Wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors

Patent metadata

Field	Value
Publication number	US-RE49281-E
Application number	US-201916713457-A
Country	US
Kind code	E1
Filing date	Dec 13, 2019
Priority date	Aug 31, 2007
Publication date	Nov 8, 2022
Grant date	Nov 8, 2022

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.

First claim

The invention claimed is:

1. A method of reprogramming a population of mammalian somatic cell into a population of induced pluripotent stem (iPS) cells, comprising:
(a) introducing into the cells of the population of mammalian somatic cell cells, 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 cells of the population of mammalian somatic cells;
(b) contacting the population of mammalian somatic cell cells with
i) a Wnt-3a conditionedWnt3a-conditioned medium, wherein cells used to prepare the Wnt3a-conditioned medium are genetically engineered to increase expression of Wnt3a; or
ii) an agent that modulatesstimulates the Wnt pathway, wherein the agent is selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein, and a GSK-3 antagonist; and

(c) culturing the population of mammalian somatic cells from (b) under conditions suitable for reprogramming the mammalian somatic cell to a pluripotent state such that reprogramming into the population of iPS cells occurs.

2. The method of claim 1 , wherein the method comprises:
(a) culturing contacting the cell cells of the population of mammalian somatic cells with the agent in culture mediumcontaining the agent;
(b) culturing contacting the cell in culture medium comprising cells of the population of mammalian somatic cells with the Wnt-3 a conditioned Wnt3a-conditioned medium or the agent in culture medium for at least 10 days;
(c) contacting the cell cells of the population of mammalian somatic cells with the Wnt-3a conditioned Wnt3a-conditioned medium or the agent that modulates the Wnt pathway, thereby enhancing such that the number of ES-like cell colonies is enhanced by at least 5-fold;
(d) contacting the cell cells of the population of mammalian somatic cells with the Wnt-3a conditioned Wnt3a-conditioned medium or the agent that modulates the Wnt pathway, thereby enhancing such that the number of ES-like cell colonies is enhanced by at least 10-fold;
(e) culturing contacting the cell in cells of the population of mammalian somatic cells with the Wnt3a-conditioned medium;
(f) contacting the cell cells of the population of mammalian somatic cells with the agent and a second agent that modulates stimulates the Wnt pathway; or
(g) culturing contacting the cell in medium containing cells of the population of mammalian somatic cells with the recombinant, exogenous, soluble, and biologically active Wnt protein in culture medium.

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 cells of the population of mammalian somatic cells are: (i) human cells, terminally differentiated cells, or fibroblasts; or (ii) modified to express or contain at least one reprogramming factor of Oct4, Klf4, and Sox2 at levels a level 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 differentiating the population of iPS cells into a desired cell type.

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 (i) the population of iPS cells is identified using morphological criteria; (ii) the method does not comprise chemical selection of iPS cells; or (iii) the method further comprises separating iPS cells from non-iPS cells.

7. A composition comprising:
(a) an a population of iPS cell cells; and
(b) a Wnt-3 a conditioned Wnt3a-conditioned medium, wherein the cells used to prepare the Wnt3a-conditioned medium are genetically engineered to increase their expression of Wnt3a; or an agent that increases activity of a stimulates the Wnt pathway, wherein the agent is 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.

Assignees

• Whitehead Inst Biomedical Res

Inventors

• Chevalier Brett
• Marson Alexander
• Young Richard A
• Foreman Ruth
• Jaenisch Rudolf

Classifications

• A61P7/06

  Antianaemics · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2502/45

  Artificially induced pluripotent stem cells · CPC title

• A61P25/14

  for treating abnormal movements, e.g. chorea, dyskinesia · CPC title

• C12N2501/602

  Sox-2 · CPC title