WO2010059806A2 - Génération de cellules souches pluripotentes induites sans utiliser de vecteurs viraux - Google Patents

Génération de cellules souches pluripotentes induites sans utiliser de vecteurs viraux Download PDF

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WO2010059806A2
WO2010059806A2 PCT/US2009/065117 US2009065117W WO2010059806A2 WO 2010059806 A2 WO2010059806 A2 WO 2010059806A2 US 2009065117 W US2009065117 W US 2009065117W WO 2010059806 A2 WO2010059806 A2 WO 2010059806A2
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cell
polypeptide
penetration peptide
linked
cell penetration
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PCT/US2009/065117
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WO2010059806A3 (fr
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Anthony J. White
Raj R. Makkar
Eduardo Marban
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Cedars-Sinai Medical Center
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Priority to US13/130,560 priority Critical patent/US20110300111A1/en
Publication of WO2010059806A2 publication Critical patent/WO2010059806A2/fr
Publication of WO2010059806A3 publication Critical patent/WO2010059806A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/605—Nanog
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/608—Lin28

Definitions

  • reprogrammed mammalian cells e.g., induced pluripotent stem cells
  • the methods involve contacting a differentiated somatic cell with transducible polypeptides comprising a reprogramming factor polypeptide linked to a cell penetration peptide so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • transducible polypeptides comprising a reprogramming factor polypeptide linked to a cell penetration peptide so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • methods for cardiac differentiation of a mammalian cell without the use of viral or plasmid vectors such methods involve contacting a mammalian cell exhibiting at least one characteristic of pluripotency with a transducible polypeptide so that cardiac differentiation of the cell occurs.
  • pluripotent stem cells The reprogramming of adult cells into a less differentiated state, e.g., into pluripotent stem cells, is of great medical importance.
  • pluripotent cells generated from readily available adult somatic cells e.g., skin
  • creation of disease-specific cell lines would greatly facilitate research, for example high throughput drug screening. Such cells would provide the additional benefit of avoiding the legal and ethical issues presently associated with embryo destruction.
  • iPS induced pluripotent stem
  • iPS cells have the morphology of ES cells (formation of colonies), express a gene profile characteristic of ES cells, and are pluripotent in vivo (teratoma formation) and in vitro (embryoid body formation). Similarly, it has been reported that human cells can be induced to pluripotency by these factors, and it appears that the c-Myc gene may not be an essential factor for iPS cell creation. See e.g.
  • the methods presented herein generally provide for the generation of a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency.
  • the methods provided herein generate induced pluripotent stem cells.
  • the methods comprise the step of contacting a differentiated mammalian cell with one or more transducible polypeptides, wherein a transducible polypeptide comprises the amino acid sequence of a reprogramming factor linked to the amino acid sequence of a cell penetration peptide so that a reprogrammed mammalian cell, e.g., an induced pluripotent stem cell, is generated.
  • the reprogramming factor is selected from the group consisting of Oct3/4, Sox2, Nanog, Lin28, c-myc and Klf4, e.g., human Oct 3/4, human Sox2, human Nanog, human Lin28, human c- myc and human Klf4, and active forms thereof.
  • at least one reprogramming factor is a human polypeptide.
  • each of the reprogramming factors is a human polypeptide.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian somatic cell, e.g., a somatic adult cell, with one or more different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell in vitro, for example, ex vivo, with one or more different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian somatic cell, e.g., an adult somatic cell, in vitro, for example, ex vivo, with one or more different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • a differentiated mammalian somatic cell e.g., an adult somatic cell
  • in vitro for example, ex vivo
  • the transducible polypeptide comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide.
  • the differentiated mammalian cell is contacted with at least 2, 3, 4, 5 or 6 different transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and a Klf4 polypeptide linked to a cell penetration peptide.
  • a transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and a Kl
  • the cell penetration peptide is selected from the group consisting of herpes simplex virus (HSV) type I protein VP22 (“VP22”), human immunodeficiency virus (HIV-I) transactivator protein TAT (“TAT”), a homeodomain from the Antennapedia polypeptide (“AntP HD”), a polymer of L-arginine or D-arginine amino acid residues (“poly-arginine”), or transducing fragments thereof.
  • HSV herpes simplex virus
  • HAT human immunodeficiency virus
  • TAT human immunodeficiency virus
  • AntP HD Antennapedia polypeptide
  • poly-arginine a polymer of L-arginine or D-arginine amino acid residues
  • the reprogramming factor polypeptide is linked to the amino terminus, that is, the amino terminal amino acid, of the cell penetration peptide. In other embodiments, the reprogramming factor polypeptide is linked to the carboxy terminus, that is, the carboxy terminal amino acid, of the cell penetration peptide. Linkage can be direct or indirect, e.g., via a linker, such as via a stretch of one or more amino acid residues.
  • a transducible polypeptide can comprise a single cell penetration peptide or multiple, e.g., 2, 3, 4, 5, or 6 cell penetration peptides. In instances where a transducible polypeptide comprises multiple cell penetration peptides, the cell penetration peptides can be each be of the same sequence, or can vary in sequence.
  • a transducible polypeptide in addition to a reprogramming factor and a cell penetration peptide, can further comprise a purification moiety that can be used in isolation and/or purification of the transducible polypeptide.
  • a transducible polypeptide can further comprise a polyhistidine moiety, e.g., six histidine residues, which can, for example, be incorporated at the amino terminal end of the transducible protein.
  • the polyhistidine moiety can be used in conjunction with well known nickel-chelate chromatography to isolate and purify the transducible polypeptide.
  • a transducible polypeptide in addition to a reprogramming factor and a cell penetration peptide, a transducible polypeptide further comprises a nuclear localization signal (NLS) which enhances nuclear localization of the transducible polypeptide.
  • NLS nuclear localization signal
  • the NLS comprises an SV40 large T antigen sequence, e.g., PKKKRKV (SEQ ID NO: 41).
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated.
  • the cell penetration peptides of the transducible polypeptides can each be of the same sequence or can vary in sequence.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell with a plurality of different transducible polypeptides, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, and iii) a Klf4 polypeptide linked to a cell penetration peptide.
  • the cell penetration peptide is selected from the group consisting of VP22, TAT, AntP HD, poly-arginine, or transducible fragments thereof.
  • the reprogrammed mammalian cell is an induced pluripotent stem cell.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, and iv) a Klf4 polypeptide linked to a cell penetration peptide.
  • the cell penetration peptide is selected from the group consisting of VP22, TAT, AntP HD, poly-arginine, or transducible fragments thereof.
  • the reprogrammed mammalian cell is an induced pluripotent stem cell.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a Nanog polypeptide linked to a cell penetration peptide, and iv) a Lin28 polypeptide linked to a cell penetration peptide.
  • the cell penetration peptide is selected from the group consisting of VP22, TAT, AntP HD, poly-arginine, or transducing fragments thereof.
  • the reprogrammed mammalian cell is an induced pluripotent stem cell.
  • a method for generating a reprogrammed mammalian cell comprising contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that a reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, iv) a Klf4 polypeptide linked to a cell penetration peptide, v) a Nanog polypeptide linked to a cell penetration peptide, and vi) a Lin28 polypeptide linked to a cell penetration peptide.
  • the cell penetration peptide is selected from the group consisting of VP22, TAT, AntP HD, poly-arginine, or transducible fragments thereof.
  • the reprogrammed cell is an induced pluripotent stem cell.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency, wherein the isolated reprogrammed mammalian cell is generated by a method provided herein.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency, wherein the isolated reprogrammed mammalian cell is generated by a method comprising: contacting a differentiated mammalian cell with one or more different transducible polypeptides so that the reprogrammed mammalian is generated, wherein the one or more different transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked
  • the isolated reprogrammed mammalian cell is generated by contact with at least 2, 3, 4, 5 or 6 different transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and a Klf4 polypeptide linked to a cell penetration peptide.
  • a transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide,
  • the reprogrammed mammalian cell comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell does not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibits at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency
  • the isolated reprogrammed mammalian cell is generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, and iii) a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell does not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibits at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency
  • the isolated reprogrammed mammalian cell is generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, and iv) a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell does not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibits at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency
  • the isolated reprogrammed mammalian cell is generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a Nanog polypeptide linked to a cell penetration peptide, and iv) a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, and/or a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell does not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, or a Lin28 polypeptide linked to a cell penetration peptide, but nonetheless exhibits at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated reprogrammed mammalian cell that exhibits at least one characteristic of pluripotency
  • the isolated reprogrammed mammalian cell is generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, iv) a Klf4 polypeptide linked to a cell penetration peptide, v) a Nanog polypeptide linked to a cell penetration peptide, and vi) a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell comprises an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, and/or a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cell does not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, or a Lin28 polypeptide linked to a cell penetration peptide, but nonetheless exhibits at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method provided herein.
  • the isolated population of cells comprises about IxIO 5 , 5xlO 5 , IxIO 6 , 5xl0 6 ,lxl0 7 , 5xlO 7 , IxIO 8 , or 5xlO 8 reprogrammed cells or total cells.
  • the isolated population of cells is present in a formulation suitable for administration to a human.
  • the isolated population of cells is present in a bag, e.g., a plastic bag, such as a plastic bag suitable for use in administration of the cells to a human.
  • the isolated population of cells is present in a syringe, such as a sterile syringe suitable for administration of the cells to a human.
  • an isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method comprising: contacting a differentiated mammalian cell with one or more different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the one or more different transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, or a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells are generated by contact with at least 2, 3, 4, 5 or 6 different transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide.
  • a transducible polypeptides selected from the group consisting of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the reprogrammed mammalian cells of such isolated populations comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells do not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, a Lin28 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibit at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, and iii) a Klf4 polypeptide linked to a cell penetration peptide.
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the reprogrammed mammalian cells of such populations of cells comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells do not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibit at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • a isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, and iv) a Klf4 polypeptide linked to a cell penetration peptide.
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the reprogrammed mammalian cells of such populations of cells comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, and/or a Klf4 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells do not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, or a Klf4 polypeptide linked to a cell penetration peptide, but nonetheless exhibit at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • a isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a Nanog polypeptide linked to a cell penetration peptide, and iv) a Lin28 polypeptide linked to a cell penetration peptide.
  • isolated ed populations of cells further comprising at least one other isolated population of cells, e.g., stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the reprogrammed mammalian cells of such populations of cells comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, and/or a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells do not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, or a Lin28 polypeptide linked to a cell penetration peptide, but nonetheless exhibit at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • an isolated population of cells comprising at least 70%, at least 80%, at least 90%, or at least 95% reprogrammed mammalian cells that exhibit at least one characteristic of pluripotency, wherein the reprogrammed mammalian cells are generated by a method comprising: contacting a differentiated mammalian cell with a plurality of different transducible polypeptides so that the reprogrammed mammalian cell is generated, wherein the different transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide, ii) a Sox2 polypeptide linked to a cell penetration peptide, iii) a c-myc polypeptide linked to a cell penetration peptide, iv) a Klf4 polypeptide linked to a cell penetration peptide, v) a Nanog polypeptide linked to a cell penetration peptide, and vi) a Lin28 polypeptide linked
  • the reprogrammed mammalian cells comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, and/or a Lin28 polypeptide linked to a cell penetration peptide.
  • the reprogrammed mammalian cells of such populations of cells do not comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a c-myc polypeptide linked to a cell penetration peptide, a Klf4 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, or a Lin28 polypeptide linked to a cell penetration peptide, but nonetheless exhibit at least one characteristic of pluripotency, e.g., the reprogrammed cell is a cell that has undergone one or more cell divisions, cell doublings or expansions after being generated via the methods presented herein.
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the invention provides a method for cardiac differentiation of a mammalian cell comprising: contacting a mammalian cell which exhibits at least one characteristic of pluripotency with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs.
  • the transducible polypeptide comprises an islet 1 (ISLl) polypeptide, e.g., a human ISLl polypeptide, linked to a cell penetration peptide.
  • the mammalian cell is a pluripotent cell, e.g., an induced pluripotent stem cell, an embryonic stem cell, or an adult stem cell, such as a cardiac stem cell, e.g., a cardiosphere-derived stem cell.
  • the cell is a reprogrammed cell generated via methods provided herein.
  • the mammalian cell is a human cell.
  • a method for generating a reprogrammed mammalian cell without the use of a virus comprises contacting a differentiated mammalian cell with transducible polypeptides.
  • the transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a NANOG polypeptide linked to a cell penetration peptide, and a Lin28 polypeptide linked to a cell penetration peptide.
  • the transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, and a Klf4 polypeptide linked to a cell penetration peptide.
  • a c-myc polypeptide linked to a cell penetration peptide is further provided in some embodiments.
  • the cell penetration peptide comprises an amino terminus and a carboxy terminus.
  • the method generates reprogrammed cells that exhibit at least one characteristic of pluripotency.
  • a virus-free composition for generating a reprogrammed mammalian cell exhibiting at least one characteristic of pluripotency.
  • Virus-free compositions include, but are not limited to, compositions that do not contain significant amounts of (i) replication-competent viruses; and/or (ii) viruses that randomly integrate into the host genome; and/or (iii) whole viruses, and/or (iv) pathogenic viruses; and/or (v) immunogenic viruses.
  • Virus-free compositions may include viral particles that (i) are not replication-competent; and/or (ii) do not randomly integrate into the host genome; and/or (iii) are not pathogenic; and/or (iv) are not immunogenic.
  • the composition comprises transducible polypeptides for contacting a differentiated mammalian cell.
  • the transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a NANOG polypeptide linked to a cell penetration peptide, and a Lin28 polypeptide linked to a cell penetration peptide.
  • the transducible polypeptides comprise an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, and a Klf4 polypeptide linked to a cell penetration peptide.
  • a c-myc polypeptide linked to a cell penetration peptide is further provided in some embodiments.
  • the cell penetration peptide comprises an amino terminus and a carboxy terminus.
  • the polypeptides are joined and/or synthesized (e.g., in frame) with one another (or are linked with optional linker molecules), and include one or more cell penetration peptides.
  • the invention provides a method for cardiac differentiation of a mammalian cell comprising: contacting a mammalian cell which exhibits at least one characteristic of pluripotency in vitro, for example ex vivo, with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs.
  • the transducible polypeptide comprises an islet 1 (ISLl) polypeptide, e.g., a human ISLl polypeptide, linked to a cell penetration peptide.
  • the mammalian cell is a pluripotent cell, e.g. , an induced pluripotent stem cell, an embryonic stem cell, or an adult stem cell, such as a cardiac stem cell, e.g., a cardiosphere-derived stem cell.
  • the cell is a reprogrammed cell generated via methods provided herein.
  • the mammalian cell is a human cell.
  • Figure 1 presents quantitative RT-PCR data demonstrating marked upregulation of collagen transcription (COLlA) in cardiac tissue following LAD ligation surgery. The data confirms myocardial infarction of the cardiac tissue.
  • Figure 2A presents Western blot data of ISLl protein. Lanes 1 and 2 are control mouse hearts, and lanes 3 and 4 are fourteen day post-myocardial infarction hearts.
  • Figure 2B presents densitometry data of ISL-I Western blot signal from five control and five 14-day post-infarction hearts. p ⁇ 0.01.
  • Figure 3 presents immunofluorescence data of peri-infarct tissue. The cells that produce IsIl within the peri-infarct tissue are c-kit positive cells. Red labels c-kit, green indicates IsIl-I and blue labels the nucleus (DAPI).
  • Figure 4 A presents PCR amplification of the ISLl gene.
  • the mouse ISLl gene was amplified by PCR (Taq polymerase, Invitrogen), using plasmid DNA as the template (Open Biosystems cat# EMM1002-99258597).
  • Figure 4B presents sequencing chromatograms for the region spanning the junction between the VP22-coding region and the ISLl -coding region of a polynucleotide encoding ISL1-VP22.
  • the PCR product of Figure 4A was ligated into the expression plasmid by the topoisomerase reaction (Voyager protein expression kit, Invitrogen)
  • the resultant plasmid was sequenced to confirm that the PCR product had inserted in frame with the VP22 gene, and in the correct orientation.
  • Figure 4C presents the expression of the ISL1-VP22 hybrid protein by induction of E coli by isopropyl ⁇ -D-thiogalactoside. The resulting protein was then purified using ProBond elution columns (Invitrogen).
  • Figure 4D presents Western blot data of VP22 and ISL1-VP22 purified proteins.
  • the synthesized VP22 and ISL1-VP22 proteins were confirmed to be the correct size (23 and 62 kDa respectively).
  • Figure 5 presents the percentage of beating embryoid bodies in proportion of beating embryoid bodies was markedly increased by exposure of the cells to ISL1-VP22 hybrid protein, compared to exposure to VP22 protein alone, or no additional protein.
  • a cell exhibits "at least one characteristic of pluripotency” if the cell: expresses at least one marker, as detected by RT-PCR, cell sorting, or immunocytochemistry techniques, of pluripotency selected from the group consisting of SSEA-3, SSEA-4 , TRA-1-60, Nanog, and Oct3/4; displays the ability to form embryoid bodies in vitro; can form tightly packed colonies on culture plates; displays the ability to differentiate into cells having characteristics of endoderm, mesoderm or ectoderm when injected into SCID mice; displays the ability to form teratomas if injected into animals; exhibits an exponential pattern of growth in cell culture, without senescence; exhibits telomerase activity; exhibits the ability to undergo at least between 10-40 population doublings in culture; comprises unmethylated DNA characteristic of pluripotent clones; or displays germline competence.
  • pluripotency selected from the group consisting of SSEA-3, SSEA-4 , TRA-1-60, Nanog, and Oct3/4
  • a cell that exhibits at least one characteristic of pluripotency can include, for example, a multipotent cell or a pluripotent cell. It is further noted, in the context of reprogrammed cells, that in instances whereby the differentiated mammalian cell used in generating the reprogrammed cell itself exhibits at least one characteristic of pluripotency, the resulting reprogrammed mammalian cell exhibits at least one additional characteristic of pluripotency relative to the differentiated mammalian cell, and/or exhibits quantitatively more of at least one characteristic of pluripotency relative to the differentiated mammalian cell. [0054] As used herein, the term "pluripotent cell” refers to a cell that has complete differentiation versatility, i.e., the capacity to grow into any of the mammalian body's approximately 260 cell types.
  • multipotent cell refers to a cell that has the capacity to grow into any of subset of the mammalian body's approximately 260 cell types. Certain multipotent cells can differentiate into at least one cell type of ectoderm, mesoderm, and endoerm germ layers.
  • cell penetration peptide refers to an amino acid sequence that, when linked to a polypeptide, e.g., a reprogramming factor, causes or enhances the ability of the polypeptide to cross the cell membrane of a cell when the cell is contacted by the cell penetration peptide linked to the polypeptide.
  • a "transducing fragment" of a cell penetration peptide refers to a portion of a full-length cell penetration peptide, e.g., a portion of a VP22, TAT, or ANTP HD sequence, that, when linked to a polypeptide, e.g., a reprogramming factor, causes or enhances the ability of the polypeptide to cross the cell membrane of a cell when the cell is contacted by the transducing fragment linked to the polypeptide.
  • a "reprogramming factor” refers to a factor, e.g., a polypeptide, that when introduced to a differentiated mammalian cell causes, induces, enhances, or contributes to generation of a reprogrammed cell from the contacted differentiated cell.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide linked with an Oct 3/4 polypeptide, e.g., a human or mouse Oct3/4 polypeptide.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse Oct3/4 have been described previously. See, e.g., Yeom et ⁇ l., Mech. Dev. 35 (3), 171-179 (1991); Takeda et ⁇ l., Nucleic Acids Res. 20 (17), 4613-4620 (1992).
  • transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of Oct3/4, such as the transducible polypeptide provided herein as SEQ ID NO:25.
  • the transducible polypeptide comprises the carboxy terminus of Oct3/4 linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:26.
  • the linkage can be direct or indirect.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide fused with a Sox2 polypeptide, e.g., a human or mouse Sox2 polypeptide.
  • a Sox2 polypeptide e.g., a human or mouse Sox2 polypeptide.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse Sox2 have been described previously. See, e.g., Gubbay et al., Nature, 6281 :245-50 (1990); Stevanovic et al, Mamm. Genome 5 (10), 640-642 (1994).
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of Sox2, such as the transducible polypeptide provided herein as SEQ ID NO:27.
  • the transducible polypeptide comprises the carboxy terminus of Sox2 linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:28.
  • the linkage can be direct or indirect.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide linked with a Nanog polypeptide, e.g. , a human or mouse Nanog polypeptide.
  • Nanog is a homeodomain-bearing transcription factor.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse Nanog have been described previously. See, e.g., Mitsui et al., Cell. 2003 May 30;l 13(5):631-42; Chambers et al., Cell. 2003 May 30; 113(5):643-55.
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of Nanog, such as the transducible polypeptide provided herein as SEQ ID NO:29.
  • the transducible polypeptide comprises the carboxy terminus of Nanog linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:30.
  • the linkage can be direct or indirect.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide linked with a Lin28 polypeptide, e.g., a human or mouse Lin28 polypeptide.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse Lin28 have been described previously. See, e.g., Moss et al, Dev. Biol. 258 (2), 432-442 (2003); Sempere et al, Genome Biol. 5 (3), Rl 3 (2004).
  • Representative cDNA and amino acid sequences of human Lin28 are provided herein as SEQ ID NOS: 9 and 10, respectively.
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of Lin28, such as the transducible polypeptide provided herein as SEQ ID NO: 31.
  • the transducible polypeptide comprises the carboxy terminus of Lin28 linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:32.
  • the linkage can be direct or indirect.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide linked with a c-myc polypeptide, e.g. , a human or mouse c-myc polypeptide.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse c-myc have been described previously. See, e.g., Hirning- FoIz et al, Cytogenet. Cell Genet. 61 (4), 289-294 (1992); Takahashi et al, Cytogenet. Cell Genet. 57 (2-3), 109-111 (1991).
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of c-myc, such as the transducible polypeptide provided herein as SEQ ID NO:33.
  • the transducible polypeptide comprises the carboxy terminus of c-myc linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:34.
  • the linkage can be direct or indirect.
  • the transducible polypeptide useful for the methods provided herein comprises a cell penetration peptide linked with a Klf4 polypeptide, e.g., a human or mouse Klf4 polypeptide.
  • the cell penetration peptide is selected from the group comprising VP22, TAT, Antp HD, and poly-arginine.
  • the sequences of human and mouse Klf4 have been described previously. See e.g., Shields et al, J. Biol. Chem. Ill (33), 20009-20017 (1996); Conkright et al, Nucleic Acids Res. 27 (5), 1263-1270 (1999).
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of Klf4, such as the transducible polypeptide provided herein as SEQ ID NO:35.
  • the transducible polypeptide comprises the carboxy terminus of Klf4 linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:36.
  • the linkage can be direct or indirect.
  • HSVl Herpes simplex type I virus
  • SEQ ID NO: 15 A representative full length VP22 sequence (aa 1-301) is depicted herein (SEQ ID NO: 15).
  • Sequences of VP22 that can be utilized as transducing fragments of VP22 are also well known. See, e.g., PCT International patent applications WO 97/05265, WO 98/04708, and WO 98/32866, each of which is incorporated herein by reference.
  • Such sequences can include, for example, amino acid sequences corresponding to amino acids 60- 301 and 159-301 of the full-length HSVl VP22 sequence (SEQ ID NO: 16).
  • VP22 sequences that can be used in conjunction with the methods described herein extend to homologous proteins and transducing fragments thereof based on sequences of VP22 protein homologs from other herpesviruses.
  • VP22-homolog sequences have been obtained from VZV ⁇ e.g., all or homologous parts of the sequence from aa 1-302), from MDV ⁇ e.g., all or homologous parts of the sequence from aa 1-249), and from BHV ⁇ e.g., all or homologous parts of the sequence from aa 1-258) ⁇ see PCT International Publication Nos. WO 97/05265, WO 98/04708, and WO 98/32866).
  • the sequences of the corresponding proteins from HSV2, VZV, BHV and MDV are well known and available in public protein/nucleic acid sequence databases.
  • a VP22 sequence from HSV2 is available as gene item UL49 under accession no. Z86099 containing the complete genome of HSV2 strain HG52; the complete genome of VZV including the homologous gene/protein is available under accession numbers X04370, M14891, Ml 6612; the corresponding protein sequence from BHV is available as "bovine herpesvirus 1 virion tegument protein" under accession number U21137; and the corresponding sequence from MDV is available as gene item UL49 under accession number LI 0283 for "gallid herpesvirus type 1 homologous sequence genes.”
  • corresponding deletions can be made, e.g.
  • Transducing fragments of VP22 can also, for example, contain one or a plurality of amino acid sequence motifs or their homologs from the C-terminal sequence of VP22 of HSl or other herpesviruses, which can be selected from RSASR (SEQ ID NO: 17), RTASR (SEQ ID NO: 18), RSRAR (SEQ ID NO: 19), RTRAR (SEQ ID NO:20), ATATR (SEQ ID NO:21), and wherein the third or fourth residue A can be duplicated, e.g., as in RSAASR (SEQ ID NO:22).
  • HIV-I TAT protein the human immunodeficiency virus (HIV-I) TAT protein.
  • the sequences of HIV-I TAT polypeptides are well known. See, e.g., (Frankel & Pabo, Cell 55:1189-93 (1988); Green & Loewenstein, Cell 55:1179-88(1988)).
  • a cell penetration peptide the YGRKKRRQRRR (SEQ ID NO:23) HIV-I TAT amino acid sequence.
  • YGRKKRRQRRR SEQ ID NO:23
  • HIV-I TAT amino acid sequence a cell penetration peptide useful for the methods provided herein is the homeodomain of the Drosophila melanogaster protein Antennapedia (Antp HD) (Lindsay, Curr. Op. Pharmacol. 2:587-94 (2002); Derossi et al, J. Biol. Chem. 269:10444-50 (1994)), described, e.g., in PCT Publication Nos.
  • Antp HD Sequences of Antp HD that can be utilized as transducing fragments are also well known. For example, among such sequences is RQIKIWFQNRRMKWKK (SEQ ID NO: 24), corresponding to the third helix of the Antp HD homeodomain.
  • RQIKIWFQNRRMKWKK SEQ ID NO: 24
  • Arg arginine
  • poly-arginine poly-arginine
  • such cell penetration peptides can comprise contiguous or partially contiguous segments of at least 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 1000 arginine residues, wherein the arginine residues may be of the D-form, the L-form, or mixtures of each.
  • a transducible polypeptide comprises a reprogramming factor linked to a cell penetration peptide, and can optionally comprise a nuclear localization signal and/or a purification moiety.
  • Transducible polypeptides provided herein can be made using any of a variety of methods, e.g., recombinant or synthetic methods, well known to those of skill in the art.
  • a transducible polypeptide can be made using standard recombinant DNA techniques.
  • a polynucleotide comprising the coding sequence of a transducible polypeptide, e.g., the coding sequence for a cell penetration peptide joined in- frame with the coding sequence of a reprogramming factor in an expression vector, e.g., a plasmid vector, and can be expressed in any suitable cell, e.g., a bacterial or mammalian cell.
  • Techniques are also well known for isolating and purifying polypeptides expressed via such methods from the expressing cells and from the media used during culture of the expressing cells.
  • a cell is used to express a single transducible polypeptide.
  • a cell is engineered to express greater than one form of transducible polypeptide, e.g. , is engineered to express a transducible polypeptide comprising a Oct3/4 reprogramming factor, and also a transducible polypeptide comprising a Sox2 reprogramming factor.
  • the coding sequence in addition to the coding sequence of a reprogramming factor and one or more cell penetration peptides, the coding sequence further comprises the amino acid sequence of a purification moiety.
  • the location of the coding sequence of the purification moiety can be placed in any position that does not interfere with the expression or activity of the reprogramming factor, the cell penetration peptide, or the optional nuclear localization sequence if it is to be present on the transducible polypeptide.
  • the coding sequence can be placed upstream (5') or downstream (3') of the coding sequence of the coding sequence of the reprogramming factor such that the purification moiety is amino or carboxy to the reprogramming factor in the expressed transducible polypeptide, respectively.
  • the coding sequence can be placed upstream (5') or downstream (3') of the coding sequence of the coding sequence of the cell penetration peptide such that the purification moiety is amino or carboxy to the cell penetration peptide in the expressed transducible polypeptide, respectively.
  • the purification moiety is present at the amino terminal end of the transducible polypeptide.
  • the coding sequence in addition to the coding sequence of a reprogramming factor and one or more cell penetration peptides, the coding sequence further comprises the amino acid sequence of a nuclear localization sequence (NLS).
  • the location of the coding sequence of the NLS can be placed in any position that does not interfere with the expression or activity of the reprogramming factor, the cell penetration peptide, or the optional purification moiety, if it is to be present on the transducible polypeptide.
  • the coding sequence can be placed upstream (5') or downstream (3') of the coding sequence of the coding sequence of the reprogramming factor such that the NLS is amino or carboxy to the reprogramming factor in the expressed transducible polypeptide, respectively.
  • the coding sequence can be placed upstream (5') or downstream (3') of the coding sequence of the cell penetration peptide such that the NLS is amino or carboxy to the cell penetration peptide in the expressed transducible polypeptide, respectively.
  • the transducible polypeptide comprises a reprogramming factor linked to the amino terminus of a cell penetration peptide.
  • the coding sequence of the transducible polypeptide is arranged accordingly.
  • the coding sequence of the reprogramming factor is positioned in- frame with the coding sequence of the cell penetration peptide such that the carboxy-most amino acid residue of the reprogramming factor is adjacent to the amino-most amino acid residue of the cell penetration peptide in the expressed transducible polypeptide.
  • the coding sequence of the reprogramming factor is positioned in- frame with the coding sequence of an amino acid linker sequence, which is, in turn, positioned in- frame with the coding sequence of the cell penetration peptide.
  • the amino acid sequence of the reprogramming factor is also linked to the amino terminus of the cell penetration peptide, but is linked via the linker sequence.
  • the transducible polypeptide comprises a reprogramming factor linked to the carboxy terminus of a cell penetration peptide.
  • the coding sequence of the transducible polypeptide is arranged accordingly.
  • the coding sequence of the cell penetration peptide is positioned in- frame with the coding sequence of the reprogramming factor such that the carboxy-most amino acid residue of the cell penetration peptide is adjacent to the amino-most amino acid residue of the reprogramming factor in the expressed transducible polypeptide.
  • the coding sequence of the cell penetration peptide is positioned in- frame with the coding sequence of an amino acid linker sequence, which is, in turn, positioned in-frame with the coding sequence of the reprogramming factor.
  • the amino acid sequence of the reprogramming factor is also linked to the carboxy terminus of the cell penetration peptide, but is linked via the linker sequence.
  • Useful promoters for use in expression vectors include, but are not limited to, a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone- inducible MMTV promoter, a SV40 promoter, a MRP pol III promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), and a constitutive CMV promoter.
  • the expression vectors should contain expression and replication signals compatible with the cell in which the transducible polypeptides are expressed.
  • Expression vectors useful for transducible constructs include viral vectors such as retroviruses, adenoviruses and adenoassociated viruses, plasmid vectors, cosmids, and the like. Viral and plasmid vectors are preferred for transfecting the expression vectors into mammalian cells.
  • the expression vector pcDNAl Invitrogen, San Diego, CA
  • the expression control sequence comprises the CMV promoter
  • Transducible polypeptides can also be made, for example, using chemical synthetic methods, or a combination of synthetic and recombinant methods.
  • transducible polypeptides can be synthetically produced using standard polypeptide synthesis techniques well known by those of skill in the art.
  • portions of a transducible polypeptide can be purified, or recombinantly expressed using, e.g., techniques such as those described herein, and the portions can be linked using synthetic techniques to yield complete transducible polypeptides.
  • the linkage can be via covalent, e.g., peptide bond, or non- covalent linkage, and can be direct or via a linker moiety, e.g., a linker moiety that links a reprogramming factor with a cell penetration peptide.
  • linkages can be utilized, including, but not limited to ether, ester, thioether, thioester, amide, imide, disulfide, peptide, or other linkages.
  • Linkage can be likewise be via any of a variety of functional groups, for example, sulfhydryl (-S), carboxylic acid (COOH) or free amine (-NH2) groups.
  • S sulfhydryl
  • COOH carboxylic acid
  • -NH2 free amine
  • the linker can directly link portions of the transducible polypeptide, e.g., a cell penetration peptide and a reprogramming factor polypeptide.
  • the linker itself can comprises two or more molecules that associate to link portions of the transducible polypeptide, e.g., a cell penetration peptide and a reprogramming factor
  • linkage may be via a biotin molecule attached, e.g., to a cell penetration peptide and streptavidin attached to the reprogramming factor polypeptide.
  • Exemplary linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, substituted carbon linkers, unsaturated carbon linkers, aromatic carbon linkers, peptide linkers, etc.
  • the linkers can be attached to the cell penetration peptide and/or the reprogramming factor polypeptide by any means or method known by one of skill in the art without limitation.
  • the linker can be attached to the cell penetration peptide and/or the reprogramming factor polypeptide with an [0089]
  • portions of the transducible polypeptide to be linked e.g., a cell penetration peptide and a reprogramming factor, can be derivatized as appropriate to facilitate linkage to another portion of the transducible polypeptide, or to a linker.
  • derivatization can be accomplished, for example, by attaching a suitable derivative or derivatives such as those available from Pierce Chemical Company, Rockford, Illinois.
  • derivatization may involve chemical treatment of one or more portions of the transducible polypeptide to be linked, e.g. , a cell penetration peptide and/or a reprogramming factor.
  • the skilled artisan can routinely generate free sulfhydryl groups on proteins to provide a reactive moiety for making a disulfide, thioether, thioester, etc. linkage. See, e.g., U.S. Pat. No. 4,659,839.
  • any of the linking methods described herein can be used to link portions of transducible polypeptides, e.g., a cell penetration peptide and a reprogramming factor, in various configurations.
  • the carboxy terminus of the cell penetration peptide may be linked, directly or indirectly, to the amino terminus of the reprogramming factor polypeptide.
  • the carboxy terminus of the reprogramming factor may be linked to the amino terminus of the cell penetration peptide, either directly or indirectly.
  • the amino terminus of the cell penetration peptide may be linked, either directly or indirectly, to the amino terminus of the reprogramming factor.
  • the carboxy terminus of the cell penetration peptide may be linked, either directly or indirectly, to the carboxy terminus of the reprogramming factor.
  • "linked to" an amino terminus or a carboxy terminus does not necessarily connote a direct linkage to the amino-most, or carboxy-most amino acid of the polypeptide, but can also be via a linker, e.g., an amino acid sequence of one or more residues, e.g., 2, 3, 4, 5, 10, 15, 20, 25, or more amino acid residues.
  • any transducible polypeptide made via methods described above can be utilized as part of the methods described herein.
  • transducible polypeptides can be utilized in conjunction with the methods presented herein.
  • a cell for example, a differentiated mammalian cell or a cell exhibiting at least one characteristic of pluripotency
  • supernatants, extracts, or co-cultures of cells producing transducible polypeptides useful for the methods described herein can be used to contact transducible polypeptides to a cell.
  • transducible polypeptides can be purified using standard techniques in the art, and added to a culture medium, e.g. , as a medium supplement, to contact cells present in or added to the culture medium.
  • Cells may be contacted with a composition comprising transducible polypeptides for varying periods of time.
  • differentiated mammalian cells are contacted in vitro with the composition for a period of time sufficient to generate reprogrammed cells that exhibit at least one characteristic of pluripotency.
  • the cells are contacted with the composition for a period of time between 1 hour and 30 days.
  • the period may be 1 day, 3 days, 5 days, 7 days, 10 days, 12 days, 15 days or more.
  • the period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days.
  • the period of contact is 5 days.
  • differentiated cells contacted with one or more transducible polypeptides for a particular duration of time may receive more than one, e.g., repeated, administrations of the one or more transducible polypeptides during the contact period.
  • differentiated cells can be contacted with one or more transducible polypeptides for a total period of 10, 11, 12, 13, 14 or 15 days, with culture medium comprising the transducible polypeptides being replaced every 2 nd , 3 rd , 4 th or 5 th day, or later.
  • the differentiated cells are contacted with one or more transducible polypeptides for a first period of time, followed by contact with one or more transducible polypeptides for a second period of time.
  • the differentiated cells are contacted with the same transducible polypeptide or combination of transducible polypeptides for a first period of time and a second period of time.
  • the differentiated cells are contacted with a first transducible polypeptide or combination of transducible polypeptides, for a first period of time, followed by contact with a second, different transducible polypeptide or combination of transducible polypeptides for a second period of time.
  • the differentiated cells are contacted with each transducible polypeptide of the combination concurrently, that is, each of the transducible polypeptides is contacted to the cells or the culture containing the cells simultaneously.
  • the differentiated cells are contacted with each of the transducible polypeptides of within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
  • the differentiated cells are contacted with each of the transducible polypeptides or a subcombination of the transducible polypeptides sequentially.
  • sequential contacting of each transducible polypeptide comprises contacting the differentiated cells with a first transducible polypeptide or combination of transducible polypeptides, then contacting the differentiated cells with a second transducible polypeptide or combination of transducible polypeptides at a later time point, etc., until each transducible polypeptide of the entire combination has been contacted to the cells.
  • each transducible polypeptide or combination of transducible polypeptides is contacted about 12, 14, 16, 18, 20 , 22 or 24 hours apart until each transducible polypeptide of the entire combination has been contacted to the cells.
  • each transducible polypeptide or combination of transducible polypeptides is contacted about 1, 2, 3, 4, 5, 6 or more days apart until each of the transducible polypeptides of the entire combination has been contacted to the cells.
  • the differentiated cells may be contacted with one or more transducible polypeptides at varying concentrations. In some embodiments, the cells are contacted with an equimolar amount of each transducible polypeptide.
  • the cells are contacted with a plurality of transducible polypeptides comprising equimolar amounts of an Oct 3/4 polypeptide linked to a cell penetration peptide, a Sox2 polypeptide linked to a cell penetration peptide, a Nanog polypeptide linked to a cell penetration peptide, and a Lin28 polypeptide linked to a cell penetration peptide.
  • the concentration of one or more transducible polypeptides may be differ (may be higher or lower) relative to the concentration of one or more of the other transducible polypeptides, e.g., increased or decreased to enhance the overall efficiency of generating reprogrammed cells.
  • the differentiated cells are contacted with a concentration of between about 0.01-10, 0.1-50, 5-100, 50-100, 100-150, 150-200 ⁇ g/ml or more of total transducible polypeptides for a duration of time sufficient to reprogram the cell to exhibit at least one characteristic of pluripotency.
  • the differentiated mammalian cells are contacted with a concentration of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 ⁇ g/ml of total transducible polypeptides for a duration of time sufficient to generate a reprogrammed cell that exhibits at least one characteristic of pluripotency.
  • the differentiated mammalian cells are contacted with a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ⁇ g/ml of total transducible polypeptides for a duration of time sufficient to generate reprogrammed cells that exhibit at least one characteristic of pluripotency. In some embodiments, the differentiated cells are contacted with a concentration of about 10, 15, 20, 25, 30, 35, 40, 45 or 50 ⁇ g/ml of total transducible polypeptides for a duration of time sufficient to generate reprogrammed cells that exhibit at least one characteristic of pluripotency.
  • the differentiated cells are contacted with a concentration of about 50, 75, 100, 125, 150 or 200 ⁇ g/ml of total transducible polypeptides for a duration of time sufficient to generate reprogrammed cells that exhibit at least one characteristic of pluripotency.
  • Cells may be maintained in culture for varying periods of time prior to assessing the cells for characteristics of pluripotency.
  • differentiated cells which have been contacted with one or more transducible polypeptides are maintained in culture for 1, 2, 3, 4, 5 days, or more than 5 days prior to identifying or selecting for reprogrammed cells.
  • differentiated cells contacted with one or more transducible polypeptides are maintained in culture for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more days (e.g., between 3-5 weeks) prior to identifying or selecting for reprogrammed cells.
  • differentiated cells which have been contacted with one or more transducible polypeptides are cultured using methods well known in the art, for example, by culturing on feeder cells, such as irradiated fibroblasts, or in conditioned media obtained from cultures of such feeder cells, in order to obtain continued long-term cultures of the induced pluripotent stem cells.
  • the putative reprogrammed cells to be expanded can be exposed to, or cultured in the presence of, an agent that suppresses cellular differentiation.
  • Such agents include, but are not limited to, human Delta 1 and human Serrate 1 polypeptides (see, Sakano et ah, U.S. Patent No. 6,337,387 entitled “Differentiation suppressive polypeptide,” issued January 8, 2002), leukemia inhibitory factor (LIF) and stem cell factor.
  • LIF leukemia inhibitory factor
  • Methods for the expansion of cell populations are also known in the art (see e.g., Emerson et al., U.S. Patent No.
  • the putative reprogrammed cells are cultured in the presence of LIF and Bone Morphogenetic Protein 4 (BMP4), as described by Chou et al., Cell 135:449-461 (2008).
  • BMP4 Bone Morphogenetic Protein 4
  • the treated fibroblasts can be plated on mitomycin C treated SNL feeder cells (a mouse cell line stably transfected with leukemia inhibitory factor (LIF) and a neomycin resistance gene) in medium designed for culture of primate embryonic stem cells supplemented with bFGF.
  • mitomycin C treated SNL feeder cells a mouse cell line stably transfected with leukemia inhibitory factor (LIF) and a neomycin resistance gene
  • the putative reprogrammed cells may be assessed for viability, proliferation potential, and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate uptake assay, propidium iodide uptake assay (to assess viability); and thymidine uptake assay, MTT cell proliferation assay (to assess proliferation). Longevity may be determined by methods well known in the art, such as by determining the maximum number of population doubling in an extended culture.
  • Differentiated cells that can be used in accordance with the methods provided herein can be any differentiated cells known in the art.
  • the differentiated cells in instances where the differentiated cells are to be administered to a subject once reprogrammed or once differentiated after reprogramming, can be reprogrammed and/or differentiated ex vivo, that is, the cells to be reprogrammed and/or differentiated can be obtained from the subject, reprogrammed outside the body of the subject, and then administered back to the subject.
  • the differentiated cells are to be administered to a subject once reprogrammed or once differentiated after reprogramming, the can be autologous or heterologous to the subject.
  • the differentiated cell can be any differentiated cell of a vertebrate species.
  • the differentiated cell is a differentiated mammalian cell.
  • the differentiated cell is a differentiated cell derived from a primate.
  • the species of the differentiated cell is human, murine, e.g., mouse, porcine, bovine, canine, equine or feline.
  • the differentiated cells are somatic cells.
  • the somatic cells are adult somatic cells.
  • the somatic cells are native somatic cells.
  • the somatic cells have been genetically engineered or altered.
  • Suitable mammalian somatic cells can also include, but are not limited to, Sertoli cells, endothelial cells, granulosa epithelial, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiac muscle cells, and other muscle cells, etc.
  • the differentiated cell is a fibroblast, e.g., an adult fibroblast or an embryonic fibroblast.
  • the differentiated cell is a cell of hematopoietic lineage. In some embodiments, the differentiated cell is derived from peripheral blood. In some embodiments, the differentiated cell is a cell of the immune system, including, but limited to, a macrophage, a lymphoid cell, an immature B cell (e.g., pro-B cell or pre-B cell), and a mature B cell (e.g., a non-naive B-cell).
  • a macrophage e.g., a lymphoid cell, an immature B cell (e.g., pro-B cell or pre-B cell), and a mature B cell (e.g., a non-naive B-cell).
  • the somatic cells are adult stem cells, e.g., cells that are capable of giving rise to all the cell types of a particular tissue.
  • adult stem cells include hematopoietic stem cells, neural stem cells, cardiac stem cells, e.g., cardiosphere derived cells (CDCs; see e.g., PCT International Publication No. WO 06/052925), and mesenchymal stem cells.
  • the differentiated cells can be primary cells, e.g., non- immortalized cells, such as those newly isolated from a subject, and the cells can be maintained in cell culture following their isolation from the subject and prior to performing a method of the invention.
  • the differentiated cells are passaged at least once or more than once prior to their use in the methods provided herein.
  • the cells are passaged between 2-10, 10-20, 20, 30, 30-40, 40-50 or more than 50 times prior to being subjected to the methods of the invention.
  • the cells will have been passaged no more than 1, 2, 5, 10, 20, or 50 times prior to being subjected to the methods of the invention.
  • Differentiated cells suitable for the methods provided herein can be obtained by any method known in the art, and can be obtained from any organ or tissue containing live somatic cells, e.g., blood, bone marrow, skin, lung, pancreas, liver, stomach, intestine, heart, pancreas, reproductive organs, bladder, kidney, urethra and other urinary organs, etc. 6.2.6 Characteristics of Reprogrammed Cells
  • the reprogrammed cells generated by the methods provided herein exhibit at least one characteristic of pluripotency.
  • the reprogrammed cell displays at least one characteristic of pluripotency if the cell: expresses at least one marker, as detected by RT-PCR or cell sorting techniques, of pluripotency selected from the group consisting of SSEA-3 , SSEA-4 , TRA- 1-60, Nanog, and Oct3/4; displays the ability to form embryoid bodies in vitro; can form tightly packed colonies on culture plates; displays the ability to differentiate into cells having characteristics of endoderm, mesoderm or ectoderm when injected into SCID mice; displays the ability to form teratomas if injected into animals; exhibits an exponential pattern of growth in cell culture, without senescence; exhibits telomerase activity; exhibits the ability to undergo at least between 10-40 population doublings in culture; comprises unmethylated DNA characteristic of pluripotent clones; or displays germline competence.
  • a cell that exhibits at least one characteristic of pluripotency can include, for example, a multipotent cell or a pluripotent cell. It is further noted, in the context of reprogrammed cells, that in instances whereby the differentiated mammalian cell used in generating the reprogrammed cell itself exhibits at least one characteristic of pluripotency, the resulting reprogrammed mammalian cell exhibits at least one additional characteristic of pluripotency relative to the differentiated mammalian cell, and/or exhibits quantitatively more of at least one characteristic of pluripotency relative to the differentiated mammalian cell.
  • Determination that a reprogrammed cell has been generated that displays at least one characteristic of pluripotency may be accomplished by methods well-known in the art, e.g., measuring changes in morphology and cell surface markers using techniques such as flow cytometry or immunocytochemistry ⁇ e.g., staining cells with tissue-specific or cell- marker specific antibodies), by examination of the morphology of cells using light or confocal microscopy, or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene-expression profiling
  • differentiated cells subjected to the methods provided herein can be characterized by examining the expression of genes which are normally expressed in undifferentiated cells and are indicative of a pluripotent state.
  • the gene expression pattern of the reprogrammed cells may be compared to the gene expression pattern of embryonic cells or other undifferentiated cells.
  • the embryonic or undifferentiated cell to which the putative reprogrammed cell is compared to is from the same species as the original differentiated cell subjected to reprogramming.
  • the absence of the expression of genes normally associated with a differentiated state can be examined to monitor the extent of reprogramming.
  • a reprogrammed cell that exhibits at least one characteristic of pluripotency can be identified by the presence of any one of the following cell surface markers: SSEA3, SSAE4, Nanog, Sox2 and Tra-1-60. In some embodiments, such cells can be identified by the presence of at least two, three, or all four of these cell surface markers.
  • Expression of such cell surface markers are routinely determined according to methods well known in the art, e.g. by flow cytometry, followed by washing and staining with an anti-cell surface marker antibody.
  • an anti-cell surface marker antibody For example, to determine the presence of SSEA-3 or SSEA-4, cells may be washed in PBS and then double-stained with an anti-SSEA-3 antibody labeled with, for instance, phycoerythrin, and an anti-SSEA-4 antibody labeled with, for instance, fluorescein isothiocyanate.
  • Other techniques known in the art for examining protein expression e.g., immunofluorescence microscopy, Western blot, protein microarrays, and the like, can be used to determine the presence of the cell surface markers.
  • RT-PCR can also be used to assess mRNA expression of SSEA3, SSAE4, Nanog, Sox2 and Tra-1-60 in differentiated cells subjected to the methods provided herein.
  • a reprogrammed cell that exhibits at least one characteristic of pluripotency can be identified based on the expression of a reporter construct within the cells, wherein a reporter gene, e.g., green fluorescent protein (GFP), is operably linked to a promoter sequence of a gene, the expression of which is typically associated with a state of pluripotency, such as SSEA3, SSEA4, Nanog, Sox2, or Tral-60.
  • a reporter gene e.g., green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the reporter construct comprises GFP under the control of a Nanog promoter (SYSTEM BIOSCIENCE, Inc. CA). See Yamanaka et al, Nature 448:313-7 (2007).
  • a reprogrammed cell that exhibits at least one characteristic of pluripotency can be identified by evaluating certain morphological criteria with reference to the morphological characteristics of an embryonic stem (ES) cell or an ES cell colony.
  • the morphological criteria includes any visually detectable aspect of the size, shape, structure, organization, and/or physical form of the putative reprogrammed cells or colonies.
  • Morphological criteria include, e.g., the shape of the colonies, the sharpness of colony boundaries (with sharp boundaries characterizing colonies of ES-like cells) , the density of the cells in the colonies (with increased density characterizing colonies of ES-like cells) , and/or the small size and distinct shape of the putative reprogrammed cells relative to naive differentiated cells.
  • Reprogrammed cells can be used for a variety of purposes.
  • reprogrammed cells can be utilized for cell transplantation and cell therapy, can be used to generate one or more differentiated cell types, and can be useful in the treatment of diseases or disorders, including, but not limited to, vascular disease, neurological diseases or disorders, autoimmune diseases or disorders, diseases or disorders involving inflammation, and cancer or the disorders associated therewith.
  • the populations of reprogrammed cells are used to renovate and repopulate tissues and organs, thereby replacing or repairing diseased tissues, organs or portions thereof.
  • the methods provided herein generate induced pluripotent stem cells, and thus provide an important advance in the art of creating inducible pluripotent stem cells.
  • the methods since differentiated cells are contacted with reprogramming factors which are provided in the form of recombinant transducible polypeptides, the methods obviate the need for viral transduction or plasmid transfection of the cell, thereby eliminating the possibility of permanent genetic modification of the cell with oncogenic or immortalizing gene sequences. This alleviates concerns over the prolonged exogenous expression of pluripotency genes which have been shown to also have oncogenic potential, e.g., c-myc.
  • differentiated cell types are derived from the reprogrammed cell generated by the methods provided herein.
  • differentiated cells may be obtained by culturing reprogrammed cells in the presence of at least one differentiation factor and selecting differentiated cells from culture. Selection of differentiated cells may be based on phenotype, such as the expression of particular cell markers normally present on differentiated cells. Alternatively, functional assays which screen for the performance of one or more functions associated with a particular differentiated cell type may be performed.
  • reprogrammed cells generated by the methods provided herein may be differentiated into any of the cells in the body including, without limitation, skin, cartilage, bone skeletal muscle, cardiac muscle, renal, hepatic, blood and blood forming, vascular precursor and vascular endothelial, pancreatic beta, neurons, glia, retinal, inner ear follicle, intestinal, lung, cells.
  • isolated populations of cells comprising at least 70%, at least 80%, at 90%, or at least 95% cells that have been differentiated using reprogrammed cells generated by the methods provided herein.
  • the cells that have been differentiated are cardiac cells.
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., reprogrammed cells generated via the methods provided herein, stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the isolated population of cells comprises about IxIO 5 , 5xlO 5 , lxl0 6 , 5xl0 6 ,lxl0 7 , 5xlO 7 , IxIO 8 , or 5xlO 8 cells, either cells that have been differentiated using reprogramed cells generated by the methods provided herein or total cells.
  • the isolated population of cells is present in a formulation suitable for administration to a human.
  • the isolated population of cells is present in a bag, e.g.
  • a plastic bag such as a plastic bag suitable for use in administration of the cells to a human.
  • the isolated population of cells is present in a syringe, such as a sterile syringe suitable for administration of the cells to a human.
  • these cells are present on a solid support, e.g., a scaffold or matrix, such as a synthetic matrix or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the reprogrammed cells are exposed to inducers of differentiation to yield other therapeutically useful cells, such as retinal pigment epithelium, definitive endoderm, pancreatic beta cells and precursors to pancreatic beta cells, hematopoietic precursors and hemangioblastic progenitors, neurons, respiratory cells, muscle progenitors, cartilage and bone-forming cells, cells of the inner ear, neural crest cells and their derivatives, gastrointestinal cells, liver cells, kidney cells, smooth and cardiac muscle cells, dermal progenitors including those with a prenatal pattern of gene expression useful in promoting scarless wound repair, as well as many other useful cell types of the endoderm, mesoderm, and endoderm.
  • other therapeutically useful cells such as retinal pigment epithelium, definitive endoderm, pancreatic beta cells and precursors to pancreatic beta cells, hematopoietic precursors and hemangioblastic progenitors, neurons, respiratory cells, muscle progenitors, cartilage and bone-forming cells, cells of
  • Such inducers include but are not limited to: cytokines such as interleukin- alpha A, interferon-alpha A/D, interferon-beta, interferon-gamma, interferon- gamma-inducible protein- 10, interleukin- 1-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony- stimulating factor, and macrophage inflammatory protein- 1 alpha, 1-beta, 2, 3 alpha, 3 beta, and monocyte chemotactic protein 1-3, 6kine, activin A, amphiregulin, angiogenin, B-endothelial cell growth factor, beta eellulin, brain- derived neurotrophic factor, ClO, cardiotrophin-1, ciliary neurotrophic factor, cytokine- induced neutrophil chemoattractant-1, eotaxin, epidermal growth factor, epithelial neutrophil activating peptide-78, erythropoiet
  • inducers include cells or components derived from cells from defined tissues used to provide inductive signals to the differentiating cells derived from the reprogrammed cells of the invention.
  • inducer cells may derive from human, nonhuman mammal, or avian, such as specific pathogen-free (SPF) embryonic or adult cells.
  • SPF specific pathogen-free
  • the reprogrammed cells are induced to undergo cardiac differentiation by sequentially exposing the cells to Activin A and Bone Morphogenic Protein -4 (BMP4), followed by Percoll centrifugation, as described by Laflamme et al., Nat Biotechnology 25:1015-24 (2007); and Schuldiner et al, Proc Natl Acad Sd USA 97:11207- 12 (2007), the contents of which are incorporated herein in their entireties.
  • BMP4 Bone Morphogenic Protein -4
  • the reprogrammed cells are induced to undergo cardiac differentiation by sequentially exposing the cells to Activin A and Bone Morphogenic Protein -4 (BMP4), followed by Percoll centrifugation, then enriching for cells that express flk-1 and CXCR4 biomarkers, as described by Yang et al., Nature 453:524-8 (2008); and Nelson et al, Stem Cells 26:1464-73 (2008), the contents of which are incorporated herein in their entireties.
  • BMP4 Bone Morphogenic Protein -4
  • the CXCR4/Flk-1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, thus enabling the targeted selection of cells having a cardiopoietic lineage.
  • Cells expressing flk-1 and CXCR4 markers can be routinely determined according to methods well known in the art, e.g. by flow cytometry.
  • the reprogrammed cells are induced to undergo cardiac differentiation by exposing the cells to dickkopf homo log 1 (Dkk-1).
  • isolated populations of cells comprising at least 70%, at least 80%, at 90%, or at least 95% cells that have been differentiated using reprogrammed cells generated by the methods provided herein.
  • the cells that have been differentiated are cardiac cells.
  • isolated populations of cells further comprising at least one other isolated population of cells, e.g., reprogrammed cells generated via the methods provided herein, stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • stem cells for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells.
  • isolated populations of cells present on a solid support e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the isolated population of cells comprises about IxIO 5 ,
  • the isolated population of cells is present in a formulation suitable for administration to a human.
  • the isolated population of cells is present in a bag, e.g., a plastic bag, such as a plastic bag suitable for use in administration of the cells to a human.
  • the isolated population of cells is present in a syringe, such as a sterile syringe suitable for administration of the cells to a human.
  • these cells are present on a solid support, e.g., a scaffold or matrix, such as a synthetic matrix or previously decellularized matrix, for example, a three- dimensional scaffold or matrix.
  • a mammalian cell that exhibits at least one characteristic of pluripotency, e.g., a pluripotent or multipotent cell, towards a cardiac lineage.
  • the cells include, but are not limited to, reprogrammed cells generated by the methods provided herein, embryonic stem cells, embryonic-like stem cells, cardiac stem cells, e.g., cardiosphere derived cells, and induced pluripotent stem cells.
  • reprogrammed cells generated by the methods provided herein can be induced to differentiate into cardiac cells.
  • Mammalian cells exhibiting cardiac differentiation generated via methods provided herein can be utilized in treatment of cardiac disorders and/or in amelioration of symptoms relating to cardiac disorders such as cardiac ischemia, myocardial infarction, and heart failure, including congestive heart failure.
  • the cells are induced to undergo cardiac differentiation by contacting the cells with a transcription factor capable of inducing cardiac differentiation.
  • the transcription factor is provided in the form of a transducible polypeptide comprising a cell penetration peptide as described herein, linked to the amino acid sequence of the transcription factor.
  • cells e.g., induced pluripotent stem cells
  • transducible polypeptides comprising a cell penetration peptide, e.g., herpes viral VP22 protein, HIV-I TAT, AntP HD, or poly-arginine, and a transcription factor having cardiac-differentiating activity for a time sufficient to induce cardiac differentiation of the cell, that is, to generate a cell that exhibits at least one characteristic of a cardiac cell.
  • the transcription factor having cardiac-differentiating activity is islet 1 (IsIl).
  • a method for cardiac differentiation of a mammalian cell exhibiting at least one characteristic of pluripotency comprising: contacting the mammalian cell with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs.
  • the transducible polypeptide comprises an islet 1 (ISLl) polypeptide linked to a cell penetration peptide.
  • the transducible polypeptide comprises a human ISLl polypeptide.
  • the cell penetration peptide comprises HIV-I TAT.
  • the cell penetration peptide comprises AntP HD.
  • the cell penetration peptide comprises poly-arginine.
  • the cell penetration peptide comprises herpes viral VP22 protein.
  • a method for cardiac differentiation of a mammalian cell exhibiting at least one characteristic of pluripotency comprising: contacting the mammalian cell in vitro, for example, ex vivo, with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs.
  • the transducible polypeptide comprises an islet 1 (ISLl) polypeptide linked to a cell penetration peptide.
  • the transducible polypeptide comprises a human ISLl polypeptide.
  • the cell penetration peptide comprises HIV-I TAT.
  • the cell penetration peptide comprises AntP HD.
  • the cell penetration peptide comprises poly-arginine.
  • the cell penetration peptide comprises herpes viral VP22 protein.
  • the transducible polypeptide comprises the carboxy terminus of VP22 linked to the amino terminus of IsIl, such as the transducible polypeptide provided herein as SEQ ID NO:39.
  • the transducible polypeptide comprises the carboxy terminus of IsIl linked to the amino terminus of VP22, such as the transducible polypeptide provided herein as SEQ ID NO:40.
  • the linkage can be direct or indirect.
  • the transcription factor having cardiac-differentiating activity is selected from the group consisting of ISLl, GATA-4, MEF2C, Nkx2.5, Hand-1, Hand-2, TBX5 and Twist- 1, e.g., human ISLl, GATA-4, MEF2C, Nkx2.5, Hand-1, Hand-2, TBX5 and Twist- 1.
  • a method for cardiac differentiation of a mammalian cell exhibiting at least one characteristic of pluripotency comprising: contacting the mammalian cell with one or more transducible polypeptides so that cardiac differentiation of the mammalian cell occurs, wherein the transducible polypeptide comprises an ISLl polypeptide linked to a cell penetration peptide, GATA-4 polypeptide linked to a cell penetration peptide, a MEF2C polypeptide linked to a cell penetration peptide, a Nkx2.5 polypeptide linked to a cell penetration peptide, a Hand-1 polypeptide linked to a cell penetration peptide, a Hand-2 polypeptide linked to a cell penetration peptide, a TBX5 polypeptide linked to a cell penetration peptide, or a Twist- 1 polypeptide linked to a cell penetration peptide.
  • the transducible polypeptide comprises an ISLl polypeptide linked to a cell penetration peptide, GATA-4 polypeptide linked to a cell penetration
  • the mammalian cell exhibiting at least one aspect of pluripotency is contacted in vitro, for example ex vivo.
  • the differentiated mammalian cell is contacted with at least 2, 3, 4, 5, 6, 7 or 8 different transducible polypeptides selected from the group consisting of an ISLl polypeptide linked to a cell penetration peptide, GATA-4 polypeptide linked to a cell penetration peptide, a MEF2C polypeptide linked to a cell penetration peptide, a Nkx2.5 polypeptide linked to a cell penetration peptide, a Hand-1 polypeptide linked to a cell penetration peptide, a Hand-2 polypeptide linked to a cell penetration peptide, a TBX5 polypeptide linked to a cell penetration peptide, or a Twist- 1 polypeptide linked to a cell penetration peptide.
  • the cell penetration peptide is selected from the group consisting of VP22, TAT, AntP HD, poly-arginine, or transducing fragments thereof.
  • a transducible polypeptide comprising a transcription factor having activity, e.g., ISLl, linked to a cell penetration peptide can be made using any of a variety of methods well known to those of skill in the art, including the methods of making a transducible peptide described in Section 5.2.3 above.
  • transcription factor having cardiac-differentiating activity is linked to the cell penetration peptide via a peptide bond.
  • a transducible polypeptide comprising a transcription factor having cardiac-differentiating activity linked to a cell penetration peptide may optionally comprise a nuclear localization signal, e.g., PKKKRKV (SEQ ID NO:37) of SV40 large T antigen, to enhance nuclear localization of the peptide.
  • a transducible polypeptide comprising a transcription factor having cardiac-differentiating activity linked to a cell penetration peptide may optionally comprise a purification moiety, e.g., a polyhistidine moiety, to facilitate isolation and purification of the transducible peptide.
  • any of the linking configurations or methods described in Section 5.2.3 may be used to link a transcription factor having cardiac-differentiating activity to a cell penetration peptide in a variety of configurations.
  • the carboxy terminus of the cell penetration peptide may be linked, directly or indirectly, to the amino terminus of the cardiac-differentiating factor polypeptide.
  • the carboxy terminus of the cardiac-differentiating factor may be linked to the amino terminus of the cell penetration peptide, either directly or indirectly.
  • the amino terminus of the cell penetration peptide may be linked, either directly or indirectly, to the amino terminus of the cardiac-differentiating factor.
  • the carboxy terminus of the cell penetration peptide may be linked, either directly or indirectly, to the carboxy terminus of the cardiac-differentiating factor.
  • an isolated mammalian cell that exhibits at least one characteristic of cardiac differentiation, wherein the isolated mammalian cell is generated by a method provided herein.
  • an isolated mammalian cells that exhibits at least one characteristic of cardiac differentiation, wherein the isolated mammalian cell is generated via a method comprising: contacting a mammalian cell that exhibits at least one characteristic of pluripotency, for example, contacting the in vitro, for example ex vivo, with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs, wherein the transducible polypeptide comprises an islet 1 (ISLl) polypeptide linked to a cell penetration peptide.
  • ISLl islet 1
  • the invention provides an isolated population of cells comprising at least 70%, 80%, 90%, 95% or 98% cells that exhibit at least one characteristic of cardiac differentiation, wherein the isolated cells are generated by a method provided herein. Also provided herein are such isolated populations of cells further comprising at least one other isolated population of cells, e.g., reprogrammed cells generated via the methods provided herein, stem cells, for example hematopoietic stem cells, stromal cells and/or isolated differentiated cells, for example adult cells. Further provided are such isolated populations of cells present on a solid support, e.g., a scaffold or matrix, such as a synthetic or previously decellularized matrix, for example, a three-dimensional scaffold or matrix. [00140] In one embodiment, the isolated population of cells comprises about IxIO 5 ,
  • the isolated population of cells is present in a formulation suitable for administration to a human.
  • the isolated population of cells is present in a bag, e.g., a plastic bag, such as a plastic bag suitable for use in administration of the cells to a human.
  • the isolated population of cells is present in a syringe, such as a sterile syringe suitable for administration of the cells to a human.
  • these cells are present on a solid support, e.g., a scaffold or matrix, such as a synthetic matrix or previously decellularized matrix, for example, a three-dimensional scaffold or matrix.
  • the invention provides an isolated population of cells comprising at least 70%, 80%, 90%, 95% or 98% cells that exhibit at least one characteristic of cardiac differentiation, wherein the isolated cells are generated by a method comprising: contacting a mammalian cell that exhibits at least one characteristic of pluripotency, e.g., contacting the cell in vitro, for example, ex vivo, with a transducible polypeptide so that cardiac differentiation of the mammalian cell occurs, wherein the transducible polypeptide comprises an islet 1 (ISLl) polypeptide linked to a cell penetration peptide.
  • ISLl islet 1
  • the cell displays at least one characteristic of cardiac differentiation if the cell: expresses at least one marker of cardiac differentiation selected from the group consisting of a-myosin heavy chain protein, natriuretic precursor A (ANP), ryanodine receptor and SERCA; forms sarcomeres in culture; appears visibly to be visibly beating in vitro; or demonstrates spontaneous membrane depolarization.
  • ABP natriuretic precursor A
  • SERCA natriuretic precursor A
  • An exemplary method for cardiac differentiation of a pluripotent mammalian cell comprising contacting the cell with a transducible polypeptide comprising ISLl and the cell penetration peptide VP22 is provided in the example of Section 6.2.
  • This example provides an exemplary method for generating a reprogrammed cell that exhibits at least one characteristic of pluripotency from a differentiated somatic cell using a plurality of different transducible polypeptides.
  • transducible polypeptides comprise: i) an Oct 3/4 polypeptide linked to a cell penetration peptide (e.g., SEQ ID NO: 25 or SEQ ID NO: 26), ii) a Sox2 polypeptide linked to a cell penetration peptide (e.g., SEQ ID NO: 27 or SEQ ID NO: 28), iii) a c-myc polypeptide linked to a cell penetration peptide (e.g., SEQ ID NO: 33 or SEQ ID NO: 34), iv) a Klf4 polypeptide linked to a cell penetration peptide (e.g., SEQ ID NO: 35 or SEQ ID NO: 36)e, v) a Nanog polypeptide linked to a cell penetration peptide (e.g., SEQ ID NO: 29 or
  • the cells are contacted with the transducible polypeptides for a two-week period, wherein cell media containing the transducible polypeptides is replaced every 2-3 days with fresh media comprising about 0.01 mg/ml of total transducible polypeptides.
  • the treated fibroblasts are plated on mitomycin C treated SNL feeder cells (a mouse cell line stably transduced with leukemia inhibitory factor (LIF) and a neomycin resistance gene) in medium designed for culture of primate embryonic stem cells, and supplemented with bFGF2. Three to four weeks later, putative reprogrammed cell clones are identified on the basis of their morphology (flat, tightly packed colonies).
  • LIF leukemia inhibitory factor
  • ISLl Promotes Cardiac Differentiation of ES cells in vitro
  • mice 22-28g Male C57B1/6 mice 22-28g (Jackson Laboratory) underwent anesthesia, analgesia, tracheal intubation, pulmonary ventilation (2cm H 2 O pressure, 120 min "1 , IITC Life Science, Woodland Hills, CA), intercostal thoracotomy and ligation of the left anterior descending (LAD) coronary artery (7-0 monofilament suture, Ethicon) to create experimental myocardial infarction.
  • Isll-VP22 recombinant protein was synthesized by inserting the IsIl gene in frame with VP22 in the plasmid pCR®T7/VP-22-l-TOPO®, then expressing the hybrid gene product in E. coli (VoyagerTM protein production kit, cat# K4860-01, Invitrogen, CA).
  • VP22 is a structural protein of herpes simplex virus that translocates to the nucleus of mammalian cells.
  • the method involved PCR amplification of a mouse IsIl cDNA clone (Open Biosystems Catalog #: EMM1002-99258597)(Fig.
  • Plasmid DNA was sequenced to confirm that the IsIl gene was inserted in the correct orientation, and in frame with VP22 (Fig. 4B).
  • the resulting plasmid construct was transformed into BL21(DE3)pLysS E. Coli and expression of the T7-regulated hybrid gene was induced by isopropyl ⁇ -D-thiogalactoside (IPTG) (Fig. 4C).
  • the expressed protein was purified by binding of His x6 amino acid residues to a nickel resin column (ProBondTM purification, cat# K850-01, Invitrogen, CA) followed by elution. Molecular weights of purified VP22 and ISL1-VP22 proteins were confirmed by Western Blot (Fig. 4D).
  • Rl mouse ES cells (Cat# SCRC-1036; ATCC, VA) were maintained in collagen-coated flasks, without a feeder layer, in medium containing leukemia-inhibitory factor (LIF - ESGROTM, Millipore, MA). The ES cells were passaged daily to prevent overcrowding of colonies.
  • Embryoid bodies (EBs) were generated by harvesting ES cells (0.5% trypsin-EDTA, cat# 59417C, Sigma-Aldrich), and culturing 10 6 cells in 1.5 mL of medium without LIF in ultra- low attachment plates. Medium was refreshed on alternate days. After 7 days, EBs were plated onto collagen-coated dishes.
  • IsIl Increased cardiac expression of IsIl by c-kit positive cells following myocardial infarction
  • IsIl protein was significantly increased in infarcted and peri-infarct cardiac tissue (18.3 ⁇ 4.6 fold, p ⁇ 0.01, Figs. 2A and 2B), accompanied by an increase in IsIl mRNA expression compared to baseline (Fig. 2C).
  • Mouse ES cells (Rl cell line) were differentiated into embryoid bodies by withdrawal of LIF from the culture medium in low-attachment wells. The cells were treated with VP22-Isll recombinant protein from day 1 to 10.
  • the results demonstrate that the embryonic transcription factor IsIl is re- expressed in injured adult cardiac tissue. Furthermore, the source of expression of IsIl within the infarcted cardiac tissue is demonstrated to be a subset of c-kit positive cells within the tissue. These results further demonstrate that a form of the IsIl protein specifically engineered to localize to the nucleus clearly promotes cardiac differentiation of ES cells in vitro.

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Abstract

L'invention porte ici, de manière générale, sur des procédés de génération de cellules mammifères reprogrammées, par exemple de cellules souches pluripotentes induites, à partir de cellules de mammifère différenciées sans utiliser de vecteurs viraux ou de plasmides. Sous un aspect, les procédés entraînent la mise en contact d'une cellule différenciée avec des polypeptides transducteurs comportant un polypeptide de facteur de reprogrammation lié à un peptide de pénétration de cellule de telle sorte qu'une cellule mammifère reprogrammée qui présente au moins une caractéristique pluripotente est générée. L'invention porte également sur des procédés pour la différenciation cardiaque d'une cellule mammifère sans utiliser de vecteurs viraux ou plasmides. Sous un aspect, de tels procédés entraînent la mise en contact d'une cellule de mammifère présentant au moins une caractéristique pluripotente avec un polypeptide transducteur, de telle sorte qu'une différenciation cardiaque de la cellule se produit.
PCT/US2009/065117 2008-11-20 2009-11-19 Génération de cellules souches pluripotentes induites sans utiliser de vecteurs viraux WO2010059806A2 (fr)

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US8846396B2 (en) 2003-07-31 2014-09-30 Universita Degli Studi Di Roma “La Sapienza” Methods for the isolation of cardiac stem cells
US11660317B2 (en) 2004-11-08 2023-05-30 The Johns Hopkins University Compositions comprising cardiosphere-derived cells for use in cell therapy
US9845457B2 (en) 2010-04-30 2017-12-19 Cedars-Sinai Medical Center Maintenance of genomic stability in cultured stem cells
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US20130295064A1 (en) * 2010-10-14 2013-11-07 University Of Central Florida Research Foundation, Inc. Cardiac induced pluripotent stem cells and methods of use in repair and regeneration of myocardium
WO2012051515A3 (fr) * 2010-10-14 2012-06-28 University Of Central Florida Research Foundation, Inc. Cellules souches pluripotentes cardio-induites et procédés d'utilisation pour la réparation et la régénération du myocarde
CN103857797A (zh) * 2011-07-19 2014-06-11 帷幄生物技术公司 用于修复软骨损伤的非遗传修饰性重编程细胞的组合物和方法
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US9884076B2 (en) 2012-06-05 2018-02-06 Capricor, Inc. Optimized methods for generation of cardiac stem cells from cardiac tissue and their use in cardiac therapy
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US9828603B2 (en) 2012-08-13 2017-11-28 Cedars Sinai Medical Center Exosomes and micro-ribonucleic acids for tissue regeneration
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US9644185B2 (en) 2013-08-28 2017-05-09 Snu R&Db Foundation Cell permeable fusion protein for facilitating reprogramming induction and use thereof
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US11351200B2 (en) 2016-06-03 2022-06-07 Cedars-Sinai Medical Center CDC-derived exosomes for treatment of ventricular tachyarrythmias
US11541078B2 (en) 2016-09-20 2023-01-03 Cedars-Sinai Medical Center Cardiosphere-derived cells and their extracellular vesicles to retard or reverse aging and age-related disorders
US11759482B2 (en) 2017-04-19 2023-09-19 Cedars-Sinai Medical Center Methods and compositions for treating skeletal muscular dystrophy
US11660355B2 (en) 2017-12-20 2023-05-30 Cedars-Sinai Medical Center Engineered extracellular vesicles for enhanced tissue delivery

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