Wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors

• US 9,593,311 B2
• Filed: 08/10/2015
• Issued: 03/14/2017
• Est. Priority Date: 08/31/2007
• Status: Expired

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Chevalier, Brett, Marson, Alexander, Young, Richard A., Foreman, Ruth, Jaenisch, Rudolf
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US14/822,653
• Publication Number:
  US 20160068819A1
• Time in Patent Office: 582 Days
• US Class Current: 1/1
• 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 mammalian somatic cell, comprising:

(a) introducing into the mammalian somatic cell, one or more retroviral vectors encoding Oct4, and optionally encoding one or more reprogramming factors selected from the group consisting of;

Klf4 and Sox2, wherein c-Myc is not introduced into the cell;

(b) contacting the mammalian somatic cell with a Wnt-3a conditioned medium;

or an agent that modulates the Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist; and

(c) culturing the cells under conditions suitable for reprogramming the mammalian somatic cell to a pluripotent state.

2. 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 Wnt-3 a conditioned medium or the agent for at least 10 days;

(c) contacting the cell with the Wnt-3a conditioned medium or 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 Wnt-3a conditioned medium or 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 the Wnt3a-conditioned medium;

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

or (g) culturing the cell in medium containing the recombinant, exogenous, soluble, and biologically active Wnt protein.

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

(a) is a human cell;

(b) is a terminally differentiated cell;

(c) is a fibroblast;

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

4. The method of claim 2, wherein the recombinant, exogenous, soluble, and biologically active Wnt protein is Wnt3a.

5. 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.

6. 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.

7. A composition comprising:

(a) an iPS cell; and

(b) a Wnt-3 a conditioned medium;

or an agent that increases activity of a Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist.

8. The composition of claim 7, wherein the recombinant, exogenous, soluble, and biologically active Wnt protein is Wnt3a.