WNT PATHWAY STIMULATION IN REPROGRAMMING SOMATIC CELLS

• US 20100310525A1
• Filed: 08/29/2008
• Published: 12/09/2010
• Est. Priority Date: 08/31/2007
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Marson, Alexander, Young, Richard A., Chevalier, Brett, Jaenisch, Rudolf, Foreman, Ruth
• Granted Patent: US 9,102,919 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• 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 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• 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 7/06 Antianaemics
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 2502/45 Artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2502/99 : genetically modified cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides compositions and methods of use in reprogramming somatic cells. Compositions and methods of the invention are of use, e.g., for generating or modulating (e.g., enhancing) generation of induced pluripotent stem cells by reprogramming somatic cells. The reprogrammed somatic cells are useful for a number of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a pluripotent state and/or enhances the speed and/or efficiency of reprogramming. Certain of the compositions and methods relate to modulating the Wnt pathway.

Claims

1. A method of reprogramming a somatic mammalian cell, comprising contacting the somatic mammalian cell with an agent that modulates a Wnt pathway, so the cell becomes reprogrammed to a pluripotent state.

3. The method of claim 1, wherein the method comprises:

(a) culturing the cell in culture medium containing the agent;

(b) culturing the cell in culture medium comprising the agent for at least 10 days;

(c) contacting the cell with the agent that modulates the Wnt pathway, thereby enhancing the number of ES-like cell colonies by at least 5-fold;

(d) contacting the cell with the agent that modulates the Wnt pathway, thereby enhancing the number of ES-like cell colonies by at least 10-fold;

(e) culturing the cell in Wnt3a-conditioned medium

(f) culturing the cell in Wnt-conditioned medium;

(g) contacting the cell with a second agent that modulates the Wnt pathway;

or

(h) culturing the cell in medium containing an exogenous soluble, biologically active Wnt protein.

9. The method of claim 1, wherein the cell:

(a) is a human cell;

(b) is a terminally differentiated cell;

(c) is a fibroblast;

(d) is modified to express or contain at least one reprogramming factor at levels greater than normally present in cells of that type;

(e) is not genetically modified;

or

(f) is not genetically modified to express c-Myc at levels greater than normally present in a cell of that cell type.

17. The method of claim 3, wherein the Wnt protein is Wnt3a.

18. The method of claim 1, further comprising:

(a) confirming that the reprogrammed cell is pluripotent;

(b) administering the reprogrammed cell to a subject;

or

(c) differentiating the cell to a desired cell type in vitro after reprogramming the cell.

19. The method of claim 1, wherein the method is practiced on:

(a) a population of cells and the method further comprises identifying ES-like cells by morphological criteria;

(b) a population of cells and the method does not comprise imposing chemical selection to select reprogrammed cells;

or

(c) a population of cells and the method further comprises separating cells that are reprogrammed to a pluripotent state from cells that are not reprogrammed to a pluripotent state.

24. The method of claim 18, further comprising administering the differentiated cell to a subject.

25. A method of treating an individual in need thereof comprising:

(a) obtaining somatic cells from the individual;

(b) reprogramming at least some of the somatic cells according to the method of claim 1; and

(c) administering at least some of the reprogrammed cells to the individual, optionally after differentiating the cells into one or more desired cell types.

2. (canceled)

4-8. -8. (canceled)

10-16. -16. (canceled)

20-23. -23. (canceled)

26-28. -28. (canceled)

29. A composition comprising (i) a somatic mammalian cell that has been modified or treated so that it expresses or contains at least one reprogramming factor at levels greater than would be the case without such modification or treatment;

and (ii) an agent that increases activity of a Wnt pathway and contributes to reprogramming the somatic cell to a pluripotent state.

30. The composition of claim 29, wherein the somatic cell:

(a) is modified to express or contain at least one reprogramming factor at levels greater than normally present in somatic cells of that type;

(b) is not genetically modified

(c) is not genetically modified to express c-Myc at levels greater than normally present in somatic cells of that type;

or

(d) is modified to express or contain at least one reprogramming factor at levels greater than normally present in somatic cells of that type.

31-34. -34. (canceled)

35. A method of identifying an agent useful for modulating the reprogramming of mammalian somatic cells comprising:

(a) culturing a population of mammalian somatic cells in medium containing an agent that modulates activity of a Wnt pathway and a candidate agent;

(b) determining, after a suitable period of time, whether cells having one or more characteristics of ES cells are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the candidate agent is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels different than would be expected had the medium not contained the candidate agent.

36-44. -44. (canceled)

45. A method of identifying an agent useful for reprogramming mammalian somatic cells to an ES-like state comprising:

(a) contacting a population of mammalian somatic cells with an agent that increases Wnt pathway activity and a candidate agent;

(b) maintaining the cells in a cell culture system for a suitable period of time; and

(c) determining whether cells having one or more characteristics of ES cells are present in said culture system, wherein the agent is identified as being useful for reprogramming mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels greater than would be expected had the cells not been contacted with the candidate agent.

46-50. -50. (canceled)

51. A method of reprogramming a somatic mammalian cell comprising culturing the cell in the presence of an extracellular signaling molecule so that the cell becomes reprogrammed.

52. The method of claim 51, wherein said extracellular signaling molecule is a molecule whose binding to an extracellular domain of a cellular receptor initiates or modifies a signal transduction pathway within the cell.

53. The method of claim 52, wherein the signal transduction pathway is the Wnt pathway.

54. A method of identifying a Wnt pathway modulator useful for modulating the reprogramming of mammalian somatic cells to a pluripotent state comprising:

(a) culturing a population of mammalian somatic cells in medium containing the Wnt pathway modulator;

(b) determining, after a suitable period of time, whether cells having one or more characteristics of ES cells are present after maintaining the cells and their progeny in culture for a suitable time period, wherein the Wnt pathway modulator is identified as being useful for modulating the reprogramming of mammalian somatic cells to an ES-like state if cells having one or more characteristics of ES cells are present at levels different than would be expected had the medium not contained the Wnt pathway modulator.

55-59. -59. (canceled)

60. A composition comprising:

(a) cell culture medium containing a Wnt pathway modulator; and

(b) a plurality of mammalian somatic cells, wherein (i) the cells are genetically modified or transiently transfected to express one or more reprogramming factors;

(ii) the cells are genetically modified to contain a nucleic acid sequence encoding a selectable marker, operably linked to a promoter for an endogenous pluripotency gene;

or (iii) the cell culture medium contains one or more small molecules, nucleic acids, or polypeptides that substitute for a reprogramming factor other than c-Myc.

61. The composition of claim 60, wherein:

(a) the cell culture medium comprises Wnt-3a CM; and

/or

(b) the one or more reprogramming factors are selected from;

Oct4, Nanog, Sox2, Lin28, and Klf4.

62. (canceled)

63. A composition comprising:

an iPS cell and an agent that activates the Wnt pathway.

64-65. -65. (canceled)