WNT pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors
First Claim
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1. A method of reprogramming a mammalian somatic cell, comprising:
- (I) 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;
(II) 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
(III) culturing the cells under conditions suitable for reprogramming the mammalian somatic cell to a pluripotent state.
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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.
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Citations
12 Claims
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1. A method of reprogramming a mammalian somatic cell, comprising:
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(I) 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;(II) 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(III) culturing the cells under conditions suitable for reprogramming the mammalian somatic cell to a pluripotent state. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A composition comprising (i) a mammalian somatic cell that has been modified or treated by introducing one or more polynucleotides 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;
wherein the cell expresses an Oct4 polypeptide and at least one reprogramming factor selected from the group consisting of Klf4 polypeptide, and a Sox2 polypeptide at levels greater than would be the case without such modification or treatment; and
(ii) a Wnt-3a 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 that contributes to reprogramming the somatic cell to a pluripotent state. - View Dependent Claims (8)
- Klf4, and Sox2, wherein c-Myc is not introduced into the cell;
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9. A composition comprising:
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(a) cell culture medium containing a Wnt-3a conditioned medium;
or a Wnt pathway modulator selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein; and
a GSK-3 antagonist; and(b) a plurality of mammalian somatic cells, wherein (i) the cells are genetically modified or transiently transfected with one or more polynucleotides 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;
(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. - View Dependent Claims (10)
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11. A composition comprising:
- an iPS cell and a Wnt-3a conditioned medium;
an agent that activates the Wnt pathway selected from the group consisting of a recombinant, exogenous, soluble, and biologically active Wnt protein; and
a GSK-3 antagonist. - View Dependent Claims (12)
- an iPS cell and a Wnt-3a conditioned medium;
Specification