF between about 50-200 ng/ml further comprises serum replacement.
  • a non-limiting example of a culture medium which comprises bFGF at a concentration between about 50-200 ng/ml is the YF100 medium which comprises a basic medium (e.g., DMEM/F12, 85%), serum replacement (15%), bFGF (100 ng/ml), L- glutamine (2 mM), P — mercaptoethanol (0.1 mM) and non-essential amino acid stock (1%).
  • a basic medium e.g., DMEM/F12, 85%
  • serum replacement e.g., fetal
  • bFGF 100 ng/ml
  • L- glutamine 2 mM
  • P — mercaptoethanol 0.1 mM
  • non-essential amino acid stock 1%
  • the serum-free culture medium which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three-dimensional culture systems in the absence of feeder cell support consists of a basic medium, ascorbic acid at a concentration range of about 50 pg/ml to about 500 pg/ml, bFGF at a concentration range between about 2 ng/ml to about 20 ng/ml, L-glutamine, and serum replacement.
  • the serum-free culture medium which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three-dimensional culture systems in the absence of feeder cell support consists of a basic medium, ascorbic acid at a concentration range of about 50 pg/ml to about 500 pg/ml, bFGF at a concentration range between about 2 ng/ml to about 20 ng/ml, L-glutamine, serum replacement and a lipid mixture.
  • the serum replacement can be any xeno-free serum replacement (devoid of animal contaminants) at a concentration range from 1-20% depending on the serum replacement used.
  • Non-limiting examples of such a culture medium include the modified HA13(a) medium [DMEM/F12 (95%), L-glutamine 2 mM, ascorbic acid 500 pg/ml, bFGF — 4 ng, and SR3 — 1%]; the modified HA13(b) medium [DMEM/F12 (95%), L-glutamine 2 mM, ascorbic acid 500 pg/ml, bFGF — 4 ng, SR3 — 1% and a lipid mixture (1%)]; the modified HA13(c) medium [DMEM/F12 (95%), L- glutamine 2 mM, ascorbic acid 50 pg/ml, bFGF — 4 ng, and SR3 — 1%]; and the modified HA13(d) medium [DMEM/F12 (95%), L-glutamine 2 mM, ascorbic acid 50
  • These culture media were capable of maintaining pluripotent stem cells (e.g., hESCs and hips cells) in a proliferative, pluripotent and undifferentiated state for at least 20 passages when cultured in a two- dimensional (e.g., on a feeder-layer free culture system; data not shown) and for at least 20 passages when cultured on a three-dimensional culture system (e.g., suspension culture without adherence to an external substrate, cell encapsulation or to protein carrier; data not shown).
  • pluripotent stem cells e.g., hESCs and hips cells
  • the serum-free culture medium which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three-dimensional culture systems in the absence of feeder cell support comprises an IL6RIL6 chimera at a concentration range of about 50-200 picogram per milliliter (pg/ml).
  • the concentration of the IL6RIL6 chimera in the culture medium is in the range from about 55 pg/ml to about 195 pg/ml, e.g., from about 60 pg/ml to about 190 pg/ml, e.g., from about 65 pg/ml to about 185 pg/ml, e.g., from about 70 pg/ml to about 180 pg/ml, e.g., from about 75 pg/ml to about 175 pg/ml, e.g., from about 80 pg/ml to about 170 pg/ml, e.g., from about 85 pg/ml to about 165 pg/ml, e.g., from about 90 pg/ml to about 150 pg/ml, e.g., from about 90 pg/ml to about 140 pg/ml,
  • the IL6RIL6 chimera-containing culture medium further comprises bFGF.
  • concentration of bFGF in the IL6RIL6 chimera-containing culture medium is in the range of from about 1 ng/ml to about 10 pg/ml, e.g., from about 2 ng/ml to about 1 pg/ml, e.g., from about 2 ng/ml to about 500 ng/ml, e.g., from about 5 ng/ml to about 150 ng/ml, e.g., from about 5 ng/ml to about 100 ng/ml, e.g., from about 5 ng/ml to about 80 ng/ml, e.g., from about 5 ng/ml to about 50 ng/ml, e.g., from about 5 ng/ml to about 30 ng/ml, e.g., about 5 ng/ml
  • the IL6RIL6 chimera-containing culture medium further comprises serum replacement.
  • the concentration of KNOCKOUTTM Serum Replacement in the IL6RIL6 chimera-containing culture medium is in the range from about 1% (v/v) to about 50% (v/v), e.g., from about 5% (v/v) to about 40% (v/v), e.g., from about 5% (v/v) to about 30% (v/v), e.g., from about 10% (v/v) to about 30% (v/v), e.g., from about 10% (v/v) to about 25% (v/v), e.g., from about 10% (v/v) to about 20% (v/v), e.g., about 15% (v/v).
  • the culture medium comprises IL6RIL6 chimera at a concentration range of about 50-200 pg/ml, bFGF at a concentration range of about 5-50 ng/ml and serum replacement at a concentration of about 5-40%.
  • the serum-free culture medium which can maintain pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in two-dimensional and three- dimensional culture systems in the absence of feeder-cells support comprises LIF at a concentration of at least 2000 units/ml.
  • LIF Leukemia inhibitory factor
  • the LIF used in the culture medium of some embodiments of the invention can be a purified, synthetic or recombinantly expressed LIF protein [e.g., human LIF polypeptide GenBank Accession No. NP — 002300.1. It should be noted that for the preparation of a xeno-free culture medium LIF is preferably purified from a human source or is recombinantly expressed.
  • Recombinant human LIF can be obtained from various sources such as Chemicon, USA (Catalogue No. LIF 10100) and AbD Serotec (MorphoSys US Inc, Raleigh, N.C. 27604, USA).
  • Murine LIF ESGRO® LIF
  • Millipore, USA Catalogue No. ESG1107
  • the concentration of LIF in the culture medium is from about 2000 units/ml to about 10,000 units/ml, e.g., from about 2000 units/ml to about 8,000 units/ml, e.g., from about 2000 units/ml to about 6,000 units/ml, e.g., from about 2000 units/ml to about 5,000 units/ml, e.g., from about 2000 units/ml to about 4,000 units/ml.
  • the concentration of LIF in the culture medium is at least about 2000 units/ml, e.g., at least about 2100 units/ml, e.g., at least about 2200 units/ml, e.g., at least about 2300 units/ml, e.g., at least about 2400 units/ml, e.g., at least about 2500 units/ml, e.g., at least about 2600 units/ml, e.g., at least about 2700 units/ml, e.g., at least about 2800 units/ml, e.g., at least about 2900 units/ml, e.g., at least about 2950 units/ml, e.g., about 3000 units/ml.
  • the LIF-containing culture medium further comprises bFGF.
  • concentration of bFGF in the LIF-containing culture medium is in the range of about 0.1 ng/ml to about 10 pg/ml, e.g., from about 2 ng/ml to about 1 pg/ml, e.g., from about 2 ng/ml to about 500 ng/ml, e.g., from about 5 ng/ml to about 150 ng/ml, e.g., from about 5 ng/ml to about 100 ng/ml, e.g., from about 5 ng/ml to about 80 ng/ml, e.g., from about 5 ng/ml to about 50 ng/ml, e.g., from about 5 ng/ml to about 30 ng/ml, e.g., about 5 ng/ml, e.g., about 10 ng/ml, e.g.
  • the LIF- containing culture medium further comprises serum replacement.
  • the culture medium comprises LIF at a concentration of about 2000-10,000 units/ml, bFGF at a concentration range from about 0.1 ng/ml to about 10 pg/ml and KNOCKOUTTM Serum Replacement at a concentration range from about 1% (v/v) to about 50% (v/v).
  • the culture medium comprises LIF at a concentration of about 2000-5,000 units/ml, bFGF at a concentration of about 5-50 ng/ml and serum replacement at a concentration of about 5- 30%.
  • the ingredients included in the culture medium of some embodiments of the invention are substantially pure, with a tissue culture and/or a clinical grade.
  • a cell culture which comprises the pluripotent stem cell of some embodiments of the invention and the culture medium of some embodiments of the invention.
  • cell culture is feeder cells free (e.g., being devoid of feeder cells or feeder cell conditioned medium).
  • the pluripotent stem cells which are included in the cell culture of some embodiments of the invention exhibit a stable karyotype (chromosomal stability) during the culturing period, e.g., for at least 2 passages, e.g., at least 4 passages, e.g., at least 8 passages, e.g., at least 15 passages, e.g., at least 20 passages, e.g., at least 25 passages, e.g., at least 30 passages, e.g., at least 35 passages, e.g., at least 40 passages, e.g., at least 45 passages, e.g., at least 50 passages.
  • a stable karyotype chromosomal stability
  • the invention provides the administration of agents capable of acting as senolytic agents together with regenerative cells such as the regenerative cells generated through the current invention.
  • Regenerative cells may be stem cells, progenitor cells, monocytes, T cells, B cells, or other cells capable of directly or indirectly inducing healing of the body, organs, or cells.
  • enhancement of endogenous regenerative activity is enhanced through administration of senolytic agents.
  • Some senolytic agents include least one of tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, tigecycline, pyrvinium, atovaquone, bedaquiline, irinotecan, sorafenib, niclosamide, stirpentol, chloroquine, rapamycin, a mitoriboscin, a mitoketoscin, a mitoflavoscin, 2-butene-l,4-bis-TPP; a derivative of 2-butene-l,4-bis-TPP; 2- chlorobenzyl-TPP; a derivative of 2-chlorobenzyl-TPP; 3-methylbenzyl-TPP; a derivative of 3-methylbenzyl-TPP; 2,4-dichlorobenzyl-TPP; a derivative of 2,4-dichlorobenzyl-TPP; 1
  • senolytic agents are used together with approaches that stimulate neurogenesis for example electroconvulsive therapy [2, 3], transcranial magnetic stimulation [4-31], low intensity transcranial ultrasound stimulation [32-34], transcranial direct current stimulation [35],
  • senolytic agents are used together with approaches that suppress inflammation, including approaches that reduce Macrophage 2 [36], astrocyte 2, or neutrophil 2 activity.
  • agents that suppress activity of senescent cells are combined with suppressors of interleukin- 17 for treatment of inflammatory diseases [37], [0672] It is known that patients with liver failure have elevated systemic levels of IL-17 as well as IL-17 made by T cells [38, 39], IL-17 levels correlate with severity of liver failure [40-44], Levels of Thl7 cells which produce IL-17 correlate with liver failure [45-48], L-17 induces liver failure in animal models [49-52] and patients [53], Suppressing IL-17 reduces liver failure in animal models [54-60],
  • IL-17 agents that increase IL-17 cause heart failure in people [78]
  • Blocking IL-17 reduces animal models of heart failure [79]
  • Thl7 cells which are T cells that make IL- 17
  • IL- 17 stimulates monocytes to make inflammatory agents that block hematopoiesis in aplastic anemia [83]
  • senolytic agents are utilized to augment efficacy of regenerative stem cell therapies such as adult stem cells, or combinations of cells.
  • Senolytic agents may include, BTSA1 [84], vilazodone [85], a SGLT2 inhibitor such as ca[86]nagliflozin which directly is senolytic but also stimulates immune mediated clearing of senolytic cells [87], a casein kinase 2 inhibitor such as 4,5,6,7-tetrabromo-2-azabenzamidazole [88], LY-D6/2 [88], PI3K-inhibitors such as wortmannin and its clinical derivative, PX-866 [89], USP7 inhibitors such as P5091 [90], bcl-2 inhibitors such as the Bh3 mimetic ABT-263 [91-147], ABT-737 [148-151], piperlongumine [152], fisetin [153-170], EF24 [171],
  • adipose derived regenerative cells One such combination of cells that are utilized in the context of the current invention is adipose derived regenerative cells.
  • One such population is the stromal vascular fraction (SVF), which is comprised of adipose-derived mononuclear cells, pericytes, EPCs with several cell types, including MSC, hematopoietic stem cells [176], Treg [177], and alternatively activated monocytes.
  • SSF stromal vascular fraction
  • MSC are rare in bone marrow, comprising only 0.01-0.001% of bone marrow cells [178], these cells are found at 100-500 fold higher frequencies in the SVF [179],
  • Cell quantification of SVF harvested from middle-aged humans revealed a range of approximately 400 000 cells/mL of aspirate [180] of which approximately 20% of the cells are endothelial and hematopoietic cells [179],
  • the cell yield from SVF is highly dependent upon the site of adipose tissue collection and the methods used for enzymatic digestion [181]
  • ASC are located in the adipose perivascular niche [182]
  • the extent of vascularization strongly influences the MSC numbers that can be recovered from different subcutaneous body sites [183]
  • MSC are generated as immature MSC from more immature cellular sources such as the pluripotent stem cells described in the current invention.
  • MSC are dedifferentiated. Generation of dedifferentiated MSC can be found in numerous publications, for example, valproic acid, a histone deacetylase inhibitor has been used previously to endow a “younger” state onto MSC.
  • MSC immune suppressive and immune modulatory effects have led to their widespread application in pre-clinical and clinical studies for treating autoimmune disorders [226], MSC are considered to be poorly immunogenic cells, exhibiting low expression levels of HLA class I and negligible expression of HL A class II or costimulatory molecules [227, 228], although expression of HLA molecules is upregulated upon exposure of MSC to pro-inflammatory stimuli such as the cytokine IFN-D [227, 229], MSC possess immune modulatory abilities that are mediated by direct contact with immune cells and/or through secretion of soluble factors such as prostaglandin E2 (PGE2), nitric oxide (NO), transforming growth factor (TGF-D), leukemia inhibitory factor (LIF), and indolamine 2.3 -dioxygenase (IDO) [230-233], BMSCs inhibit the differentiation of dendritic cells from their precursors [234], inhibit B cell maturation [235], and stimulate the release of the immune suppressive cytokine
  • senolytic agents are utilized to enhance activity of T regulatory cells.
  • Feuerer et al. [177] examined adipose tissue for content of Treg cells based on functionality and expression of the CD4+, CD25+, FoxP3+ phenotype. Increased numbers of these cells were observed in adipose compared to other peripheral tissues.
  • the authors made a case for the role of Treg in controlling inflammation associated with obesity.
  • the adipose Treg’s appeared to have a “primed” phenotype, as witnessed by highly elevated IL- 10 transcript and protein levels in adipose Treg.
  • the invention discloses augmentation of T regulatory cell activity by senolytic agents regardless if the T regulatory cells are endogenously derived or are exogenous. In some cases T regulatory cells are generated by treatment of the patient with means that expand T regulatory cells in vivo.
  • the cell culture of the invention exhibit a doubling time of at least 20 hours, e.g., a doubling time which is between 20 to 40 hours (e.g., about 36 hours), thus representing a non-tumorigenic, genetically stable pluripotent stem cells (e.g., hESCs and iPS cells).
  • the cell culture of the invention is characterized by at least 40%, at least 50%, at least 60%, e.g., at least 70%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95% of undifferentiated pluripotent stem cells.
  • a method of expanding and maintaining pluripotent stem cells in a pluripotent and undifferentiated state Furthermore, for generation of iPSC for creating MSC to use in orthopedic conditions, we present multiple culture conditions and media, which permit the indefinite culture and robust proliferation of primate pluripotent stem cells in an undifferentiated state with continued expression of characteristic pluripotency markers. Also, the media described are prepared in the complete absence of both feeder cells and conditioned medium. As described here, the defined culture conditions and media are suitable for use with human pluripotent stem cells. Pluripotent cells express one or more pluripotent cellspecific marker, such as Oct-4, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.
  • iPS cells include, but are not limited to human ES cells (e.g., Hl, H7, H9 and H14), iPS cells (e.g., iPS- Foreskin and iPS-IMR90), and vector-free iPS cells (e.g., iPS-DF19-9, iPS-DF4-3, and iPS-DF6-9), which are all available through WiCell® International Stem Cell (WISC) Bank (Madison, Wis.).
  • WISC WiCell® International Stem Cell
  • primate pluripotent stem cells including iPS and vector- free iPS cells
  • certificate of analyses for primate pluripotent stem cells cultured on growth matrix using the fully defined medium disclosed here is available on the WISC website.
  • Additional pluripotent stem cell lines include, but are not limited to, disease model cell lines and genetically modified lines containing marker genes.
  • the culture conditions and media are entirely free of non-human animal products and all proteins used are of human origin. The development of these media and culture conditions make possible the derivation and maintenance of human pluripotent stem cell lines in defined and controlled conditions without direct or indirect exposure to non-human animal cells of any kind.
  • the media and culture conditions described here enable the derivation of new lines of human pluripotent stem cells which have never been exposed to non-human cells or a to medium in which animal cells were cultured.
  • the medium is free of animal products or proteins. This medium has been demonstrated to support undifferentiated pluripotent stem cell proliferation through at least twenty -five passages, which is firm evidence that it will support such cultures indefinitely.
  • a suitable medium is capable of supporting the derivation of new human ES and iPS cell lines, and derived using the media described herein after as “new lines”. These lines have passed through more than ten passages in culture. In the past, use of conditioned medium has sometimes been referred to as creating “feeder-free” culture conditions.
  • a defined and humanized medium for the culture and proliferation of human pluripotent stem cells typically includes salts, vitamins, lipids, an energy source such as glucose, minerals, amino acids, growth factors and other components.
  • stem cell media have included serum from one source or another.
  • the serum replacement additive can be a commercially available product sold for that purpose or can be a formulated mixture of proteins, including but not limited to serum albumin, vitamins, minerals, a transferrin or a transferrin substitute, and insulin or an insulin substitute.
  • the albumin, insulin and transferrin may be recombinant proteins.
  • This serum replacement additive may also be supplemented with, but is not limited to, selenium and a mixture of lipids.
  • a defined serum replacement mix is used in lieu of serum from any source in culturing human pluripotent stem cells, to avoid variation in serum constituents and to use media that are as defined as possible.
  • TGFP transforming growth factor beta
  • GABA gamma-aminobutyric acid
  • PA pipecolic acid
  • LiCl lithium chloride
  • TGFP transforming growth factor beta
  • other lithium salts can substitute for LiCl in the cell culture medium.
  • These may include lithium salts, wherein the anion includes, but is not limited to, chloride, bromide, carbonate, citrate, sulfate, or other biologically compatible monovalent anion (see, for example, US 2004/0028656 and WO 2008/055224).
  • additives By selective deletion of these additives, it may be empirically determined if one or more of them is not required to achieve this result for a given medium. However, it is clear that the combination is sufficient to enable a variety of media that will support the long-term culture and proliferation of undifferentiated human pluripotent stem cells without feeder cells or conditioned medium.
  • GABA GABA receptor
  • scientific literature includes the identification of several molecules which are agonists of that same receptor and might be substituted for GABA in the medium as an equivalent.
  • PA also interacts with the GABA receptor. While both PA and GABA were found to be helpful in the medium at the concentrations used here, it is also envisioned that one or the other of these constituents could be increased in concentration to obviate the need for the other.
  • the FGF in higher concentrations (40 to 100 ng/ml) seems to obviate the need for feeder cells.
  • the inventors also found it advantageous to include in the culture vessel of human pluripotent stem cells a biological matrix.
  • a biological matrix is composed of a blend of four human proteins: collagen isolated from human placenta, fibronectin isolated from human plasma, vitronectin isolated from human plasma or from a recombinant source, and laminin isolated from human placenta.
  • extracellular matrices may be suitable for use in the present invention, which include, but are not limited to, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
  • Other suitable extracellular matrices may include, but are not limited to, GeltrexTM.
  • the major components of GeltrexTM matrix include laminin, collagen IV, entactin, and heparin sulfate proteoglycan.
  • Preferred matrices of the present invention include collagen, fibronectin, vitronectin, and laminin derived matrices.
  • Peripheral blood mononuclear cells were isolated by Ficoll from a health volunteer and separated into Vasa positive or Vasa negative cells using magnetic activated cell sorting according to the manufacturers instructions. Cells are subsequently exposed to a “dedifferentiation cocktail” of OCT4, NANOG and KLF4 (R&D Systems) according to the manufacturers instructions. Cells were cultured on feeder cells and were assessed for OCT4 gene expression after 1, 2, and 4 weeks of culture using quantitative RT-PCR and expressed as relative units compared to internal control (GAPDH). As a positive control a standard iPSC cell line was used. Results are shown in FIG. 1.
  • PBMC Peripheral blood mononuclear cells
  • monocytes were isolated by selection for CD14 using magnetic activated cell sorting.
  • VASA expressing monocytes were further purified using MACS based on detection of extracellular VASA.
  • Cells were treated with valproic acid and tricostatin A prior to transfection with cytoplasmic extract from induced pluripotent stem cells.
  • Cells were treated with 1 microMolar of valproic acid and 100 nanoMolar of tricostatin A.
  • Subsequent to transfection cells were assessed for expression of pluripotency markers by flow cytometry at days 7, 14 and 21. Results are shown in FIGs. 2A and 2B, and the tables below.
  • PBMC Peripheral blood mononuclear cells
  • T cells were isolated using Ficoll. T cells were isolated using magnetic activated cell sorting for CD3 positive selection. Cultured T cells were treated with valproic acid and tricostatin A prior to transfection with OCT4, KLF4, SOX2 and c-Myc. Cells were treated with 1 microMolar of valproic acid and 100 nanoMolar of tricostatin A. Subsequent to transfection cells were assessed for expression of pluripotency markers by flow cytometry at days 7, 14 and 21. Results are shown in FIGs. 3 A and 3B and the tables below.
  • Control NANOG CD9 CD24 1.0 21.0 5458.0 5545.0 4544.0 2.0 22.0 5546.0 4347.0 3453.0 3.0 24.0 5535.0 4347.0 4585.0 4.0 21.0 5866.0 4432.0 3247.0 5.0 21.0 4573.0 5735.0 4573.0 6.0 27.0 5753.0 4348.0 4434.0 7.0 26.0 5638.0 4352.0 4234.0 8.0 27.0 5325.0 4573.0 3263.0 9.0 23.0 6548.0 4537.0 3238.0 10.0 23.0 6547.0 4853.0 3246.0
  • PBMC Peripheral blood mononuclear cells
  • Example 5 Superior Activity of Combined Senolytic Immunotherapy with Mesenchymal Stem Cells and Hepatic Progenitor Cells
  • Non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice on BALB/c background were divided into the following groups, with each group consisting of 12 mice per group (based on previous experience immune deficient mice have a high rate of contamination, we are adding 2 mice per group to account for this).
  • NOD-SCID mice received purified IgG antibodies from wildtype BALB/c mice adoptively transferred Based on previous studies a concentration of 2 micrograms of antibody protein per mouse was injected. The following groups were used:
  • Carbon tetrachloride was purchased from Sigma Aldrich (ThermoFisher) and dissolved in vegetable oil at 10% concentration and administered to animals by gavage at a dose of 0.28 mL/kg. Animals received treatment interventions listed above 24 hours after CCL4 administration. Cells were administered at a concentration of 500,000 cells per mouse. After the cellular transplantation, blood samples were collected at 24, 48 and 72 hours and assessed for liver function by AST quantification. Results are shown in FIG. 5.
  • rTMS Improves Functional Recovery by Enhancing Neurogenesis and Activating BDNF/TrkB Signaling in Ischemic Rats. Int J Mol Sci, 2017. 18(2).
  • Thl7 cells may indicate liver injury in patients infected with HBV. Liver Int, 2014. 34(2): p. 266-73.
  • Target Platelet Toxicity ofBcl-x(L) Inhibitors PROTACs, SNIPERs and Prodrug-Based Approaches. Chembiochem, 2022. 23(12): p. e202100689.

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Abstract

L'invention concerne des procédés, des compositions de matière et des protocoles pour la génération de cellules dédifférenciées possédant une capacité pluripotente ou multipotente. Les procédés comprennent l'extraction de cellules avec une proclivité pour la dédifférenciation à partir du sang périphérique, la culture desdites cellules dans un milieu pour assurer une multiplication stable desdites cellules, et ensuite l'application d'un protocole de dédifférenciation en plusieurs étapes pour obtenir une population de cellules pluripotentes et/ou multipotentes. Des cellules sanguines périphériques sont enrichies pour des cellules exprimant Vasa et/ou Dazl, et/ou Stella et multipliées dans des conditions pour prendre en charge la prolifération sans perte de ces marqueurs. Ensuite, des cellules sont traitées avec des cocktails de dédifférenciation en plusieurs étapes afin de générer des cellules souches pluripotentes. Lesdites cellules souches pluripotentes sont capables de générer des cellules multi-lignées ainsi que d'exprimer des marqueurs caractéristiques de pluripotence tels que OCT4 et NANOG.
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US10383895B2 (en) * 2016-02-18 2019-08-20 Viera Bioscience, Inc. Stimulation of therapeutic angiogenesis by T regulatory cells
US20230226119A1 (en) * 2021-10-11 2023-07-20 Therapeutic Solutions International, Inc. Umbilical cord derived regenerative and immune modulatory stem cell populations
WO2023173370A1 (fr) * 2022-03-17 2023-09-21 Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences Milieux et procédés pour la production de cellules et de tissus humains à partir de tératomes, d'organoïdes et de corps embryoïdes

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