Reprogramming of somatic cells

• US 9,714,414 B2
• Filed: 08/29/2014
• Issued: 07/25/2017
• Est. Priority Date: 04/07/2007
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Jaenisch, Rudolf, Hanna, Yaqub, Wernig, Marius, Lengner, Christopher J., Meissner, Alexander, Brambrink, Oliver Tobias, Welstead, G. Grant, Foreman, Ruth
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US14/473,250
• Publication Number:
  US 20150118755A1
• Time in Patent Office: 1,061 Days
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/02 of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• CPC Class Codes: A61P 5/14 of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 5/18 of the parathyroid hormones
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/4702 : Regulators; Modulating activity
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/8775 : Murine embryos
• CPC Class Codes: C12N 2501/602 : Sox-2
• CPC Class Codes: C12N 2501/603 : Oct-3/4
• CPC Class Codes: C12N 2501/604 : Klf-4
• CPC Class Codes: C12N 2501/605 : Nanog
• CPC Class Codes: C12N 2501/606 : c-Myc
• CPC Class Codes: C12N 2506/11 : from blood or immune system cells
• CPC Class Codes: C12N 2506/13 : from connective tissue cells, from mesenchymal cells
• CPC Class Codes: C12N 2800/30 : Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/003 : tet inducible
• CPC Class Codes: C12N 2830/006 : tet repressible
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/91011 : Methyltransferases (general) (2.1.1.)
• CPC Class Codes: G01N 33/5023 : on expression patterns

Abstract

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

Claims

1. A method of reprogramming a cell to a pluripotent state comprising:

(a) introducing into the cell exogenous factors comprising (i) polynucleotides encoding Oct-4, Sox-2, and Klf-4, but not c-Myc;

or (ii) Oct-4, Sox-2, and Klf-4 polypeptides, but not a c-Myc polypeptide, and (b) culturing the cell for a sufficient amount of time to reprogram the cell to the pluripotent state, wherein the reprogrammed cell does not express a selectable marker operably linked to a pluripotency gene.

2. The method of claim 1, wherein the polynucleotides are introduced by transfection or viral infection.

3. The method of claim 1, wherein step (a) comprises introducing said polynucleotides into the cell;

and the method further comprising inactivating at least one of the polynucleotides after the cell obtains the pluripotent state, wherein said inactivating optionally comprises excising at least a portion of a coding sequence from the at least one of the polynucleotides, wherein the portion of the coding sequence is flanked by sequences that are recognized by a site-specific recombinase, and wherein said excising comprises introducing or expressing the site-specific recombinase in the cell after the cell obtains the pluripotent state, wherein the site-specific recombinase removes the portion of the coding sequence from the at least one of the polynucleotides.

4. A method of reprogramming a human adult somatic cell to a pluripotent state comprising the steps of:

(a) introducing into a human adult somatic cell reprogramming agents that contribute to reprogramming of the cell, wherein the reprogramming agents comprise Oct-4, Sox-2, and Klf-4, but not c-Myc; and

(b) culturing the cell in a medium comprising cell proliferation factors and factors that support reprogramming for a period of time sufficient to reprogram the cell to the pluripotent state, wherein the cell does not comprise a selectable marker operably linked to an endogenous pluripotency gene.

5. The method of claim 4, wherein the reprogramming agents are introduced as (i) polynucleotides encoding Oct-4, Sox-2, and Klf-4, but not c-Myc;

or (ii) Oct-4, Sox-2, and Klf-4 polypeptides, but not a c-Myc polypeptide.

6. The method of claim 4, wherein the somatic cell is a partially differentiated cell or a fully differentiated cell.

7. A method of generating a reprogrammed human pluripotent cell comprising:

introducing into a human somatic cell, exogenous polynucleotides encoding Oct-4, Sox-2, and Klf-4, but not c-Myc;

or exogenous Oct-4, Sox-2, and Klf-4 polypeptides, but not a c-Myc polypeptide, culturing said human somatic cell for a sufficient amount of time to obtain a reprogrammed human pluripotent that (i) is resistant to DNA demethylation;

(ii) expresses endogenous Oct-4;

(iii) differentiates into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; and

(iv) does not comprise a selectable marker operably linked to an endogenous pluripotency gene.

8. The method of claim 7, wherein the exogenously introduced polynucleotides are introduced into the human somatic cell by transfection or viral infection.

9. The method of claim 7, wherein step (a) comprises introducing said polynucleotides into the human somatic cell;

and the method further comprising inactivating at least one of the polynucleotides after the human somatic cell becomes the reprogrammed human pluripotent cell, wherein said inactivating optionally comprises excising at least a portion of a coding sequence from the at least one of the polynucleotides, wherein the portion of the coding sequence is flanked by sequences that are recognized by a site-specific recombinase, and wherein said excising comprises introducing or expressing the site-specific recombinase in the cell after the human somatic cell becomes the reprogrammed human pluripotent cell, wherein the site-specific recombinase removes the portion of the coding sequence from the at least one of the polynucleotides.