Combined chemical and genetic approaches for generation of induced pluripotent stem cells

Abstract

To provide identification and use of small molecules for inducing pluripotency in mammalian cells as well as methods for inducing pluripotency.SOLUTION: The method comprises the steps of: (a) introducing into a non-pluripotent mammalian cell: i) one or more expression cassettes comprising a polynucleotide encoding Oct polypeptide, a polynucleotide encoding Klf polypeptide and a polynucleotide encoding Sox polypeptide or ii) exogenous Oct polypeptide, exogenous Sox polypeptide and exogenous Klf polypeptide; and (b) contacting the non-pluripotent mammalian cell with GSK3 inhibitor, MEK inhibitor and TGFβ receptor / ALK5 inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 61 / 069,956, filed March 17, 2008, and 61 / 197,986, filed October 31, 2008, each of which is incorporated by reference. [Background technology]

[0002] 2. Background of the Invention Stem cells are often classified as totipotent or pluripotent. Totipotent stem cells have the ability to differentiate into anything, i.e., give rise to all the different types of cells in the body. An example of a totipotent stem cell is a fertilized egg cell. Pluripotent stem cells can give rise to all the cell types in the body that come from the three main germ cell layers, or the embryo itself.

[0003] Pluripotent stem cells, such as embryonic stem cells (ESCs), proliferate rapidly while maintaining pluripotency, i.e., the ability to differentiate into various cell types. Embryonic stem cells are a promising donor source for cell transplantation therapy. However, human ESCs are also associated with ethical issues regarding the use of human embryos and rejection after allogeneic transplantation. These issues could be overcome by generating pluripotent stem cells directly from the patient's somatic cells. The existence of "pluripotency-inducing" factors has been suggested, as somatic cell nuclei acquire a state similar to that of embryonic stem cells by fusing with ESCs. Recently, previous studies have shown that retroviral-mediated transfection of four transcription factors (Oct-3 / 4, Sox2, KLF4, and c-Myc), which are highly expressed in ESCs, into mouse fibroblasts leads to the generation of induced pluripotent stem (iPS) cells. Takahashi, K. and J Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006)(Non-Patent Document 1); Okita, K., Ichisaka, T. and J Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313-317 (2007)(Non-Patent Document 2); Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 (2007)(Non-Patent Document 3); Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.Cell Stem Cell 1, 55-70 (2007)(Non-Patent Document 4); Meissner, A., Wernig, M. and Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature Biotechnol. 25, 1177-1181 (2007)(Non-Patent Document 5); Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007)(Non-Patent Document 6); Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 (2007)(Non-Patent Document 7); Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnol. 26, 101-106 (2007); Wernig, M., Meissner, A., Cassady, JP and Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10-12 (2008). iPS cells resemble ESCs in morphology, proliferation and pluripotency as judged by teratoma formation and chimera contribution.

[0004] The recent breakthrough of using defined genetic manipulation, i.e., viral transduction of several genes highly and / or specifically expressed in mouse or human embryonic stem (ES) cells, in the reprogramming of mouse and human somatic cells into induced pluripotent stem (iPS) cells has opened up great opportunities to generate patient-specific stem cells for various applications (e.g., cell therapy or drug discovery) as well as to study epigenetic reverse processes, regardless of the arguments related to traditional human ES cells. The eventual clinical application of iPS cell techniques will primarily require methods for directed differentiation of human PS cells to generate homogenous populations of lineage-specific cell types as well as to eliminate the risks and low efficiency / slow in vivo kinetics associated with the shortcomings of current iPS cell genetic manipulation. Recent studies have shown that cMyc, one of the four previously required genes, is not necessarily required for overexpression in iPS cell generation. See Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnol. 26, 101-106 (2007); Wernig, M., Meissner, A., Cassady, JP and Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10-12(2008). However, in the absence of cMyc, reprogramming efficiency was quite low and reprogramming kinetics was quite slow. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takahashi, K. and J Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006) [Non-Patent Document 2] Okita, K., Ichisaka, T. and J Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313-317 (2007) [Non-Patent Document 3] Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324 (2007) [Non-Patent Document 4] Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55-70 (2007) [Non-Patent Document 5] Meissner, A., Wernig, M. and Jaenisch, R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature Biotechnol. 25, 1177-1181 (2007) [Non-Patent Document 6] Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007) [Non-Patent Document 7] Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 (2007) [Non-Patent Document 8] Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnol. 26, 101-106 (2007) [Non-Patent Document 9] Wernig, M., Meissner, A., Cassady, JP and Jaenisch, R. c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10-12(2008) Summary of the Invention

[0006] BRIEF SUMMARY OF THE INVETION The present invention provides a method for screening for an agent that induces reprogramming or dedifferentiation of a mammalian cell into a pluripotent stem cell. In some embodiments, the method comprises: (a) introducing at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into a non-pluripotent cell to generate a transfected cell; (b) contacting the transfected cells with a library of different agents; (c) screening the contacted cells for characteristics of pluripotent stem cells; and (d) correlating the occurrence of stem cell characteristics with specific agents from the library, thereby identifying agents that stimulate dedifferentiation of cells into pluripotent stem cells. Includes.

[0007] In some embodiments, step (a) comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide.

[0008] In some embodiments, step (a) comprises introducing at least one, but not all, of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Myc polypeptide, and an exogenous Sox polypeptide into the non-pluripotent cell.

[0009] In some embodiments, the particular agent is between 50 and 1500 daltons.

[0010] In some embodiments, step (a) comprises introducing two expression cassettes into the cell, where each expression cassette comprises a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and the remaining members of the group are not introduced into the cell.

[0011] In some embodiments, step (a) comprises introducing three expression cassettes into the cell, where each expression cassette comprises a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and where the remaining members of the group are not introduced into the cell.

[0012] In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human mammalian cell. In some embodiments, the non-pluripotent cell is a progenitor cell. In some embodiments, the progenitor cell is a neural progenitor cell, a skin progenitor cell, or a hair follicle progenitor cell.

[0013] In some embodiments, the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

[0014] The present invention also provides a method for screening mammalian cells having characteristics of pluripotent stem cells. In some embodiments, the method comprises: (a) contacting a cell with a MAPK / ERK kinase (MEK) inhibitor such that proliferation of non-pluripotent cells is inhibited and proliferation of pluripotent stem cells is promoted; and (b) screening the contacted cells for characteristics of pluripotent stem cells. Includes.

[0015] In some embodiments, the method further comprises: Prior to step (a), contacting the cells with a library of agents; and After step (b), selecting an agent that induces pluripotent stem cells based on the results of step (b). Includes.

[0016] In some embodiments, the cells are human cells, hi some embodiments, the cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells.

[0017] In some embodiments, the MEK inhibitor is PD0325901.

[0018] The present invention also provides a method for generating induced pluripotent stem cells from mammalian non-pluripotent cells. In some embodiments, the method comprises: (a) introducing one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into a non-pluripotent cell; (b) contacting the cells with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby generating induced pluripotent stem cells. Includes.

[0019] In some embodiments, step (a) comprises contacting the non-pluripotent cells with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide. i. contacting a non-pluripotent cell with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; ii. subsequently culturing the cells in the absence of said exogenous polypeptide. at least two (e.g., two, three, four, five, or more) cycles of

[0020] In some embodiments, step (a) comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

[0021] In some embodiments, the method further comprises screening the contacted cells for a characteristic of a pluripotent stem cell.

[0022] In some embodiments, an expression cassette for expression of an Oct polypeptide and an expression cassette for expression of a Sox polypeptide are introduced into the non-pluripotent cell.

[0023] In some embodiments, the introducing step comprises introducing one or more expression cassettes for expression of a KLF polypeptide and an Oct polypeptide into the non-pluripotent cell, and no expression cassettes for a Myc polypeptide and / or a Sox polypeptide are introduced into the cell.

[0024] In some embodiments, the Klf polypeptide is Klf4 and the Oct polypeptide is Oct4.

[0025] In some embodiments, the non-pluripotent cell is a somatic cell.

[0026] In some embodiments, the non-pluripotent cells are fibroblasts.

[0027] In some embodiments, neither an expression cassette for expression of a Myc polypeptide nor an expression cassette for expression of a Klf polypeptide is introduced into the non-pluripotent cell.

[0028] In some embodiments, the introducing step occurs in vivo. In some embodiments, the introducing step occurs in vitro.

[0029] In some embodiments, the method further comprises: (c) selecting cells that exhibit characteristics of pluripotent stem cells Further includes:

[0030] In some embodiments, non-pluripotent cells are obtained from an animal and the induced pluripotent stem cells are differentiated into a desired cell type.

[0031] In some embodiments, the desired cell type is introduced into an animal. In some embodiments, the animal is a human. In some embodiments, the animal is a non-human animal.

[0032] In some embodiments, the selected cells do not contain an exogenous expression cassette for expression of Oct4.

[0033] In some embodiments, the agent inhibits H3K9 methylation. In some embodiments, the agent that inhibits H3K9 methylation is BIX01294.

[0034] In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell. In some embodiments, the non-pluripotent cell is a progenitor cell. In some embodiments, the progenitor cell is a neural progenitor cell.

[0035] In some embodiments, the introducing step comprises: a first vector comprising a promoter operably linked to a first expression cassette, the first expression cassette comprising a polynucleotide encoding Klf4; a second vector comprising a promoter operably linked to a second expression cassette, the second expression cassette comprising a polynucleotide encoding Sox2; and A third vector comprising a promoter operably linked to a third expression cassette, the third expression cassette comprising a polynucleotide encoding c-Myc. This includes introducing:

[0036] In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, a standard non-viral plasmid vector, or an episomal expression vector.

[0037] The present invention also includes a mixture of mammalian cells and an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, wherein the cells express at least one or more of an Oct polypeptide, a Klf polypeptide, a Sox polypeptide, and a Myc polypeptide; and / or are in contact with at least one or more of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Sox polypeptide, and an exogenous Myc polypeptide.

[0038] In some embodiments, the cell comprises a first recombinant expression cassette, a second recombinant expression cassette, and a third recombinant expression cassette, wherein the first expression cassette comprises a promoter operably linked to a polynucleotide encoding a Klf polypeptide, the second expression cassette comprises a promoter operably linked to a polynucleotide encoding a Sox polypeptide, and the third expression cassette comprises a promoter operably linked to a polynucleotide encoding a Myc polypeptide.

[0039] In some embodiments, the agent inhibits H3K9 methylation. In some embodiments, the agent that inhibits H3K9 methylation is BIX01294.

[0040] In some embodiments, the cell comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors, wherein the one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors comprise the first expression cassette, the second expression cassette, and the third expression cassette.

[0041] In some embodiments, the mixture comprises a first, a second, and a third retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector, wherein the first retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the first expression cassette, the second retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the second expression cassette, and the third retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the third expression cassette.

[0042] In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell comprises a progenitor cell. In some embodiments, the progenitor cell is a neural progenitor cell, a skin progenitor cell, or a hair follicle progenitor cell.

[0043] In some embodiments, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

[0044] The present invention also provides a mammalian cell that endogenously expresses at least one protein selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, wherein the cell does not endogenously express at least one protein of the group, and the protein that is not endogenously expressed is expressed from an RNA encoded by a heterologous recombinant expression cassette present in the cell, wherein the cell endogenously or heterologously expresses the Oct polypeptide, the Klf polypeptide, the Myc polypeptide, and the Sox polypeptide, respectively, and expression of the protein from the heterologous expression cassette results in reprogramming or dedifferentiation of the cell from a non-pluripotent cell to a pluripotent stem cell.

[0045] In some embodiments, the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.In some embodiments, the cell endogenously expresses Sox polypeptide and Myc polypeptide, and heterologously expresses Oct polypeptide and Klf polypeptide.In some embodiments, the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

[0046] The present invention also provides a method for inducing Oct4 expression in a cell. In some embodiments, the method comprises contacting a cell with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby inducing Oct4 expression in the cell.

[0047] In some embodiments, immediately prior to the contacting step, the cells do not express Oct4. In some embodiments, the contacted cells are not pluripotent cells. In some embodiments, after the contacting step, the cells are induced to become pluripotent.

[0048] The present invention also provides a method for inducing non-pluripotent cells into pluripotent cells. In some embodiments, the method comprises contacting non-pluripotent cells with one or more agents that induce pluripotency, and / or introducing an expression cassette into the cells to express a protein that induces pluripotency, wherein the cells are not cultured on feeder cells, and the cells are attached to a solid culture surface. In some embodiments, the method further comprises screening the contacted cells for characteristics of pluripotent stem cells.

[0049] In some embodiments, the cells are attached to the solid culture surface by a molecular tether selected from the group consisting of matrigel, extracellular matrix (ECM) or ECM analog, laminin, fibronectin, and collagen.

[0050] In some embodiments, the contacting step comprises: (a) introducing into a non-pluripotent cell one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; (b) contacting the cells with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby generating induced pluripotent stem cells. Includes.

[0051] The present invention also provides a method for generating induced pluripotent stem cells from mammalian non-pluripotent cells. In some embodiments, the method comprises: (a) the cells; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation (including, but not limited to, BIX); L-type Ca channel agonists (including but not limited to BayK); cAMP pathway activators (including but not limited to forskolin); DNA methyltransferase (DNMT) inhibitors (including but not limited to RG108 or 5-Aza-C); Nuclear receptor ligands (including but not limited to dexamethasone); GSK3 inhibitors (including but not limited to CHIR99021); MEK inhibitors; TGFβ receptor / ALK5 inhibitors (including but not limited to SB431542, A-83-01, or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine); HDAC inhibitors (including but not limited to TSA, VPA, sodium butyrate, SAHA, etc.); and Erk inhibitors At least one of (e.g., one, two, three, four, or more) thereby producing induced pluripotent stem cells.

[0052] In some embodiments, the method further comprises screening the contacted cells for a characteristic of a pluripotent stem cell.

[0053] In some embodiments, the method further comprises: (a) the cells; i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, and ii. L-type Ca channel agonists (including but not limited to BayK); cAMP pathway activators (including but not limited to forskolin); DNA methyltransferase (DNMT) inhibitors (including but not limited to RG108 or 5-Aza-C); Nuclear receptor ligands (including but not limited to dexamethasone); GSK3 inhibitors (including but not limited to CHIR99021); MEK inhibitors; TGFβ receptor / ALK5 inhibitors (including but not limited to SB431542, A-83-01, or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine); HDAC inhibitors (including but not limited to TSA, VPA, sodium butyrate, SAHA, etc.); and Erk inhibitors and one agent selected from the group consisting of The method includes the step of contacting.

[0054] In some embodiments, the method further comprises: (b) further comprising the step of introducing into the non-pluripotent cell one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and / or a Sox polypeptide.

[0055] In some embodiments, Klf4 and Oct4 are introduced into the cells (optionally, Myc and Sox are not introduced).

[0056] The present invention also provides A mammalian cell; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitor, HDAC inhibitors; and Erk inhibitors At least one of (e.g., one, two, three, four, or more) A mixture of:

[0057] In some embodiments, the mixture comprises: i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and Erk inhibitors and one agent selected from the group consisting of Includes.

[0058] In some embodiments, the cells comprise a heterologous expression cassette for expression of an Oct polypeptide and a heterologous expression cassette for expression of a Sox polypeptide.

[0059] In some embodiments, the cell is a non-pluripotent cell. In some embodiments, the cell is a fibroblast cell.

[0060] In some embodiments, neither an expression cassette for expression of a Myc polypeptide nor an expression cassette for expression of a Klf polypeptide is introduced into the non-pluripotent cell.

[0061] The present invention also provides one agent that inhibits H3K9 methylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and Erk inhibitors and one agent selected from the group consisting of The present invention provides a composition comprising:

[0062] The present invention also provides i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and Erk inhibitors and one agent selected from the group consisting of A kit is provided, comprising:

[0063] In some embodiments, the kit further comprises a mammalian cell.

[0064] Certain aspects of the present invention are set forth immediately below in claim form.

[0065] 1. A method for producing induced pluripotent stem cells from mammalian non-pluripotent cells, comprising: (a) introducing into the non-pluripotent cell one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide; (b) contacting the cells with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby generating induced pluripotent stem cells. A method comprising: 2. The method of claim 1, wherein step (a) comprises contacting the non-pluripotent cells with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide. 3. Step (a) i. contacting the non-pluripotent cell with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; ii. subsequently culturing the cells in the absence of said exogenous polypeptide. 3. The method of claim 2, comprising at least two cycles of: 4. The method of claim 1, wherein step (a) comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide. 5. The method of claim 1, wherein an expression cassette for expressing an Oct polypeptide and an expression cassette for expressing a Sox polypeptide are introduced into the non-pluripotent cell. 6. The method of claim 1, wherein the introducing step comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of a KLF polypeptide and an Oct polypeptide, and no expression cassettes for a Myc polypeptide and / or a Sox polypeptide are introduced into the cell. 7. The method of any one of claims 1 to 6, wherein the Klf polypeptide is Klf4 and the Oct polypeptide is Oct4. 8. The method of any one of claims 1 to 7, wherein the non-pluripotent cells are somatic cells. 9. The method of any one of claims 1 to 7, wherein the non-pluripotent cells are fibroblasts. 10. The method according to any one of claims 1 to 7, wherein neither an expression cassette for expression of a Myc polypeptide nor an expression cassette for expression of a Klf polypeptide is introduced into the non-pluripotent cell. 11. The method of any one of claims 1 to 10, wherein the introducing step is carried out in vivo. 12. The method of any one of claims 1 to 10, wherein the introducing step is carried out in vitro. 13. The method of any one of claims 1 to 12, further comprising the step of (c) selecting cells that exhibit characteristics of pluripotent stem cells. 14. The method of any one of claims 1 to 13, wherein the non-pluripotent cells are obtained from an animal and the induced pluripotent stem cells are differentiated into a desired cell type. 15. The method of claim 14, wherein the desired cell type is introduced into an animal. 16. The method of claim 15, wherein the animal is a human. 17. The method of claim 15, wherein the animal is a non-human animal. 18. The method of claim 13, wherein the selected cells do not contain an exogenous expression cassette for expression of Oct4. 19. The method of any one of claims 1 to 8, wherein the agent inhibits H3K9 methylation. 20. The method of claim 19, wherein the agent that inhibits H3K9 methylation is BIX01294. 21. The method of any one of claims 1 to 20, wherein the cell is a human cell. 22. The method of any one of claims 1 to 20, wherein the cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells. 23. The method of any one of claims 1 to 22, wherein the non-pluripotent cells are progenitor cells. 24. The method of claim 23, wherein the progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. 25. The process of introduction is a first vector comprising a promoter operably linked to a first expression cassette, the first expression cassette comprising a polynucleotide encoding Klf4; a second vector comprising a promoter operably linked to a second expression cassette, the second expression cassette comprising a polynucleotide encoding Sox2; and A third vector comprising a promoter operably linked to a third expression cassette, the third expression cassette comprising a polynucleotide encoding c-Myc. The method of any one of claims 1 or 4 to 24, comprising introducing: 26. The method of any one of claims 1 or 4 to 25, wherein the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, a non-viral plasmid vector, or an episomal expression vector. 27. A method for screening an agent that induces reprogramming or dedifferentiation of a mammalian cell into a pluripotent stem cell, comprising: (a) introducing at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into a non-pluripotent cell to generate a transfected cell; (b) contacting the transfected cells with a library of different agents; (c) screening the contacted cells for characteristics of pluripotent stem cells; and (d) correlating the occurrence of stem cell characteristics with specific agents from the library, thereby identifying agents that stimulate dedifferentiation of cells into pluripotent stem cells. A method comprising: 28. The method of claim 27, wherein step (a) comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide. 29. The method of claim 27, wherein step (a) comprises introducing at least one, but not all, of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Myc polypeptide, and an exogenous Sox polypeptide into the non-pluripotent cell. 30. The method of claims 27 to 29, wherein the specific active substance is 50 to 1500 daltons. 31. The method of claim 27, wherein step (a) comprises introducing into the cell two expression cassettes, each expression cassette comprising a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and the remaining members of the group are not introduced into the cell. 32. The method of claim 27, wherein step (a) comprises introducing three expression cassettes into the cell, each expression cassette comprising a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and the remaining members of the group are not introduced into the cell. 33. The method of any one of claims 27 to 32, wherein the cell is a human cell. 34. The method of any one of claims 27 to 33, wherein the cell is a non-human mammalian cell. 35. The method of any one of claims 27 to 34, wherein the non-pluripotent cells are progenitor cells. 36. The method of claim 35, wherein the progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. 37. The method of any one of claims 27 to 36, wherein the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. 38. A method for screening mammalian cells having characteristics of pluripotent stem cells, comprising: (a) contacting a cell with a MAPK / ERK kinase (MEK) inhibitor such that proliferation of non-pluripotent cells is inhibited and proliferation of pluripotent stem cells is promoted; and (b) screening the contacted cells for characteristics of pluripotent stem cells. A method comprising: 39. prior to step (a), contacting the cells with a library of agents; and After step (b), selecting an agent that induces pluripotent stem cells based on the results of step (b). 40. The method of claim 38, comprising: 40. The method of any one of claims 38 to 39, wherein the cell is a human cell. 41. The method of any one of claims 38 to 39, wherein the cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells. 42. The method of any one of claims 38 to 41, wherein the MEK inhibitor is PD0325901. 43. A mixture of mammalian cells and an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, comprising: The cells, expressing at least one or more of an Oct polypeptide, a Klf polypeptide, a Sox polypeptide, and a Myc polypeptide; and / or contacting at least one or more of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Sox polypeptide, and an exogenous Myc polypeptide; mixture. 44. The cell comprises a first recombinant expression cassette, a second recombinant expression cassette, and a third recombinant expression cassette; the first expression cassette comprises a promoter operably linked to a polynucleotide encoding a Klf polypeptide; the second expression cassette comprises a promoter operably linked to a polynucleotide encoding a Sox polypeptide; and the third expression cassette comprises a promoter operably linked to a polynucleotide encoding a Myc polypeptide; 44. The mixture of claim 43. 45. The mixture of any one of claims 43 to 44, wherein the agent inhibits H3K9 methylation. 46. ​​The method of claim 45, wherein the agent that inhibits H3K9 methylation is BIX01294. 47. The cell comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors; the one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors comprising a first expression cassette, a second expression cassette, and a third expression cassette; 47. The mixture according to any one of claims 43 to 46. 48. The mixture comprises a first, a second, and a third retroviral vector, a lentiviral vector, an adenoviral vector, a non-viral plasmid vector, or an episomal expression vector; the first retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the first expression cassette; the second retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the second expression cassette; the third retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises the third expression cassette; 48. The mixture of claim 47. 49. The mixture of any one of claims 43 to 48, wherein the cells are human cells. 50. The mixture of any one of claims 43 to 48, wherein the cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells. 51. The mixture of any one of claims 43 to 50, wherein the cells comprise progenitor cells. 52. The mixture of claim 51, wherein the progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. 53. The mixture of any one of claims 43 to 52, wherein the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. 54. A mammalian cell that endogenously expresses at least one protein selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, the cell does not endogenously express at least one protein of the group, the protein that is not endogenously expressed is expressed from an RNA encoded by a heterologous recombinant expression cassette present in the cell; The cell expresses an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide endogenously or heterologously, respectively; and Expression of the protein from the heterologous expression cassette results in reprogramming or dedifferentiation of the cell from a non-pluripotent cell to a pluripotent stem cell. mammalian cells. 55. The cell of claim 54, wherein the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. 56. The cell of any one of claims 54 to 55, which endogenously expresses a Sox polypeptide and a Myc polypeptide, and heterologously expresses an Oct polypeptide and a Klf polypeptide. 57. The cell of claim 56, wherein the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. 58. A method for inducing Oct4 expression in a cell, comprising: contacting the cell with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby inducing Oct4 expression in the cell. A method comprising: 59. The method of claim 58, wherein immediately prior to the contacting step, the cells do not express Oct4. 60. The method of any one of claims 58-59, wherein the contacted cell is not a pluripotent cell. 61. The method of any one of claims 58-59, wherein after the contacting step, the cells are induced to become pluripotent. 62. A method for inducing a non-pluripotent cell into a pluripotent cell, comprising: contacting the non-pluripotent cells with one or more agents that induce pluripotency and / or introducing an expression cassette into the cells to express a protein that induces pluripotency, wherein the cells are not cultured on feeder cells and are attached to a solid culture surface. A method comprising: 63. The method of claim 62, wherein the cells are attached to the solid culture surface by a molecular tether selected from the group consisting of matrigel, extracellular matrix (ECM) or ECM analog, laminin, fibronectin, and collagen. 64. The method of claim 62, wherein the contacting step comprises a step as described in claim 1 or any claim dependent thereon. 65. A method for producing induced pluripotent stem cells from mammalian non-pluripotent cells, comprising: (a) the cells; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least two of thereby producing induced pluripotent stem cells. A method comprising: 66. The contacting step comprises: (a) the cells; i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; and ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor One agent selected from the group consisting of At least two of 66. The method of claim 65, comprising contacting 67. (b) introducing into said non-pluripotent cells one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and / or a Sox polypeptide. 67. The method of claim 65 or 66, further comprising: 68. The method of any one of claims 65 to 67, wherein Klf4 and Oct4 are introduced into the cell. 69. A mammalian cell; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least two of A mixture of. 70. The mixture is i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of 70. The mixture of claim 69, comprising: 71. The mixture of any one of claims 69 to 70, wherein the cells comprise a heterologous expression cassette for expressing an Oct polypeptide and a heterologous expression cassette for expressing a Sox polypeptide. 72. The mixture of any one of claims 69 to 71, wherein the cells are non-pluripotent cells. 73. The mixture of any one of claims 69 to 72, wherein the cells are fibroblasts. 74. The mixture of any one of claims 69 to 73, wherein neither an expression cassette for expression of a Myc polypeptide nor an expression cassette for expression of a Klf polypeptide is introduced into the non-pluripotent cell. 75. One agent that inhibits H3K9 methylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least two of A composition comprising: 76. i. an agent that inhibits H3K9 methylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of 76. The composition of claim 75, comprising: 77. One agent that inhibits H3K9 methylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor A kit comprising at least two of the following: 78. i. One agent that inhibits H3K9 methylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of 78. The kit of claim 77, comprising: 79. The kit of claim 77 or 78, further comprising mammalian cells.

[0066] Other aspects of the invention will be apparent from reading the entire application. [The present invention 1001] 1. A method for producing induced pluripotent stem cells from mammalian non-pluripotent cells, comprising: (a) the cells; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least one of thereby producing induced pluripotent stem cells. A method comprising: [The present invention 1002] The contacting step comprises: (a) the cells; i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; and ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor One agent selected from the group consisting of At least two of The method of claim 1001, comprising contacting [The present invention 1003] (b) introducing into said non-pluripotent cells one or more expression cassettes for the expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and / or a Sox polypeptide. The method of the present invention 1001 or 1002 further comprising: [The present invention 1004] The method of claim 1003, wherein Klf4 and Oct4 are introduced into said cell. [The present invention 1005] 1. A method for producing induced pluripotent stem cells from mammalian non-pluripotent cells, comprising: (a) introducing one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into the non-pluripotent cell or modifying the expression of one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide in the non-pluripotent cell; (b) contacting the cells with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby generating induced pluripotent stem cells. A method comprising: [The present invention 1006] The method of claim 1005, wherein step (a) comprises contacting the non-pluripotent cells with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide. [The present invention 1007] Step (a) i. contacting the non-pluripotent cell with one or more exogenous polypeptides selected from a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide; ii. subsequently culturing the cells in the absence of the exogenous polypeptide. The method of the present invention 1006, comprising at least two cycles of [The present invention 1008] The method of claim 1005, wherein step (a) comprises introducing into said non-pluripotent cell one or more expression cassettes for expression of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide. [The present invention 1009] The method according to claim 1005, wherein an expression cassette for expression of an Oct polypeptide and an expression cassette for expression of a Sox polypeptide are introduced into said non-pluripotent cell. [The present invention 1010] The method of claim 1005, wherein the introducing step comprises introducing one or more expression cassettes for expression of a KLF polypeptide and an Oct polypeptide into the non-pluripotent cell, and no expression cassettes for a Myc polypeptide and / or a Sox polypeptide are introduced into the cell. [The present invention 1011] The method of any one of claims 10 to 15, wherein the Klf polypeptide is Klf4 and the Oct polypeptide is Oct4. [The present invention 1012] The method of claim 1005, wherein said non-pluripotent cells are somatic cells. [The present invention 1013] The method of claim 1005, wherein said non-pluripotent cells are fibroblasts. [The present invention 1014] The method according to claim 1005, wherein neither an expression cassette for expression of a Myc polypeptide nor an expression cassette for expression of a Klf polypeptide is introduced into said non-pluripotent cell. [The present invention 1015] The method of claim 1005, wherein the introducing step is performed in vivo. [The present invention 1016] The method of any one of claims 10 to 5, wherein the introducing step is performed in vitro. [The present invention 1017] (c) selecting cells that exhibit characteristics of pluripotent stem cells The method of the present invention 1005 further comprises: [The present invention 1018] The method of claim 1005, further comprising obtaining said non-pluripotent cells from an animal and differentiating said induced pluripotent stem cells into a desired cell type. [The present invention 1019] The method of claim 1018, wherein said desired cell type is introduced into an animal. [The present invention 1020] The method of claim 1019, wherein the animal is a human. [The present invention 1021] The method of claim 1019, wherein the animal is a non-human animal. [The present invention 1022] The method of claim 1017, wherein said selected cells do not contain an exogenous expression cassette for expression of Oct4. [The present invention 1023] The method of claim 1005, wherein said agent inhibits H3K9 methylation. [The present invention 1024] The method of claim 1023, wherein said agent that inhibits H3K9 methylation is BIX01294. [The present invention 1025] The method of claim 10, wherein the cell is a human cell. [The present invention 1026] The method of claim 1005, wherein said cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells. [The present invention 1027] The method of claim 1005, wherein said non-pluripotent cells are progenitor cells. [The present invention 1028] The method of claim 1027, wherein said progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. [The present invention 1029] The process of introducing a first vector comprising a promoter operably linked to a first expression cassette, the first expression cassette comprising a polynucleotide encoding Klf4; a second vector comprising a promoter operably linked to a second expression cassette, the second expression cassette comprising a polynucleotide encoding Sox2; and A third vector comprising a promoter operably linked to a third expression cassette, the third expression cassette comprising a polynucleotide encoding c-Myc. The method of the present invention 1005, comprising introducing [The present invention 1030] The method of claim 1005, wherein the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, a non-viral plasmid vector, or an episomal expression vector. [The present invention 1031] 1. A method for screening an agent that induces reprogramming or dedifferentiation of a mammalian cell into a pluripotent stem cell, comprising: (a) introducing at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into a non-pluripotent cell to generate a transfected cell; (b) contacting the transfected cells with a library of different agents; (c) screening the contacted cells for characteristics of pluripotent stem cells; and (d) correlating the occurrence of stem cell characteristics with specific agents from the library, thereby identifying agents that stimulate dedifferentiation of cells into pluripotent stem cells. A method comprising: [The present invention 1032] The method of claim 1031, wherein step (a) comprises introducing into the non-pluripotent cell one or more expression cassettes for expression of at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide. [The present invention 1033] The method of claim 1031, wherein step (a) comprises introducing at least one, but not all, of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Myc polypeptide, and an exogenous Sox polypeptide into the non-pluripotent cell. [The present invention 1034] The method of claim 1031, wherein the specific agent is 50 to 1500 daltons. [The present invention 1035] The method of claim 1031, wherein step (a) comprises introducing two expression cassettes into the cell, each expression cassette comprising a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and the remaining members of the group are not introduced into the cell. [The present invention 1036] The method of claim 1031, wherein step (a) comprises introducing three expression cassettes into the cell, each expression cassette comprising a polynucleotide encoding a different protein, the proteins being selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, and the remaining members of the group are not introduced into the cell. [The present invention 1037] The method of claim 1031, wherein the cell is a human cell. [The present invention 1038] The method of claim 1031, wherein said cell is a non-human mammalian cell. [The present invention 1039] The method of claim 1031, wherein said non-pluripotent cells are progenitor cells. [The present invention 1040] The method of claim 1040, wherein the progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. [The present invention 1041] The method of claim 1031, wherein the Oct polypeptide is Oct4, the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. [The present invention 1042] 1. A method for screening for mammalian cells having characteristics of pluripotent stem cells, comprising: (a) contacting a cell with a MAPK / ERK kinase (MEK) inhibitor such that proliferation of non-pluripotent cells is inhibited and proliferation of pluripotent stem cells is promoted; and (b) screening the contacted cells for characteristics of pluripotent stem cells. A method comprising: [The present invention 1043] Prior to step (a), contacting the cells with a library of agents; and After step (b), selecting an agent that induces pluripotent stem cells based on the results of step (b). The method of the present invention 1042, comprising: [The present invention 1044] The method of claim 1042, wherein the cell is a human cell. [The present invention 1045] The method of claim 1042, wherein the cells are mouse, dog, bovine, pig, rat, and non-human primate cells. [The present invention 1046] The method of claim 1042, wherein the MEK inhibitor is PD0325901. [The present invention 1047] A mixture of mammalian cells and an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, The cells, expressing at least one or more of an Oct polypeptide, a Klf polypeptide, a Sox polypeptide, and a Myc polypeptide; and / or contacted with at least one or more of an exogenous Oct polypeptide, an exogenous Klf polypeptide, an exogenous Sox polypeptide, and an exogenous Myc polypeptide; mixture. [The present invention 1048] the cell comprises a first recombinant expression cassette, a second recombinant expression cassette, and a third recombinant expression cassette; the first expression cassette comprises a promoter operably linked to a polynucleotide encoding a Klf polypeptide; the second expression cassette comprises a promoter operably linked to a polynucleotide encoding a Sox polypeptide; the third expression cassette comprises a promoter operably linked to a polynucleotide encoding a Myc polypeptide; A mixture of 1047 of the present invention. [The present invention 1049] The mixture of invention 1047, wherein said agent inhibits H3K9 methylation. [The present invention 1050] The method of claim 1049, wherein said agent that inhibits H3K9 methylation is BIX01294. [The present invention 1051] the cell comprises one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors; the one or more retroviral vectors, lentiviral vectors, adenoviral vectors, non-viral plasmid vectors, or episomal expression vectors comprising a first expression cassette, a second expression cassette, and a third expression cassette; A mixture of 1047 of the present invention. [The present invention 1052] the mixture comprises a first, a second, and a third retroviral vector, a lentiviral vector, an adenoviral vector, a non-viral plasmid vector, or an episomal expression vector; the first retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises a first expression cassette; the second retroviral, lentiviral, adenoviral, non-viral plasmid, or episomal expression vector comprises a second expression cassette; and the third retroviral vector, lentiviral vector, adenoviral vector, non-viral plasmid vector, or episomal expression vector comprises a third expression cassette; A mixture of the present invention 1051. [The present invention 1053] The mixture of claim 1047, wherein the cells are human cells. [The present invention 1054] The mixture of invention 1047, wherein said cells are mouse cells, dog cells, bovine cells, pig cells, rat cells, and non-human primate cells. [The present invention 1055] The mixture of claim 1047, wherein said cells comprise progenitor cells. [The present invention 1056] The mixture of claim 1055, wherein said progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells. [The present invention 1057] A mixture of invention 1047, wherein the Klf polypeptide is Klf4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. [The present invention 1058] A mammalian cell that endogenously expresses at least one protein selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide, the cell does not endogenously express at least one protein of the group, the protein that is not endogenously expressed is expressed from an RNA encoded by a heterologous recombinant expression cassette present in the cell; The cell expresses an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide endogenously or heterologously, respectively; and Expression of the protein from the heterologous expression cassette results in reprogramming or dedifferentiation of the cell from a non-pluripotent cell to a pluripotent stem cell. mammalian cells. [The present invention 1059] The cell according to claim 1058, wherein the Oct polypeptide is Oct 4, the Klf polypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. [The present invention 1060] The cell according to claim 1058, which endogenously expresses a Sox polypeptide and a Myc polypeptide, and heterologously expresses an Oct polypeptide and a Klf polypeptide. [The present invention 1061] The cell according to claim 1060, wherein the Oct polypeptide is Oct 4, the Klf polypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2. [The present invention 1062] 1. A method of inducing Oct4 expression in a cell, comprising: contacting the cell with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby inducing Oct4 expression in the cell. A method comprising: [The present invention 1063] The method according to claim 1062, wherein immediately prior to the contacting step, the cells do not express Oct4. [The present invention 1064] The method of claim 1062, wherein the contacted cells are not pluripotent cells. [The present invention 1065] The method according to claim 1064, wherein after the contacting step, the cells are induced to become pluripotent. [The present invention 1066] 1. A method for inducing a non-pluripotent cell into a pluripotent cell, comprising: contacting the non-pluripotent cells with one or more agents that induce pluripotency and / or introducing an expression cassette into the cells to express a protein that induces pluripotency, wherein the cells are not cultured on feeder cells and are attached to a solid culture surface. A method comprising: [The present invention 1067] The method of claim 1066, wherein said cells are attached to the solid culture surface by a molecular tether selected from the group consisting of matrigel, extracellular matrix (ECM) or ECM analog, laminin, fibronectin, and collagen. [The present invention 1068] The method of claim 1066, wherein the contacting step comprises any of the steps of claim 1005 or any of the dependent claims of claim 1005. [The present invention 1069] A mammalian cell; one agent that inhibits H3K9 methylation or promotes H3K9 demethylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least two of A mixture of. [The present invention 1070] i. an agent that inhibits H3K9 methylation or promotes H3K9 demethylation; ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of The mixture of 1069 of the present invention comprising: [The present invention 1071] The mixture of claim 1069, wherein said cells comprise a heterologous expression cassette for expressing an Oct polypeptide and a heterologous expression cassette for expressing a Sox polypeptide. [The present invention 1072] The mixture of claim 1069, wherein the cells are non-pluripotent cells. [The present invention 1073] The mixture of claim 1069, wherein the cells are fibroblasts. [The present invention 1074] The mixture of 1071 of the present invention, wherein neither an expression cassette for the expression of a Myc polypeptide nor an expression cassette for the expression of a Klf polypeptide is introduced into the non-pluripotent cells. [The present invention 1075] one agent that inhibits H3K9 methylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor At least two of A composition comprising: [The present invention 1076] i. an agent that inhibits H3K9 methylation; and ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of The composition of the present invention 1075 comprising: [The present invention 1077] one agent that inhibits H3K9 methylation; L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor A kit comprising at least two of the following: [The present invention 1078] i. an agent that inhibits H3K9 methylation; and ii. L-type Ca channel agonists; Activator of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; Nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and erk inhibitor and one agent selected from the group consisting of The kit of the present invention 1077, comprising: [The present invention 1079] The kit of claim 1077 further comprising a mammalian cell. [Brief description of the drawings]

[0067] [Figure 1] Generation of iPS cells from defined primary neural progenitor cells by Oct4 / Klf4 viral transduction and BIX01294 treatment. Comparison of the number of GFP+ iPS cell colonies generated from 3.5x104 primary OG2 neural progenitor cells by retroviral transduction of Oct4 / Klf4 / Sox2 / c-Myc, Oct4 / Klf4 / Sox2, or Oct4 / Klf4 with BIX01294 treatment, or by retroviral transduction of Oct4 / Klf4 / Sox2 / c-Myc, Oct4 / Klf4 / Sox2, or Oct4 / Klf4 without BIX01294 treatment. [Diagram 2] Generation of iPS cells from primary neural progenitor cells by Klf4 / Sox2 / c-Myc viral transduction and BIX01294 treatment. Number of GFP+ iPS cell colonies generated from 3.5x104 primary OG2 neural progenitor cells by retroviral transduction of Klf4 / Sox2 / c-Myc with or without BIX01294 treatment. [Diagram 3] Generation of OK2B iPSCs from primary OG2 MEFs. Bar graph shows the average number of GFP+ colonies derived from OG2-MEFs in three independent experiments. The graph shows data for OG2 MEF cells transduced with four factors (Oct4, Klf4, Sox2, and cMyc; 4F); OG2 MEF cells transduced with OK (OK); OG2 MEF cells transduced with OK and treated with 1 μM BIX (OK+BIX); OG2 MEF cells transduced with OK and treated with 1 μM BIX + 2 μM BayK (OK+BIX+BayK); OG2 MEF cells transduced with OK and treated with 1 μM BIX + 0.04 μM RG108 (OK+BIX+RG108). n=3. Error bars show standard deviation calculated in Excel. [Figure 4]OK2B iPSCs have a transcriptional profile similar to the one of mESCs. (A) RT-PCR analysis of OK2B iPSCs revealed that they express genes specific to pluripotent mESCs. R1 mESCs were used as a positive control, whereas OG2 MEFs were used as a negative control. GAPDH was used as a loading control. (B) Bisulfite sequencing revealed that the nanog promoter in OK2B iPSCs was demethylated, further supporting the reactivation of endogenous genes specific to mESCs. Schematic representation of cytosines present in the region of the Nanog promoter amplified for this analysis. Open circles indicate demethylated cytosines, whereas filled circles indicate methylated cytosines. [Diagram 5] Treatment with BIX, BayK, or a combination of both does not increase mES cell proliferation. Scatter graphs show the number of R1 mES cells after treatment with DMSO (control), 2 μM BayK, 1 μM BIX, and a combination of both (BayK+BIX). n=3. Error bars show standard deviation calculated in Excel. No significant differences were obtained for each treatment compared to DMSO as calculated using a t-test in Excel. [Figure 6] RT-PCR analysis of Sox2 expression after compound treatment. OG2+ / -ROSA26+ / - (OG2) MEFs were treated with DMSO (control), 1 μM BIX, 2 μM BayK, and a combination of both for 6 days. RNA was then extracted using Qiagen RNAeasy Mini kit. Sox2 expression was assessed by semi-quantitative PCR. OK2B iPSC p37 and R1 were used as positive controls, and GAPDH was used as a loading control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] definition "Oct polypeptide" refers to either a naturally occurring member of the octamer transcription factor family, or a variant thereof that maintains a similar transcription factor activity (at least within 50%, 80%, or 90% activity) compared to the closest naturally occurring family member, or a polypeptide that contains at least the DNA binding domain of a naturally occurring family member, and optionally contains a transcription activation domain. Exemplary Oct polypeptides include, for example, Oct3 / 4 (herein referred to as "Oct4"), which contains a POU domain. See Ryan, AK and Rosenfeld, MG Genes Dev. 11, 1207-1225 (1997). In some embodiments, a variant has at least 90% amino acid sequence identity over the entire sequence compared to a naturally occurring Oct polypeptide family member, such as those listed above.

[0069] "Klf polypeptide" refers to either a naturally occurring member of the Kruppel-like factor (Klfs) zinc finger protein family that contains an amino acid sequence similar to that of the Drosophila embryonic pattern regulator Kruppel, or a naturally occurring member variant that maintains a similar transcription factor activity (within at least 50%, 80%, or 90% activity) compared to the closest naturally occurring family member, or a polypeptide that contains at least the DNA binding domain of a naturally occurring family member and optionally contains a transcription activation domain. See Dang, DT, Pevsner, J. and Yang, VW. Cell Biol. 32, 1103-1121 (2000). Exemplary Klf family members include, for example, Klf1, Klf4, and Klf5, each of which has been shown to be capable of substituting for each other to obtain iPS cells. See Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In some embodiments, the variant has at least 90% amino acid sequence identity over the entire sequence compared to a natural Klf polypeptide family member, such as those listed above. To the extent that a KLF polypeptide is described herein, it can be substituted with Essrb. Thus, each Klf polypeptide embodiment described herein is intended to be equally described with respect to the use of Essrb in place of a Klf4 polypeptide.

[0070] "Myc polypeptide" refers to either a naturally occurring member of the Myc family (see, e.g., Adhikary, S. and Eilers, M. Nat. Rev. Mol Cell Biol. 6:635-645(2005)), or a variant thereof that maintains a similar (within at least 50%, 80%, or 90% activity) transcription factor activity compared to the closest naturally occurring family member, or a polypeptide that contains at least the DNA binding domain of a naturally occurring family member and, optionally, a transcription activation domain. Exemplary Myc polypeptides include, e.g., c-Myc, N-Myc, and L-Myc. In some embodiments, a variant has at least 90% amino acid sequence identity over the entire sequence compared to a naturally occurring Myc polypeptide family member, e.g., those listed above.

[0071] "Sox polypeptide" refers to either a naturally occurring member of the SRY-related HMG-box (Sox) transcription factor characterized by the presence of a high mobility group (HMG) domain, or a variant thereof that maintains a similar transcription factor activity (within at least 50%, 80%, or 90% of the activity) compared to the closest naturally occurring family member, or a polypeptide that contains at least the DNA binding domain of the naturally occurring family member and, optionally, a transcription activation domain. See, e.g., Dang, DT, et al., Int. J. Biochem. Cell Biol. 32:1103-1121(2000). Exemplary Sox polypeptides include, e.g., Sox1, Sox2, Sox3, Sox15, or Sox18, each of which has been shown to be capable of substituting for each other to obtain iPS cells. See, Nakagawa, et al., Nature Biotechnology 26:101-106(2007). In some embodiments, variants have at least 90% amino acid sequence identity over the entire sequence compared to a naturally occurring Sox polypeptide family member, such as those listed above.

[0072] "H3K9" refers to histone H3 lysine 9. H3K9 can be dimethylated at K9. See, e.g., Kubicek, et al., Mol. Cell 473-481 (2007).

[0073] The term "pluripotent" or "pluripotency" refers to cells that, under appropriate conditions, have the ability to give rise to progeny capable of differentiating into cell types that collectively exhibit characteristics associated with cell lineages derived from all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells may contribute to many or all tissues of prenatal, postnatal, or adult animals. Standard art-recognized tests, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to demonstrate the pluripotency of a cell population. However, identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells.

[0074] "Pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. A characteristic of pluripotent stem cells is their ability to produce progeny that can differentiate into cell types that collectively exhibit characteristics associated with cell lineages derived from all three germ layers (endoderm, mesoderm, and ectoderm) under appropriate conditions. Expression or non-expression of certain combinations of molecular markers is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least some, and optionally all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells.

[0075] The term "library" is used in accordance with common usage in the art to mean a collection of molecules, optionally organized and / or classified so that the individual members can be identified. Libraries can include, but are not limited to, combinatorial chemical libraries, natural product libraries, and peptide libraries.

[0076] A "recombinant" polynucleotide is a polynucleotide that is not in a natural state. For example, the polynucleotide contains a nucleotide sequence not found in nature or is in a situation other than that in nature. For example, it is separated from nucleotide sequences that are typically contiguous in nature, or it is adjacent (or contiguous) to nucleotide sequences that are not typically contiguous. For example, the sequence in question may be cloned into a vector or otherwise recombined with one or more additional nucleic acids.

[0077] "Expression cassette" refers to a polynucleotide comprising a promoter or other regulatory sequence operably linked to a protein-encoding sequence.

[0078] The terms "promoter" and "expression control sequence" are used herein to refer to a number of nucleic acid control sequences that induce nucleic acid transcription. As used herein, a promoter includes necessary nucleic acid sequences near the transcription start site, e.g., in the case of a polymerase II type promoter, a TATA element. A promoter also includes, optionally, distal enhancers or repressors, which may be located several thousand base pairs away from the transcription start site. Promoters include constitutive and inducible promoters. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to the functional linkage of a nucleic acid expression control sequence (e.g., a promoter, or a number of transcription factor binding sites) with a second nucleic acid sequence, such that the expression control sequence induces transcription of the nucleic acid corresponding to the second sequence.

[0079] As used herein, a "heterologous sequence" or "heterologous nucleic acid" is derived from a source foreign to a particular host cell, or, if derived from the same source, is modified from its original form. Thus, a heterologous expression cassette in a cell is an expression cassette that is not endogenous to a particular host cell, for example, by being linked to a nucleotide sequence derived from an expression vector rather than chromosomal DNA, by being linked to a heterologous promoter, by being linked to a reporter gene, etc.

[0080] The term "agent" or "test compound" refers to any compound useful in the screening assay methods described herein. The agent may be, for example, an organic compound (e.g., a small molecule such as a drug), a polypeptide (e.g., a peptide or an antibody), a nucleic acid (e.g., DNA, RNA, double-stranded, single-stranded, oligonucleotide, antisense RNA, small inhibitory RNA, microRNA, ribozyme, etc.), an oligosaccharide, or a lipid. Typically, the agent used in the present screening method has a molecular weight of less than 10,000 daltons, e.g., less than 8000 daltons, less than 6000 daltons, less than 4000 daltons, less than 2000 daltons, e.g., 50-1500, 500-1500, 200-2000, 500-5000 daltons. The test compound may be in the form of a test compound library, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to fusion partners, such as targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, a test compound (called a "lead compound") with a desired property or activity, such as the ability to induce pluripotency under a certain condition as described herein, is identified, and new chemical entities with useful properties are created by creating variants of the lead compound and evaluating the properties and activities of the variant compounds. Often, high-throughput screening (HTS) methods are used for such analysis.

[0081] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. This term includes synthetic, natural, and non-natural nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages, have similar binding properties as the reference nucleic acid, and are metabolized in the same manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).

[0082] Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be made by creating sequences in which the third position of one or more (or all) selected codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98(1994)).

[0083] The terms "inhibitor", "activator" and "modulator" of expression or activity are used to refer to inhibitory, activating or modulating molecules, such as ligands, agonists, antagonists, and their homologs and mimetics, identified using in vitro and in vivo assays of the expression or activity of the described target protein (or encoding polynucleotide). The term "modulator" includes inhibitors and activators. An inhibitor is, for example, an agent that inhibits the expression of the described target protein or binds to the described target protein, partially or completely blocks the stimulatory or protease inhibitor activity of the described target protein, reduces the described target protein, inhibits the described target protein, delays the activation of the described target protein, inactivates the described target protein, desensitizes the described target protein, or downregulates the activity of the described target protein, such as an antagonist. An activator is, for example, an agent, such as an agonist, that induces or activates the expression of the described target protein (or encoding polynucleotide), or binds to the described target protein, stimulates the described target protein, increases the described target protein, opens the described target protein, activates the described target protein, promotes the described target protein, enhances the activation or protease inhibitor activity of the described target protein, sensitizes the described target protein, or upregulates the activity of the described target protein. Modulators include natural and synthetic ligands, antagonists, and agonists (e.g., small chemical molecules, antibodies, etc. that function as agonists or antagonists). Such assay methods for inhibitors and activators include, for example, applying a putative modulator compound to cells expressing the described target protein, and then ascertaining the functional effect on the described target protein activity as described above.A sample or assay containing the described target protein treated with a potential activator, inhibitor, or modulator is compared to a control sample without the inhibitor, activator, or modulator to determine the extent of the effect. The control sample (not treated with a modulator) is assigned a relative activity value of 100%. The described target protein is inhibited when the activity value relative to the control is about 80%, optionally 50% or 25%, 10%, 5%, or 1%. The described target protein is activated when the activity value relative to the control is 110%, optionally 150%, optionally 200%, 300%, 400%, 500%, or 1000-3000% or more.

[0084] Detailed Description of the Invention I. Introduction The present invention is based in part on the surprising discovery that small molecules can be used to mimic the effects of transcription factors involved in the induction of induced pluripotent stem cells (iPS).For example, as detailed herein, Oct4 can be "replaced" with small molecules that reduce the methylation of histone 3 lysine 9 (H3K9).Thus, for example, contacting BIX01294, a small molecule that specifically inhibits G9a (the histone methyltransferase of H3K9), with mammalian cells expressing Klf4, c-Myc, and Sox2 induces pluripotent stem cells.

[0085] These results are not only interesting in terms of the role of H3K9 methylation and its involvement in cellular programming, but also show that it is possible to identify small molecules that replace transcription factors previously shown to be essential for the induction of pluripotent stem cells. This would be particularly interesting if the goal is to introduce (or reintroduce) induced pluripotent stem cells, or subsequently differentiated cells, such as progenitor cells derived from iPS cells, into patients. As some of the four iPS transcription factors (e.g. Oct4, Myc) have known oncogenic activity, it may be beneficial to replace these factors with other less or no oncogenic molecules. Furthermore, replacing some or each of the four factors with small molecules that do not require transformation (and therefore the potential oncogenic effects of DNA insertion into chromosomes) would further help reduce the possible cancer side effects of iPS-related therapies.

[0086] The present invention also provides induced pluripotent cells in which at least some of the iPS transcription factors are expressed at endogenous or even lower levels, but which are nevertheless pluripotent.The present invention is based in part on the discovery that certain cells that endogenously express Sox2 can be induced to become pluripotent by the introduction and heterologous expression of only Oct4 and Klf4.

[0087] Furthermore, as shown herein, non-pluripotent cells that do not endogenously or heterologously express Sox polypeptides (e.g., Sox2) can be induced to become pluripotent using the methods of the present invention.For example, pluripotency can be induced by introducing only Oct4 and Klf4 into non-pluripotent cells (e.g., fibroblasts) and contacting the cells with an agent that inhibits H3K9 methylation, and optionally with at least one of an L-type calcium channel agonist, an activator of the cAMP pathway, a DNA methyltransferase (DNMT) inhibitor, a nuclear receptor agonist, a GSK3 inhibitor, or a MEK inhibitor. The inventors have also discovered that combinations of agents such as a GSK inhibitor and an HDAC inhibitor; or a GSK inhibitor and a cAMP pathway activator; or a GSK inhibitor and an ALK5 inhibitor (with and without a G9a inhibitor) are effective in inducing pluripotency in cells heterologously expressing Oct4 alone, or heterologously expressing Oct4 / Sox2 or Sox2 / Klf4. Thus, the invention provides a mixture of cells and an agent, where the cells are not initially pluripotent cells (e.g., not stem cells) and optionally heterologously or endogenously express Oct4 and / or Klf4 or have otherwise been contacted with Oct4 and / or Klf4, and the agent is one or more of the following agents that inhibit H3K9 methylation: L-type Ca channel agonists; activators of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; and / or Erk inhibitors.

[0088] As further discussed below, the present invention also provides a novel method for screening cells having characteristics of pluripotent stem cells by culturing the cells to be screened with MEK inhibitors, agents that inhibit H3K9 methylation, L-type Ca channel agonists, cAMP pathway activators, DNA methyltransferase (DNMT) inhibitors, nuclear receptor ligands, GSK3 inhibitors, MEK inhibitors, TGFβ receptor / ALK5 inhibitors, HDAC inhibitors, or Erk inhibitors. For example, MEK inhibitors inhibit the proliferation of non-iPS cells while promoting the proliferation and stable reprogramming of iPS cells, thereby enriching cells having characteristics of pluripotent stem cells in a particular cell mixture.

[0089] II. Induction of pluripotent stem cells A. Heterologous / Endogenous Expression In some embodiments of the present invention, the non-pluripotent cell that endogenously expresses at least one (optionally, two or three) protein from the group consisting of Oct polypeptide, Klf polypeptide, Myc polypeptide, and Sox polypeptide is identified.Then, the remaining (non-endogenously expressed) protein from this group can be heterologously expressed in cell, and optionally screened for reprogramming and / or dedifferentiation into pluripotent cell in the presence of one or more of MEK inhibitor, H3K9 methylation inhibitor, L-type Ca channel agonist, cAMP pathway activator, DNA methyltransferase (DNMT) inhibitor, nuclear receptor ligand, GSK3 inhibitor, MEK inhibitor, TGFβ receptor / ALK5 inhibitor, HDAC inhibitor, and / or Erk inhibitor.

[0090] It is believed that any type of mammalian non-pluripotent cell can be screened for protein expression and then converted into a pluripotent cell. In some embodiments, the starting cell is an isolated progenitor cell. Exemplary progenitor cells include, but are not limited to, endodermal progenitor cells, mesodermal progenitor cells (e.g., muscle progenitor cells, bone progenitor cells, blood progenitor cells), and ectodermal progenitor cells (e.g., epidermal tissue progenitor cells and neural progenitor cells). Cells useful for these aspects of the invention can be easily identified by screening cell lines for expression of Oct, Klf, Myc, and Sox polypeptides, or by identifying cells with low promoter methylation (e.g., by DNA bisulfite sequencing), or by identifying cells with altered histone state to confirm the low silencing state of these genes. Transcription factors that are not endogenously expressed can then be heterologously expressed to induce pluripotency without heterologously expressing factors that are already endogenously expressed.

[0091] As shown in the examples, some cells (e.g., neural progenitor cells and fibroblasts (data not shown)) can be induced to become pluripotent by heterologously expressing only Oct4 and Klf4. This demonstrates that it is not necessary to overexpress all of the Sox and Myc proteins, perhaps Oct and Klf proteins, (e.g., using a high-expression viral vector) to become pluripotent. In fact, some of these proteins may be expressed at endogenous levels or even at lower detectable levels and still be suitable for conversion to pluripotent cells by heterologous expression of other members of this group. Thus, in some embodiments of the present invention, cells that endogenously express Sox and / or Myc polypeptides are identified, and Oct and Klf polypeptides are heterologously expressed in the cells, thereby inducing the conversion of the cells into pluripotent cells. In some embodiments, cells that endogenously express Oct and / or Klf and / or Myc polypeptides are identified, and Sox polypeptides are heterologously expressed in the cells, thereby inducing the conversion of the cells into pluripotent cells. Optionally, Sal14 (Zhang et al., Nat Cell Biol. 8(10):1114-23(2006)) can be expressed in place of any or all of Myc, Klf4, and Sox2.

[0092] The efficiency of pluripotency induction described herein can be further improved by including in the non-pluripotent cells, for example, one or more of UTF1, SV40, TERT (by introducing expression cassettes encoding these gene products or by contacting the cells with the proteins themselves), and / or by reducing p53 expression (e.g., by siRNA).See, e.g., Zhao, et al., Cell Stem Cell 3:475-479(2008).

[0093] B. Transcription factor proteins As detailed herein, many aspects of the present invention involve introducing one or more polypeptides into a cell, thereby inducing pluripotency in the cell.As discussed above, introducing a polypeptide into a cell may include introducing a polynucleotide comprising one or more expression cassettes into the cell and inducing expression, thereby introducing the polypeptide into the cell by transcription and translation from the expression cassette.Alternatively, exogenous polypeptides (i.e., proteins that are supplied from outside the cell and / or are not produced by the cell) can be introduced into a cell by many different methods that do not involve the introduction of a polynucleotide that encodes the polypeptide.

[0094] Therefore, for any embodiment of the present invention described herein that refers to the introduction of a polypeptide into a cell or the introduction of an expression cassette encoding a polypeptide into a cell, it should be understood that the present invention also expressly provides the exogenous introduction of the polypeptide as a protein into a cell.Thus, in some embodiments, a mammalian non-pluripotent cell is induced to become pluripotent by (a) exogenously introducing one or more of Klf polypeptide, Oct polypeptide, Myc polypeptide, and / or Sox polypeptide into the non-pluripotent cell, and optionally (b) contacting the cell with one or more of MEK inhibitor, H3K9 methylation inhibitor, L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor, thereby generating induced pluripotent stem cell.

[0095] In some embodiments of the present invention, the non-pluripotent cell that endogenously expresses at least one (optionally, two or three) protein from the group consisting of Oct polypeptide, Klf polypeptide, Myc polypeptide, and Sox polypeptide is identified.Then, the remaining (non-endogenously expressed) protein from this group can be exogenously introduced into cell, and optionally screened for reprogramming and / or dedifferentiation into pluripotent cell in the presence of one or more of MEK inhibitor, H3K9 methylation inhibitor, L-type Ca channel agonist, cAMP pathway activator, DNA methyltransferase (DNMT) inhibitor, nuclear receptor ligand, GSK3 inhibitor, MEK inhibitor, TGFβ receptor / ALK5 inhibitor, HDAC inhibitor, or Erk inhibitor.

[0096] In some embodiments, cells that endogenously express Sox and / or Myc polypeptides are identified, and as a second step, Oct and Klf polypeptides are exogenously introduced into the cells, thereby inducing the conversion of the cells into pluripotent cells. In some embodiments, cells that endogenously express Oct and / or Klf and / or Myc polypeptides are identified, and Sox polypeptides are exogenously introduced into the cells, thereby inducing the conversion of the cells into pluripotent cells.

[0097] Polypeptide can be exogenously introduced into cells in any number of ways.Simply, one or more proteins can be cultured in the presence of target cells under conditions in which the proteins are introduced into cells.In some embodiments, exogenous protein comprises the transcription factor polypeptide of interest linked (e.g., linked as fusion protein, or otherwise covalently or non-covalently linked) to a polypeptide that enhances the ability of transcription factor to enter cells (optionally cell nucleus).

[0098] Examples of polypeptide sequences that enhance transmembrane transport include the Drosophila homeoprotein antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3: 1121-34,1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8,1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90: 9120-4,1993), the herpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88: 223-33,1997); the HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55: 1179-1188,1988; Frankel and Pabo, Cell 55: 1 289-1193, 1988); delivery-enhancing transporters, such as those described in U.S. Pat. No. 6,730,293 (including, but not limited to, peptide sequences containing at least 7-25 consecutive arginines); and the commercially available Penetratin™ 1 peptide and the Diatos Peptide Vector ("DPV") of the Vectocell® platform available from Daitos SA of Paris, France. See also WO / 2005 / 084158 and WO / 2007 / 123667, as well as the additional transporters described therein. Not only are these proteins capable of crossing the plasma membrane, but the attachment of other proteins, such as the transcription factors described herein, is sufficient to stimulate cellular uptake of these complexes.

[0099] In some embodiments, the transcription factor polypeptides described herein are exogenously introduced as part of a lipid cocktail, such as liposomes or commercially available Fugene6 and lipofectamine. In another alternative, the transcription factor proteins may be microinjected or otherwise directly introduced into the target cells.

[0100] As discussed in the examples, the inventors have found that long-term incubation of cells with the transcription factor polypeptide of the present invention is toxic to cells.Therefore, the present invention provides for intermittent incubation of non-pluripotent mammalian cells with one or more of Klf polypeptides, Oct polypeptides, Myc polypeptides, and / or Sox polypeptides, with periods of cell incubation in the absence of one or more polypeptides.In some embodiments, the cycle of incubation in the presence of polypeptides and in the absence of polypeptides may be repeated two, three, four, five, six or more times, and is performed for a period of time (i.e., incubation in the presence of polypeptides and in the absence of polypeptides) sufficient to generate pluripotent cells.In order to improve the efficiency of this method, various agents (e.g., MEK inhibitors and / or GSK inhibitors and / or TGFβ inhibitors) may be included.

[0101] C. Using small molecules to replace iPS transcription factors In some embodiments of the present invention, the cell expresses (endogenously or heterologously) at least one protein selected from an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide (e.g., at least one, two, or three of these), and is contacted with at least one agent sufficient to induce the cell into a pluripotent stem cell in the absence of expression of one or more of the remaining non-expressed proteins, i.e., any of the Oct polypeptides, Klf polypeptides, Myc polypeptides, or Sox polypeptides that are not expressed in the cell.

[0102] As shown in the examples, contacting a cell with a certain agent "supplements" or replaces what is generally understood as the necessary expression of one of these proteins to obtain pluripotent cells.By contacting a cell with an agent that functionally replaces the expression of one of the proteins listed above, it is possible to generate a pluripotent cell that expresses all of the proteins listed above, except for the protein that is replaced or supplemented by the agent.The remaining proteins can be expressed endogenously, heterologously, or a combination of the two (e.g., Sox and Myc polypeptides can be expressed endogenously, Klf polypeptides can be expressed heterologously, and Oct polypeptides can be "substituted" by contacting a cell with a complementary agent, such as an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, instead of being expressed).

[0103] Additionally, small molecules can improve the efficiency of a process for generating pluripotent cells (e.g., iPS cells). For example, improved efficiency may be manifested by speeding up the time to generate such pluripotent cells (e.g., by reducing the development time of pluripotent cells by at least one day compared to a similar or same process without the small molecule). Alternatively, or in combination, small molecules may increase the number of pluripotent cells generated by a particular process (e.g., by increasing the number by at least 10%, 50%, 100%, 200%, 500%, etc. in a particular period of time compared to a similar or same process without the small molecule).

[0104] As described in the Examples, cells that heterologously express Klf4, Sox2, and Myc can be induced to become pluripotent by further contacting the cells with an agent that inhibits H3K9 methylation without heterologously expressing Oct4. In fact, it has been discovered that pluripotency can be induced by contacting non-pluripotent cells with an agent that inhibits H3K9 methylation and introducing Oct4 alone (e.g., without introducing a vector that expresses a Myc polypeptide, a Sox polypeptide, or a KLf polypeptide) or by introducing Oct4 and Klf4. Cells that can be induced to become pluripotent include, but are not limited to, neural progenitor cells and fibroblasts. Agents that inhibit H3K9 methylation include agents that inhibit methylases (also known as methyltransferases) that target H3K9. For example, it is known that G9a histone methyltransferase methylates H3K9, and inhibition of G9a histone methyltransferase reduces methylation of H3K9. See, e.g., Kubicek, et al., Mol. Cell 473-481(2007). One example of a G9a histone methyltransferase useful in the methods of the present invention is BIX01294 (see, e.g., Kubicek, et al., Mol. Cell 473-481(2007)), or its salts, hydrates, isoforms, racemates, solvates, and prodrug forms. Bix01294 is shown below. TIFF2025015628000002.tif48128

[0105] The Bix01294 compounds of the present invention also include salts, hydrates, solvates, and prodrugs. Bix01294 has asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers, and individual isomers are all intended to be within the scope of the present invention. For example, the compounds of the present invention may be R-isomers or S-isomers or mixtures thereof. Furthermore, the compounds of the present invention may be E-isomers or Z-isomers or combinations thereof.

[0106] In some embodiments, the agent that inhibits H3K9 methylation is a substrate analogue of histone methyltransferase.The substrate of many methyltransferases is S-adenosyl-methionine (SAM).Thus, in some embodiments, the agent that inhibits H3K9 methylation is a SAM analogue.Exemplary SAM analogues include, but are not limited to, methylthio-adenosine (MTA), sinefungin, and S-adenosyl-homocysteine ​​(SAH).In other embodiments, the agent that inhibits H3K9 methylation does not compete with SAM in histone methyltransferase.

[0107] The resulting pluripotent cells (derived from heterologous expression and / or small molecule "surrogates") can develop into many or all of the three major tissue types: endoderm (e.g., the lining of the internal digestive tract), mesoderm (e.g., muscle, bone, blood), and ectoderm (e.g., epidermal tissue and nervous system), but may optionally exhibit limitations in their developmental capacity (e.g., may not form placental tissue or other cell types of defined lineage). The cells may be human or non-human (e.g., primate, rat, mouse, rabbit, cow, dog, cat, pig, etc.).

[0108] In other embodiments, BIX01294 or other agents that inhibit H3K9 methylation or promote H3K9 demethylation can be used to induce pluripotency in previously non-pluripotent cells. In some embodiments, agents that inhibit H3K9 methylation are used to induce Oct4 expression in cells, or at least to induce changes in Oct4 promoter DNA methylation and / or histone methylation, to induce cells to become pluripotent. Thus, in some embodiments, to induce cells to become pluripotent, initially non-pluripotent cells are contacted with agents that inhibit H3K9 methylation. Indeed, without intending to limit the scope of the present invention to a particular mode of action, the inventors believe that all methods of inducing cells to become pluripotent are improved by contacting non-pluripotent cells with agents that inhibit H3K9 methylation or promote H3K9 demethylation. For example, in the method comprising contacting non-pluripotent cells with an agent that inhibits H3K9 methylation, the agent that inhibits H3K9 methylation can be contacted with non-pluripotent cells to induce pluripotency.The method optionally also comprises contacting cells with one or more of the following: L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor, each compound being included in an amount sufficient to improve induction efficiency.In some embodiments, as described in this paragraph, Oct4 alone, or Oct4 / Klf4, or Sox2 / Klf4 are further heterologously expressed in non-pluripotent cells, and pluripotency is induced after contacting with agent as described herein.

[0109] The inventors have also found that a combination of a GSK inhibitor and an HDAC inhibitor, or a GSK inhibitor and a cAMP pathway activator, or a GSK inhibitor and an ALK5 inhibitor, can induce pluripotency in mouse or human fibroblasts or keratinocytes expressing either Oct4 alone, Oct4 / Klf4, or Sox2 / Klf4 (data not shown).In other embodiments, non-pluripotent cells are induced to become pluripotent in a method comprising contacting the non-pluripotent cells with a GSK3 inhibitor. Optionally, the method also comprises contacting the cell with one or more of the following: L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor, each compound is included in an amount sufficient to improve induction efficiency.In other embodiments, the method comprises contacting the non-pluripotent cell with TGFβ receptor / ALK5 inhibitor, and the non-pluripotent cell is induced to become pluripotent. The method optionally also includes contacting the cell with one or more of the following: L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; HDAC inhibitor; or Erk inhibitor, each compound is included in an amount sufficient to improve induction efficiency.In another embodiment, the method includes contacting the non-pluripotent cell with HDAC inhibitor, and induces the non-pluripotent cell to become pluripotent.The method optionally also includes contacting the cell with one or more of the following: L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor, TGFβ receptor / ALK5 inhibitor, or Erk inhibitor, each compound is included in an amount sufficient to improve induction efficiency.In another embodiment, a non-pluripotent cell is induced to become pluripotent in a method comprising contacting the non-pluripotent cell with a MEK inhibitor.The method optionally also comprises contacting the cell with one or more of the following: an L-type Ca channel agonist; an activator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a TGFβ receptor / ALK5 inhibitor; an HDAC inhibitor; or an Erk inhibitor, each of which is included in an amount sufficient to improve induction efficiency. In other embodiments, the method comprises contacting non-pluripotent cells with two, three, four, five, six, seven, eight, nine, or each of the following: MEK inhibitor, L-type Ca channel agonist; H3K9 methylation inhibitor, cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor, and each compound is included in an amount sufficient to improve induction efficiency.In some embodiments, as described in this paragraph, Oct4 alone, or Oct4 / Klf4, or Sox2 / Klf4 are further heterologously expressed in non-pluripotent cells, and pluripotency is induced after contacting with the agent as described herein.

[0110] Exemplary L-type calcium channel agonists include, but are not limited to, BayK8644 (see, e.g., Schramm, et al., Nature 303:535-537 (1983)), dehydrodidemnin B (see, e.g., U.S. Patent No. 6,030,943), FPL64176 (FPL) (see, e.g., Liwang, et al., Neuropharmacology 45:281-292(2003)), S(+)-PN 202-791 (see, e.g., Kennedy, et al., Neuroscience 49:937-44(1992)), and CGP48506 (see, e.g., Chahine, et al., Canadian Journal of Physiology and Pharmacology 81:135-141(2003)).

[0111] Exemplary cAMP pathway activators include, but are not limited to, forskolin (see, e.g., Liang, et al., Endocrinology 146: 4437-4444(2005)), FSH (Liang, supra), milrinone (Liang, supra), cilostamide (Liang, supra), rolipram (Liang, supra), dbcAMP (Liang, supra), and 8-Br-cAMP (Liang, supra).

[0112] Exemplary DNA methyltransferase (DNMT) inhibitors may include antibodies that bind to dominant-negative variants of DNMTs, as well as siRNAs and antisense nucleic acids that suppress the expression of DNMTs.DNMT inhibitors include RG108 (e.g., available from Sigma-Aldrich), 5-aza-C (5-azacytidine or azacytidine) (see, e.g., Schermelleh, et al., Nature Methods 2:751-6(2005)), 5-aza-2'-deoxycytidine (5-aza-CdR) (see, e.g., Zhu, Clinical Medicinal Chemistry 3(3):187-199(2003)), decitabine (see, e.g., Gore, Nature Clinical Practice Oncology 2:S30-S35(2005)), doxorubicin (see, e.g., Levenson, Molecular Pharmacology 2:S30-S35(2005)), and 5-aza-C (5-azacytidine or azacytidine) (see, e.g., Schermelleh, et al., Nature Methods 2:751-6(2005)). 71:635-637(2007)), EGCG ((-)-epigallocatechin-3-gallate) (see, e.g., Fang, et al., Cancer Research 63:7563-7570(2003)), RG108 (see, e.g., Carninci, et al., WO2008 / 126932, incorporated herein by reference), and Zebularine (Carninci, supra).

[0113] Exemplary nuclear receptor ligands, i.e., agonists, antagonists, activators, and / or repressors of nuclear receptors, can regulate local gene expression or transcription at the delivery site. Nuclear receptor agonists (and nuclear receptor antagonists) can be used. In some embodiments, nuclear receptors are transcriptional coregulators. Activation or inhibition of certain nuclear receptors regulates the epigenetic state of the specific locus to which the nuclear receptor is bound. The inventors have discovered that dexamethasone (e.g., 1 μM, a glucocorticoid receptor agonist), ciglitazone and Fmoc-Leu (both used at 5 μM) (PPAR agonists), and bexarotene (e.g., (3 μM) (RXR antagonist) can enhance cell reprogramming. Representative nuclear receptor ligands include estradiol (e.g., 17-β estradiol), all-trans retinoic acid, 13-cis retinoic acid, dexamethasone, clobetasol, androgens, thyroxine, vitamin D3 glitazones, troglitazone, pioglitazone, rosiglitazone, prostaglandins, and fibrates (e.g., bezafibrate, ciprofibrate, gemfibrozil, fenofibrate, , and clofibrate). In addition, when endogenous ligands (e.g., hormones estradiol and testosterone) bind to cognate nuclear receptors, the activity of endogenous ligands usually upregulates gene expression. This upregulation or stimulation of gene expression by ligands can also be called agonist response. The agonist action of endogenous hormones can also be mimicked by certain synthetic ligands, such as the glucocorticoid receptor anti-inflammatory drug dexamethasone. Agonist ligands function by inducing receptor conformations that favor coactivator binding (see, for example, WO08011093A, which is incorporated herein by reference).

[0114] GSK3 inhibitors may include antibodies that bind to dominant negative variants of GSK3, as well as siRNA and antisense nucleic acids that target GSK3. Specific examples of GSK3 inhibitors include Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418 (see, e.g., Gould, et al., The International Journal of Neuropsychopharmacology 7:387-390(2004)), CT99021 (see, e.g., Wagman, Current Pharmaceutical Design 10:1105-1137(2004)), CT20026 (see, e.g., Wagman, supra), SB216763 (see, e.g., Martin, et al., Nature Immunology 6:777-784(2005)), AR-A014418 (see, e.g., Noble, et al., PNAS 102:6990-6995(2005)), lithium (see, e.g., Gould, et al., Pharmacological Research 48:49-53(2003)), SB 415286 (see, e.g., Frame, et al., Biochemical Journal 359:1-16(2001)), and TDZD-8 (see, e.g., Chin, et al., Molecular Brain Research, 137(1-2):193-201(2005)).Further exemplary GSK3 inhibitors available from Calbiochem (see, e.g., Dalton, et al., WO2008 / 094597, incorporated herein by reference) include BIO(2'Z,3'£)-6-bromoindirubin-3'-oxime (GSK3 inhibitor IX); BIO-acetoxime(2'Z,3'E)-6-bromoindirubin-3'-acetoxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopenadienyl ruthenium complex (GSK3 inhibitor XV); TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β inhibitor I);2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β inhibitor II);OTDZT 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β inhibitor III);α-4-dibromoacetophenone (GSK3β inhibitor VII);AR-AO14418 N-(4-Methoxybenzoyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β inhibitor VIII); 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β inhibitor XI); TWS1 19 pyrrolopyrimidine compound (GSK3β inhibitor XII); L803H-KEAPPAPPQSpP-NH2 or its myristoylated form (GSK3β inhibitor XIII); 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β inhibitor VI); GSK3b inhibitors include, but are not limited to, AR-AO144-18;SB216763;and SB415286.The GSK3b residues that interact with inhibitors have been identified.See, for example, Bertrand et al., J. Mol Biol.333(2):393-407(2003).GSK3 inhibitors can, for example, activate Wnt / β-catenin pathway.Many of the downstream genes of β-catenin co-regulate pluripotency gene network.For example, GSK inhibitor activates cMyc expression and enhances its protein stability and transcription activity.Therefore, in some embodiments, GSK3 inhibitor can be used to stimulate the endogenous Myc polypeptide expression in cells, making Myc expression unnecessary for inducing pluripotency.

[0115] MEK inhibitors may include antibodies against dominant negative variants of MEK, as well as siRNAs and antisense nucleic acids that suppress the expression of MEK. Specific examples of MEK inhibitors include PD0325901 (see, e.g., Rinehart, et al., Journal of Clinical Oncology 22: 4456-4462(2004)), PD98059 (available, e.g., from Cell Signaling Technology), UO126 (available, e.g., from Cell Signaling Technology), SL327 (available, e.g., from Sigma-Aldrich), ARRY-162 (available, e.g., from Array Biopharma), PD184161 (see, e.g., Klein, et al., Neoplasia 8:1-8(2006)), PD184352 (CI-1040) (see, e.g., Mattingly, et al., The Journal of Pharmacology and Experimental Therapeutics 316:456-465 (2006)), sunitinib (see, e.g., Voss, et al., US2008004287, incorporated herein by reference), sorafenib (Voss, see supra), vandetanib (Voss, see supra), pazopanib (Voss, see supra), axitinib (Voss, see supra), and PTK787 (Voss, see supra).

[0116] Several MEK inhibitors are currently undergoing evaluation in clinical trials. CI-1040 has been evaluated in phase I and phase II clinical trials for cancer (see, e.g., Rinehart, et al., Journal of Clinical Oncology 22(22):4456-4462(2004)). Other MEK inhibitors undergoing evaluation in clinical trials include PD184352 (see, e.g., English, et al., Trends in Pharmaceutical Sciences 23(1):40-45(2002)), BAY 43-9006 (see, e.g., Chow, et al., Cytometry (Communications in Clinical Cytometry) 23(1):40-45(2002)), and PD184352 (see, e.g., English, et al., Trends in Pharmaceutical Sciences 23(1):40-45(2002)). 46:72-78 (2001)), PD-325901 (also PD0325901), GSK1120212, ARRY-438162, RDEA119, AZD6244 (also ARRY-142886 or ARRY-886), RO5126766, XL518 and AZD8330 (also ARRY-704) (see, e.g., information from the National Institutes of Health at clinicaltrials.gov on the world wide web and information from the National Cancer Institute at cancer.gov / clinicaltrials on the world wide web).

[0117] TGFβ receptor (eg, ALK5) inhibitors may include antibodies against dominant negative variants of TGFβ receptors (eg, ALK5) and antisense nucleic acids that suppress the expression of TGFβ receptors. Exemplary TGFβ receptor / ALK5 inhibitors include SB431542 (see, e.g., Inman, et al., Molecular Pharmacology 62(1):65-74(2002)), A-83-01, also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (see, e.g., Tojo, et al., Cancer Science 96(11):791-800(2005) and commercially available products, e.g., from Toicris Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO (see, e.g., Dalton, et al., Molecular Pharmacology 62(1):65-74(2002)), which are incorporated herein by reference. WO 2008 / 094597), BMP4 (Dalton, supra), GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide) (see, e.g., Gellibert, et al., Journal of Medicinal Chemistry 49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., Cancer Research 67(5):2351-2359 (2007)), IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, e.g., Kim, et al., Xenobiotica 38(3):325-339(2008)), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., de Gouville, et al., Drug News Perspective 19(2):85-90(2006)), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology 65(3):744-752(2004)), and pyrimidine derivatives (see, e.g., those listed in Stiefl, et al., WO2008 / 006583, incorporated herein by reference). Furthermore, while "ALK5 inhibitors" are not intended to include non-specific kinase inhibitors, "ALK5 inhibitors" should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542 (see, e.g., Inman, et al., J Mol. Pharmacol. 62(1):65-74(2002)). Without intending to limit the scope of the present invention, ALK5 inhibitors are believed to affect the mesenchymal to epithelial transformation / transition (MET) process. The TGFβ / activin pathway is a driver of epithelial to mesenchymal transition (EMT). Thus, inhibiting the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0118] Considering the data herein showing the effect of inhibiting ALK5, it is believed that inhibiting TGFβ / activin pathway has a similar effect.Therefore, as described in each paragraph herein, any inhibitor of TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of ALK5 inhibitor.Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, SMAD 2 / 3 phosphorylation inhibitors, inhibitors of SMAD 2 / 3 and SMAD 4 interaction, and activators / agonists of SMAD 6 and SMAD 7.In addition, the following classifications are merely for organizational purposes, and those skilled in the art will know that a compound can affect one or more points in the pathway and therefore function in more than one of the defined categories.

[0119] TGFβ receptor inhibitors may include antibodies against dominant negative variants of the TGFβ receptor and siRNA or antisense nucleic acids targeting the TGFβ receptor.Specific examples of inhibitors include SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY21 09761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P144; soluble TBR2-Fc; and tumor cells transfected with antisense targeting the TGFβ receptor (e.g., Wrzesinski, (see, e.g., Kaminska, et al., Acta Biochimica Polonica 52(2):329-337(2005); and Chang, et al., Frontiers in Bioscience 12:4393-4401(2007)).

[0120] Inhibitors of SMAD 2 / 3 phosphorylation can include antibodies against dominant negative variants of SMAD2 or SMAD3 and antisense nucleic acids targeting SMAD2 or SMAD3. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (morin) (see, for example, Wrzesinski, supra; Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); and Kataoka, et al., EP1992360, which are incorporated herein by reference).

[0121] Inhibitors of SMAD 2 / 3 and smad4 interaction can include antibodies against dominant negative variants of SMAD2, SMAD3 and / or smad4, and antisense nucleic acids targeting SMAD2, SMAD3 and / or smad4. Specific examples of inhibitors of SMAD 2 / 3 and SMAD4 interaction include, but are not limited to, Trx-SARA, Trx-xFoxHlb, and Trx-Lef1 (see, e.g., Cui, et al., Oncogene 24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell, 17:3819-3831(2006)).

[0122] Activators / agonists of SMAD 6 and SMAD 7 include, but are not limited to, antibodies against dominant negative variants of SMAD 6 or SMAD 7 and antisense nucleic acids targeting SMAD 6 or SMAD 7. Specific examples of inhibitors include, but are not limited to, smad7-as PTO-oligonucleotides (see, e.g., Miyazono, et al., US6534476, and Steinbrecher, et al., US2005119203, both of which are incorporated herein by reference).

[0123] Exemplary HDAC inhibitors can include antibodies that bind to dominant negative variants of HDAC, as well as siRNA and antisense nucleic acids that target HDAC. HDAC inhibitors include TSA (trichostatin A) (see, e.g., Adcock, British Journal of Pharmacology 150:829-831(2007)), VPA (valproic acid) (see, e.g., Munster, et al., Journal of Clinical Oncology 25:18S(2007):1065), sodium butyrate (NaBu) (see, e.g., Han, et al., Immunology Letters 108:143-150(2007)), SAHA (suberoylanilide hydroxamic acid or vorinostat) (see, e.g., Kelly, et al., Nature Clinical Practice Oncology 2:150-157(2005)), sodium phenylbutyrate (see, e.g., Gore, et al., Cancer Research 2:150-157(2005)), and phenylbutyrate (see, e.g., Gore, et al., Cancer Research 2:150-157(2005)). 66:6361-6369(2006)), depsipeptide (FR901228, FK228) (see, e.g., Zhu, et al., Current Medicinal Chemistry 3(3):187-199(2003)), trapoxin (TPX) (see, e.g., Furumai, et al., PNAS 98(1):87-92(2001)), cyclic hydroxamic acid-containing peptide 1 (CHAP1) (Furumai, supra), MS-275 (see, e.g., Carninci, et al., WO2008 / 126932, which is incorporated herein by reference), LBH589 (see, e.g., Goh, et al., WO2008 / 108741, which is incorporated herein by reference), and PXD101 (Goh, (see above). In general, at a global level, pluripotent cells have more histone acetylation and differentiated cells have less histone acetylation. Histone acetylation is also involved in histone and DNA methylation regulation.In some embodiments, HDAC inhibitors promote the activation of silenced pluripotency genes.

[0124] Exemplary ERK inhibitors include PD98059 (see, e.g., Zhu, et al., Oncogene 23:4984-4992(2004)), UO126 (Zhu, supra), FR180204 (see, e.g., Ohori, Drug News Perspective 21(5):245-250(2008)), sunitinib (see, e.g., Ma, et al., US2008004287, incorporated herein by reference), sorafenib (Ma, supra), vandetanib (Ma, supra), pazopanib (Ma, supra), axitinib (Ma, supra), and PTK787 (Ma, supra).

[0125] Once the expression cassette has been introduced into the cells and / or the cells have been contacted with one or more agents, the cells can optionally be screened for pluripotent stem cell characteristics, thereby identifying pluripotent cells in the mixture, which can, for example, be isolated from other cells and used further, as appropriate.

[0126] III. Non-pluripotent cells As used herein, "non-pluripotent cells" refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells derived from selected tissues from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells.

[0127] In some embodiments in which an individual is treated with the resulting pluripotent cells, the individual's own non-pluripotent cells are used to generate the pluripotent cells by the methods of the invention.

[0128] The cells may be derived, for example, from a human or non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, and cows.

[0129] IV. Transformation The present invention relies on routine techniques in the field of recombinant genetics. Essential textbooks that disclose the general methods of use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0130] In some embodiments, the species of the cell and the species of the protein to be expressed are the same. For example, if a mouse cell is used, the mouse ortholog is introduced into the cell. If a human cell is used, the human ortholog is introduced into the cell.

[0131] It will be understood that when two or more proteins are expressed in a cell, one or more expression cassettes can be used. For example, when an expression cassette expresses multiple polypeptides, a polycistronic expression cassette can be used.

[0132] A. Plasmid Vectors In certain embodiments, plasmid vectors are intended to be used to transform host cells.Generally, for these hosts, plasmid vectors containing replicon and control sequences derived from species compatible with host cells are used.Vector may have a replication site and a marking sequence that allows phenotypic selection in transformed cells.

[0133] B. Viral Vectors Certain viruses can infect or enter cells by receptor-mediated endocytosis, integrate into host cell genome, and stably and efficiently express viral genes, making them attractive candidates for introducing foreign nucleic acid into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver the nucleic acid of the present invention are described below.

[0134] i. Adenovirus Vectors A particular method of delivering nucleic acid involves the use of adenovirus expression vectors. Although it is known that adenovirus vectors have a low ability to integrate into genomic DNA, this feature is offset by the high gene transfer efficiency obtained by these vectors. By "adenovirus expression vector" it is meant to include a construct that contains sufficient adenovirus sequences (a) to support packaging of the construct, and (b) to ultimately express the tissue-specific or cell-specific construct cloned into the vector. With knowledge of the genetic organization of adenovirus, a linear double-stranded DNA virus of approximately 36 kb, it is possible to replace large pieces of adenovirus DNA with foreign sequences up to 7 kb (Grunhaus et al., Seminar in Virology, 200(2):535-546, 1992).

[0135] ii. AAV vectors Nucleic acids can be introduced into cells using adenovirus-mediated transfection. High transfection efficiency has been reported in cell systems using adenovirus-coupled systems (Kelleher and Vos, Biotechniques, 17(6): 1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64, 1994.). Adeno-associated virus (AAV) is an attractive vector system because it has a high integration frequency and can infect non-dividing cells, and is therefore useful for gene delivery to mammalian cells, such as mammalian cells in tissue culture (Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992), or in vivo. Details regarding the production and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0136] iii. Retroviral vectors Retroviruses are promising gene delivery vectors because of their ability to integrate genes into the host genome, to introduce large amounts of foreign genetic material, to infect a wide range of species and cell types, and to be packaged in specialized cell lines (Miller et al., Am. J. Clin. Oncol, 15(3):216-221, 1992).

[0137] To construct retroviral vector, nucleic acid (e.g., nucleic acid encoding gene of interest) is inserted into viral genome in place of certain viral sequence to generate replication-defective virus.To produce virion, packaging cell line is constructed that contains gag, pol and env genes but does not contain LTR and packaging components (Mann et al., Cell, 33:153-159, 1983). When the recombinant plasmid containing cDNA with retroviral LTR and packaging sequence is introduced (for example, by calcium phosphate precipitation) into a specific cell line, the RNA transcript of the recombinant plasmid is packaged into viral particles by the packaging sequence, and then secreted into the medium (Nicolas and Rubinstein, Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986; Mann et al., Cell, 33:153-159, 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, integration and stable expression typically involve the division of host cells (Paskind et al., Virology, 67:242-248, 1975).

[0138] Lentivirus is a complex retrovirus that contains other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env.Lentivirus vectors are well known in the art (see, for example, Naldini et al., Science, 272(5259):263-267, 1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer et al., J Virol, 71(9):6641-6649, 1997; U.S. Patent Nos. 6,013,516 and 5,994,136).Some examples of lentivirus include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: SIV.Lentivirus vectors are made by attenuating HIV pathogenic genes. For example, the genes env, vif, vpr, vpu, and nef are deleted to render the vector biologically safe.

[0139] Recombinant lentivirus vectors can infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression in vivo and ex vivo. For example, recombinant lentiviruses can infect non-dividing cells, and suitable host cells are transfected with two or more vectors with packaging functions, i.e., gag, pol and env, rev and tat, as described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. Recombinant viruses can be targeted by linking the envelope protein with an antibody or a specific ligand for targeting a receptor on a specific cell type. Inserting a sequence of interest (including a regulatory region) into a viral vector together with another gene that codes for a ligand of a receptor on a specific target cell makes the vector target specific.

[0140] iv. Delivery using modified viruses The nucleic acid to be delivered may be housed in an infectious virus that is engineered to express a specific binding ligand. Thus, the virus particle specifically binds to the cognate receptor of the target cell and delivers its contents to the cell. A new method designed to specifically target retroviral vectors has been developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification allows specific infection of liver cells via sialoglycoprotein receptors.

[0141] Another approach to target recombinant retroviruses was designed, in which biotinylated antibodies against retroviral envelope proteins and against specific cell receptors were used. The antibodies were coupled by using streptavidin through the biotin moiety (Roux et al., Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, ecotropic viruses were demonstrated to infect various human cells bearing the surface antigens in vitro (Roux et al., 1989).

[0142] C. Vector Delivery and Cell Transformation Nucleic acid delivery methods suitable for transforming cells, tissues, or organisms for use with the present invention are intended to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into a cell, tissue, or organism, as described herein or known to those of skill in the art. Such methods include, for example, ex vivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987), optionally using Fugene6 (Roche) or Lipofecyamine (Invitrogen); microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099, incorporated herein by reference); No. 5,789,215), injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81.7161-7165, 1987; 1984); calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol, 10:689-695, 1990); the use of DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol, 5:1188-1190, 1985); direct sonic loading (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); liposome-mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982); Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979; Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene, 10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al., J Biol Chem., 266:3361-3364, These methods include, but are not limited to, direct delivery of DNA by receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; each of which is incorporated herein by reference), and any combination of such methods.

[0143] V. Cell culture Cells to be induced to become pluripotent can be cultured according to any method known in the art. Rough guidelines can be found, for example, in Maherali, et al., Cell Stem Cell 3:595-605 (2008).

[0144] In some embodiments, cells are cultured in contact with feeder cells.Exemplary feeder cells include but are not limited to fibroblasts, such as mouse embryonic fibroblasts (MEF) cells.Methods for culturing cells on feeder cells are known in the art.

[0145] In some embodiments, cells are cultured in the absence of feeder cells. For example, cells can be directly attached to a solid culture surface (e.g., a culture plate), for example, via a molecular tether. The inventors have discovered that cultured cells induced to become pluripotent are highly efficiently induced to pluripotency (i.e., more cells acquire pluripotency) when directly attached to a solid culture surface, compared to cells otherwise treated identically and cultured on feeder cells. Exemplary molecular tethers include, but are not limited to, matrigel, extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen. However, one skilled in the art will recognize that this is a non-limiting list and that other molecules can be used to attach cells to a solid surface. Methods for initially attaching tethers to a solid surface are known in the art.

[0146] As used in this "Culture" section, "cells induced to become pluripotent" may be induced by any method in the art, including but not limited to, the methods described herein.

[0147] VI. Use of Pluripotent Cells The present invention allows further research and development of stem cell technology, including but not limited to preventive or therapeutic use.For example, in some embodiments, the cells of the present invention (either pluripotent cells or cells induced to differentiate along a desired cell fate) are introduced into an individual who needs the cells of the present invention, including but not limited to an individual who needs to regenerate an organ, tissue, or cell type.In some embodiments, the cells are first obtained from an individual in a biopsy, induced to become pluripotent as described herein, and optionally induced to differentiate (e.g., into a specific desired progenitor cell), and then returned to the individual by transplantation.In some embodiments, the cells are genetically modified before being introduced into the individual.

[0148] In some embodiments, the pluripotent cells generated by the methods of the invention are subsequently induced to form, for example, hematopoietic (stem / progenitor) cells, neural (stem / progenitor) cells (optionally further differentiated cells, e.g., subtype-specific neurons, oligodendrocytes, etc.), pancreatic cells (e.g., endocrine precursor cells or pancreatic hormone-expressing cells), hepatic cells, cardiovascular (stem / progenitor) cells (e.g., cardiomyocytes, endothelial cells, smooth muscle cells), retinal cells, etc.

[0149] Various methods are known for inducing differentiation of pluripotent stem cells into desired cell types. A non-exclusive list of recent patent publications that describe methods for inducing differentiation of stem cells into various cell fates is as follows: U.S. Patent Application Publication Nos. 2007 / 0281355; 2007 / 0269412; 2007 / 0264709; 2007 / 0259423; 2007 / 0254359; 2007 / 0196919; 2007 / 0172946; 2007 / 0141703; 2007 / 0134215.

[0150] By introducing the pluripotent cells of the present invention into specific damaged tissue, and optionally by targeting the pluripotent cells of the present invention to specific damaged tissue, various diseases can be ameliorated.Examples of diseases caused by tissue damage include but are not limited to neurodegenerative disease, cerebral infarction, occlusive vascular disease, myocardial infarction, heart failure, chronic obstructive pulmonary disease, emphysema, bronchitis, interstitial lung disease, asthma, hepatitis B (liver damage), hepatitis C (liver damage), alcoholic hepatitis (liver damage), liver cirrhosis (liver damage), liver dysfunction (liver damage), pancreatitis, diabetes, Crohn's disease, inflammatory bowel disease, IgA nephritis, nephritis, kidney dysfunction, bedsore, burn, suture wound, laceration, incision, bite, dermatitis, scar keloid, keloid, diabetic ulcer, arterial ulcer, and venous ulcer.

[0151] The polypeptides described herein (e.g., one or more of Klf polypeptides, Oct polypeptides, Myc polypeptides, and Sox polypeptides) are themselves useful therapeutic agents, either alone or in combination as described herein. For example, the polypeptides or combinations thereof are useful for reducing tissue damage, and thus can be administered to treat, ameliorate, or prevent tissue damage. In some embodiments, the compounds of the present invention are administered to individuals who have tissue damage to internal organs, or who are at risk of having tissue damage to internal organs. Internal organs include, but are not limited to, the brain, pancreas, liver, intestine, lungs, kidneys, or heart, which are injured, for example, by burns or cutting. For example, in some embodiments, the compounds of the present invention are effective in reducing infarct size upon reperfusion after ischemia. Thus, the proteins of the present invention can be administered to individuals who are at risk of having a stroke, who have had a stroke, or who have had a stroke. Similarly, the proteins of the present invention can be administered to individuals who are at risk of having a heart attack or heart damage, who have had a heart attack or heart damage, or who have had a heart attack or heart damage.

[0152] The agents described herein (e.g., agents that inhibit H3K9 methylation; L-type Ca channel agonists; activators of the cAMP pathway; DNA methyltransferase (DNMT) inhibitors; nuclear receptor ligands; GSK3 inhibitors; MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; or Erk inhibitors) are also useful therapeutic agents, either alone or in combination with each other as described herein. For example, the agents or combinations thereof are useful for reducing tissue damage, and thus can be administered to treat, ameliorate, or prevent tissue damage. In some embodiments, the agents of the present invention are administered to individuals who have tissue damage to internal organs, or who are at risk of having tissue damage to internal organs. Internal organs include, but are not limited to, the brain, pancreas, liver, intestine, lungs, kidneys, or heart, for example, injured by burns or cuts. For example, in some embodiments, the agents of the present invention are effective in reducing infarct size upon reperfusion after ischemia. Thus, the agents of the invention can be administered to individuals at risk of suffering a stroke, who have suffered a stroke, or who have suffered a stroke.Similarly, the agents of the invention can be administered to individuals at risk of suffering a heart attack or heart damage, who have suffered a heart attack or heart damage, or who have suffered a heart attack or heart damage.

[0153] The active compounds described herein also include their salts, hydrates, solvates, and prodrug forms. The compounds of the present invention also include their isomers and metabolites. Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers, and individual isomers are all intended to be within the scope of the present invention. For example, the compounds of the present invention may be R-isomers or S-isomers or mixtures thereof. Furthermore, the compounds of the present invention may be E-isomers or Z-isomers or combinations thereof.

[0154] The pharma- ceutically acceptable salt of the acidic compound of the present invention is a salt formed with a base, i.e., a cationic salt, such as an alkali salt and an alkaline earth metal salt, such as a sodium salt, a lithium salt, a potassium salt, a calcium salt, a magnesium salt, and an ammonium salt, such as an ammonium salt, a trimethyl-ammonium salt, a diethylammonium salt, and a tris-(hydroxymethyl)-methyl-ammonium salt.In some embodiments, the present invention provides a hydrochloride salt.In other embodiments, the compound is ellipticine hydrochloride.

[0155] Likewise, if a basic group such as pyridyl forms part of the structure, acid addition salts are also possible, for example salts with inorganic acids, organic carboxylic acids and organic sulfonic acids, for example hydrochloric acid, methanesulfonic acid, maleic acid.

[0156] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent compounds may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent compounds for purposes of the present invention.

[0157] The compounds of the present invention can be made by a variety of methods known to those of skill in the art (see Comprehensive Organic Transformations Richard C. Larock, 1989). Those of skill in the art will appreciate that other methods of making the compounds are useful in the present invention.

[0158] Administration of the cells or compounds described herein is by any route normally used for the introduction of drugs. The pharmaceutical compositions of the invention may include a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers depend, in part, on the particular composition being administered, as well as on the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences, 17). thed. 1985).

[0159] Suitable formulations for administration include aqueous or non-aqueous solutions, isotonic sterile solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In carrying out the present invention, the compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intrathecally, or intraocularly (e.g., by eye drops or injection). The formulations of the compounds may be placed in single- or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described. The modulators can also be administered as part of a prepared food or drug.

[0160] The dose administered to a patient in the context of the present invention must be sufficient to induce a beneficial response in the subject over a period of time, i.e., to ameliorate the condition of the subject. The optimal dose level for any patient depends on a variety of factors, including the potency of the particular modulator used, the age, weight, physical activity, and diet of the patient, as well as possible combinations with other drugs. The size of the dose also depends on the existence, nature, and extent of adverse side effects associated with the administration of a particular compound or vector in a particular subject. Administration may be by single or divided doses.

[0161] VII. Screening for agents that induce the generation of pluripotent stem cells The present invention provides methods for screening for agents that can "replace" one of the four iPS transcription factors (i.e., Oct, Klf, Myc, and Sox polypeptides), or, in cells where Myc is not required for reprogramming cells into pluripotent cells, replace Oct, Klf, or Sox polypeptides (Nakagawa, M. et al. Nature Biotechnol. 26, 101-106 (2007); Wernig, M., Meissner, A., Cassady, JP, and Jaenisch, R. Cell Stem Cell 2, 10-12(2008)), or that improve the efficiency of fusion to pluripotency.

[0162] In some embodiments, the method includes introducing one or more expression cassettes for expression of at least one, but not all, of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide into a non-pluripotent cell to generate a transfected cell; then contacting the transfected cell with a library of distinct agents; screening the contacted cells for pluripotent stem cell characteristics; and correlating the occurrence of stem cell characteristics with particular agents from the library, thereby identifying agents that stimulate dedifferentiation of the cell into a pluripotent stem cell. In some embodiments, to identify library members that induce or improve the induction of cells into pluripotency, cells are contacted with at least one of an agent that inhibits H3K9 methylation; an L-type Ca channel agonist; an activator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEK inhibitor; a TGFβ receptor / ALK5 inhibitor; an HDAC inhibitor; or an Erk inhibitor, as well as with one or more members of a small molecule or other agent library. Thus, provided herein is a mixture of non-pluripotent cells and at least one (e.g., one, two, three, four, five, or more) of an agent that inhibits H3K9 methylation; an L-type Ca channel agonist; an activator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEK inhibitor; a TGFβ receptor / ALK5 inhibitor; an HDAC inhibitor; or an Erk inhibitor.

[0163] The agents in the library may be any small chemical compound or may be biological entities such as proteins, sugars, nucleic acids or lipids. Typically, the test agents are small chemical molecules and peptides. Although essentially any compound can be used as a potential agent in the assays of the present invention, most often compounds that can be dissolved in aqueous or organic (particularly DMSO-based) solutions are used. The assays are designed to screen large compound libraries by automating the assay steps and feeding the assays with compounds from any convenient source, which are typically performed in parallel (e.g., in a robotic assay, on a microtiter plate in a microtiter format). It will be understood that there are many compound suppliers, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), and the like.

[0164] In some embodiments, high-throughput screening methods involve preparing a combinatorial chemical or peptide library containing a large number of potential iPS replacement agents (potentially acting to replace one of the iPS proteins). Such "combinatorial chemical libraries" are then screened in one or more assays as described herein to identify library members (particular chemical species or subclasses) that exhibit a desired characteristic activity, i.e., an activity that induces pluripotent stem cell characteristics in cells that express some, but not all, of the Oct, Klf, Myc, and Sox polypeptides.

[0165] A combinatorial chemical library is a collection of diverse compounds produced by chemical synthesis or biological synthesis by combining a large number of chemical "building blocks", e.g., reagents. For example, a linear combinatorial chemical library, e.g., a polypeptide library, is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Through combinatorial mixing of such chemical building blocks, millions of compounds are synthesized.

[0166] The preparation and screening of combinatorial compound libraries are well known to those skilled in the art.Such combinatorial compound libraries include, but are not limited to, peptide libraries (see, for example, U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493(1991); and Houghton et al., Nature 354:84-88(1991)).Other chemistries for making chemical diversity libraries can also be used. Such chemistries include peptoid libraries (e.g., PCT Publication No. WO 91 / 19735), coded peptide libraries (PCT Publication No. WO 93 / 20242), random biooligomer libraries (PCT Publication No. WO 92 / 00091), benzodiazepine libraries (U.S. Patent No. 5,288,514), diversomers, such as hydantoin, benzodiazepine, and dipeptide libraries (Hobbs, et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptide libraries (Hagihara, et al., J. Amer. Chem. Soc. 114:6568 (1992)), non-peptidic peptidomimetic libraries with glucose backbones (Hirschmann, et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), small molecule compound libraries similar to organic synthesis libraries (Chen, et al., J. Amer. Chem. Soc. 116:2661(1994)), oligocarbamate libraries (Cho, et al., Science 261:1303(1993)), and / or peptidyl phosphonate libraries (Campbell, et al., J. Org. Chem. 59:658(1994)), nucleic acid libraries (see, e.g., Ausubel, Berger, and Sambrook).all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn, et al., Nature Biotechnology 14(3):309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (see, e.g., Liang, et al., Science 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), and small organic molecule libraries (e.g., benzodiazepines, Baum, C and EN, Jan 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, etc.).

[0167] Equipment for preparing combinatorial libraries is commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries themselves are commercially available (see, e.g., ComGenex, Princeton, NJ, Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[0168] The cells contacted with the agent and optionally expressing some, but not all, of the Oct, Klf, Myc, and Sox polypeptides (e.g., expressing a combination of one, two, or three of the Oct, Klf, Myc, and Sox polypeptides) can then be screened to develop pluripotent cells, for example, by screening for one or more pluripotent stem cell characteristics. An initial screen can be designed by transforming the cells to be screened with an expression cassette that includes a promoter element known to be activated in pluripotent stem cells (optionally not activated in other cells) operably linked to a selection or otherwise identifiable marker. For example, a detectable marker such as GFP or other reporter system can be used. Exemplary promoter elements known to be activated in pluripotent stem cells include, but are not limited to, Oct4, Nanog, SSEA1, and ALP promoter sequences. The cells can also be screened (e.g., by immunofluorescence, etc.) for the expression of other pluripotent cell markers known in the art, including, but not limited to, Nanog, SSEA1, and ALP. In some embodiments, cell morphology is examined.

[0169] In some embodiments, cells are cultured in the presence of a MAPK / ERK kinase (MEK) inhibitor. The inventors have discovered that the presence of a MEK inhibitor inhibits the proliferation of non-pluripotent cells and stimulates the proliferation of pluripotent stem cells. This effect therefore strengthens the "signal" of the screening, allowing for more efficient and sensitive detection of agents that induce reprogramming of cells into pluripotent stem cells. A wide variety of MEK inhibitors are known, including, but not limited to, PD0325901 (see, e.g., Thompson, et al., Current Opinion in Pharmacology 5(4):350-356(2005)); MEK inhibitor U0126 (Promega), ARRY-886 (AZD6244) (Array Biopharma); PD98059 (Cell Signaling Technology); and aminothioacrylonitrile (U.S. Patent No. 6,703,420). Other MEK inhibitors are described, inter alia, in US Pat. No. 6,696,440 and WO 04 / 045617.

[0170] VIII. Cell mixture As discussed herein, the present invention provides non-pluripotent cells in a mixture with one or more compounds selected from the group consisting of: H3K9 methylation inhibitor; L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor.In some embodiments, the compound is mixed at a concentration sufficient to induce pluripotency or at a concentration sufficient to improve the induction efficiency of pluripotency. For example, in some embodiments, the compound is at a concentration of at least 0.1 nM, such as at least 1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM, or 100000 nM, such as 0.1 nM to 100000 nM, such as 1 nM to 10000 nM, such as 10 nM to 10000 nM. In some embodiments, the mixture is in a synthetic container (e.g., a test tube, a petri dish, etc.). Thus, in some embodiments, the cell is an isolated cell (not part of an animal). In some embodiments, the cell is isolated from an animal (human or non-human), placed in a container, and contacted with one or more compounds as described herein. The cell may then be cultured, optionally returned to the same or a different animal, and optionally returned to the same or a different animal after stimulating the cell to a particular cell type or lineage.

[0171] As described herein, in some embodiments, the cell comprises an expression cassette for heterologous expression of at least one or more of Oct polypeptide, Myc polypeptide, Sox polypeptide, and Klf polypeptide.In some embodiments, the cell does not comprise an expression cassette for expressing any of Oct, Myc, Sox, or Klf polypeptide.The cell that comprises or does not comprise such an expression cassette is useful, for example, in the screening method described herein.

[0172] Examples of non-pluripotent cells include the non-pluripotent cells described herein, including but not limited to cells derived from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include but are not limited to fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells.

[0173] The present invention also provides a mixture (with or without cells) of an agent that inhibits H3K9 methylation (including but not limited to BIX-01294) and a compound selected from at least one of the following: L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor. In some embodiments, the agent and at least one compound listed above are at the above concentrations. Such a mixture is useful, for example, as a "premix" for inducing pluripotency of cells.

[0174] IX. Kit The present invention also provides a kit for use in, for example, inducing pluripotency in cells or improving the efficiency of inducing pluripotency.Such a kit may comprise one or more compounds selected from the group consisting of: H3K9 methylation inhibitor; L-type Ca channel agonist; cAMP pathway activator; DNA methyltransferase (DNMT) inhibitor; nuclear receptor ligand; GSK3 inhibitor; MEK inhibitor; TGFβ receptor / ALK5 inhibitor; HDAC inhibitor; or Erk inhibitor. In some embodiments, the kit comprises an agent that inhibits H3K9 methylation (including but not limited to BIX-01294) and a second compound (separate from the agent that inhibits H3K9 methylation or mixed with the agent that inhibits H3K9 methylation) selected from at least one of an L-type Ca channel agonist; an activator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEK inhibitor; a TGFβ receptor / ALK5 inhibitor; an HDAC inhibitor; or an Erk inhibitor.

[0175] In some embodiments, the kit further comprises non-pluripotent cell.The examples of non-pluripotent cell include the non-pluripotent cell described herein, including but not limited to, cells from tissue selected from bone marrow, skin, skeletal muscle, adipose tissue and peripheral blood.Exemplary cell types include but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts and T cells. EXAMPLES

[0176] Example 1 Towards identifying conditions that can replace viral introduction of oncogenic transcription factors (e.g., cMyc and Oct4 (Hochedlinger, K. et al., Cell 121, 465-477(2005)) and enhance reprogramming efficiency, we sought to exploit a combination of two approaches. One was to investigate defined progenitor cell types based on the concept that certain accessible adult progenitor cells may endogenously express some of the genes required to induce pluripotency at certain levels and / or the loci of these genes may be less silenced so that such progenitors can be reprogrammed more efficiently and / or with less genetic manipulation; the other approach was to screen small molecules that could replace viral incorporation of specific transcription factors and / or enhance the reprogramming process.

[0177] Among the various accessible adult stem / progenitor cells from various tissues, we initially focused our efforts on neural progenitor cells for the following reasons: (i) in contrast to heterogeneous primary fibroblast cultures (e.g., MEFs), which may contain various types of stem / progenitor cells, neural progenitor cells are a relatively defined population of cells and can be clonally expanded under chemically defined conditions, and (ii) neural progenitor cells endogenously express specific Sox genes (e.g., Sox1 or Sox2), which, although at lower levels than overexpression, may be sufficient to generate iPS cells. (iii) Neural progenitor cells or Sox gene expressing cells can be isolated from other tissues (Fernandes, KJL et al., Nature Cell Biology 6, 1082-1093 (2004); Seaberg, RM et al., Nature Biotechnol. 22, 1115-1124 (2004)) and expanded in vitro. Therefore, defined neural progenitor cells represent an excellent model system to address the above questions in the reprogramming process / mechanism. To establish an unlimited, highly reproducible and defined source of neural progenitor cells that can be used in high-throughput screening, we chose to use mESC-derived neural progenitor cells containing a GFP-IRES-Puro / GiP reporter under the control of Oct4 regulatory elements. Because mESCs can be expanded in large quantities and their differentiation into homogenous populations of neural progenitor cells is well defined (Conti, L. et al., PLoS Biol. 3, e283 (2005)), and their validated reporter activity (Ying, QL et al., Nature 416, 545-548 (2002)) can facilitate easy assay detection.

[0178] Reporter neural progenitors were generated using well-established procedures by differentiating Oct4-GiP mESCs grown in monolayer on gelatin for 8 days at low cell density in chemically defined medium / CDM conditions lacking serum and other growth factors / cytokines, followed by the formation of neurospheres, followed by serial passaging of single cells in monolayer for over 6 passages / 24 days in neuronal expansion medium supplemented with 10 ng / ml bFGF and EGF. The resulting neural progenitors were homogenous with respect to cell morphology and neural marker expression, and were confirmed to be GFP-negative and puromycin-sensitive. Such neural progenitors plated in monolayer in regular mESC growth medium were transfected with combinations of 4, 3, or 2 of the 4 factors, and then transfected cells were treated with individual small molecules from a small molecule known drug collection in a typical 6-well format. Compound treatment and culture were continued for an additional 10 days before the addition of puromycin. The number of green and puro-resistant colonies was counted on day 14. Compound conditions that produced more green colonies than the corresponding gene-only conditions were selected as primary hits, compared to neurons transfected with only the four genes as a positive control. To further confirm these primary hit conditions, the inventors used OG2 + / - / ROSA26 + / -We chose to use late passage mouse CNS neural progenitor cells (Do, JT et al., Stem Cells 25, 1013-1020 (2007)) derived from fetal brain of transgenic mice (containing Oct4-GFP reporter) and expanded in monolayer under the same neural CDM conditions as above with 10 ng / ml bFGF and EGF. Although unlikely with all the appropriate controls, such cells derived from truly non-pluripotent tissues would be free of any concerns of contamination with ES cells in the above screening system. Similar culture conditions and reprogramming assays were performed with OG2 neural progenitor cells, except that puromycin was not used and green colonies were picked and characterized by staining for Nanog, SSEA1, and ALP. We found that almost all of the green colonies that could be identified on days 12-14 could be expanded into long-term stable iPS cells that were indistinguishable from classical mESCs by morphology and expression of typical pluripotency markers.

[0179] We first focused our characterization efforts on two new conditions that could be reaffirmed using fetal neural progenitor cells. Just as we hypothesized that certain tissue-specific progenitor cells with endogenous expression of certain relevant reprogramming genes might require less exogenous genetic manipulation to generate iPS cells, we found that viral transduction of Oct4 and Klf4 alone was sufficient to generate iPS cells from neural progenitor cells in 10-14 days. Such a reprogramming efficiency (3.5 × 10 4 1–2 GFP colonies per cell) in conditions with additional Sox2 and cMyc viral transduction (3.5 × 10 4Although the reprogramming kinetics was lower than that of the first four genes (8-10 GFP colonies per cell) (Figure 1), it is interesting to note that the reprogramming kinetics with only two genes (Oct4 and Klf4) is not significantly slower than that with the first four genes. This contrasts with the recent observation that omitting cMyc in generating iPS cells from MEFs is significantly slower (e.g., an additional 2 weeks) than conditions with cMyc overexpression, even though embryonic fibroblasts endogenously express cMyc. Most interestingly, we found that the small molecule BIX01294 (Kubicek, S. et al., Molecular Cell 25, 473-481(2007)), which specifically inhibits G9a (a histone methyltransferase for H3K9me2), did not significantly shorten the reprogramming kinetics, but could significantly improve the reprogramming efficiency to or above the level using viral introduction of all four factors. Reprogramming events are typically assayed by the ability to identify iPS cell colonies, and are influenced by many factors, including cell culture method, cell identification and / or selection, and input cell type, number, and reprogramming efficiency and kinetics.Thus, the requirement of any given gene for reprogramming is related to its specific setting and highly dependent on reprogramming efficiency / kinetics.In this regard, this single small molecule BIX01294 functionally replaces the viral introduction of cMyc and Sox2 to a large extent.

[0180] GFP+ iPS cell colonies appeared immediately 12 days after transduction of OG2 neural progenitor cells with Oct4 / Klf4 retrovirus and treatment with BIX01294. At day 14, iPS cells generated from Oct4-Klf4 viral transduction and BIX01294 treatment could be easily expanded in the presence of LIF on MEF feeder cells in normal mESC culture conditions without the need for continued BIX01294 treatment. iPS cells generated by Oct4 / Klf4 viral transduction and BIX01294 treatment could self-renew long-term in mESC growth medium on MEF feeders without continued BIX01294 treatment. They grow as compact hemispherical colonies. These iPS cells maintain characteristic mESC colony morphology and homogeneously express typical pluripotency markers, including Oct4, Nanog, SSEA1, and ALP, at levels comparable to mESCs by immunocytochemistry, histological staining, and RT-PCR analysis. Moreover, such iPS cells serially passaged for 10 generations can be effectively differentiated into characteristic neurons (βIII-tubulin), beating cardiomyocytes (cardiac troponin), and pancreatic or hepatic cells (Pdx1 or albumin), derivatives of the three primary germ layers, under standard embryoid body or directed differentiation methods. And most importantly, such iPS cells can efficiently incorporate into the ICM of blastocysts after aggregation with 8-cell embryos, produce a high degree of chimerism after transplantation of the aggregated embryos into mice, and contribute to the germ lineage in vivo. These in vitro and in vivo characterizations confirm that iPS cells generated by Oct4 and Klf4 viral transduction with simultaneous BIX01294 treatment are indistinguishable from the original four-factor iPS cells and classical mESCs morphologically, functionally, and by expression of typical pluripotency markers.

[0181] One question is whether the expression of Oct4, Sox2, Klf4, and cMyc, whether endogenous or exogenous, is essential to generate iPS cells. Interestingly, recent reprogramming studies on the generation of human iPS cells from fibroblasts have shown that exogenous expression of Klf4 and cMyc is functionally interchangeable with Nanog and Lin28, while expression of Oct4 and Sox2 appears to be necessary so far, according to all published iPS cell studies. Interestingly, we found that viral introduction of Klf4, Sox2, and cMyc with simultaneous BIX01294 treatment in the absence of Oct4 expression can also generate iPS cells, while viral introduction of these three factors / KSM alone failed to produce iPS cell colonies under our assay conditions (Figure 2). Similarly, such KSM-BIX01294-generated iPS cells can be stably expanded without BIX01294 for several generations in normal mESC growth conditions on MEF feeders and self-renew long-term, maintain characteristic mESC morphology, homogeneously express typical pluripotency markers including ALP, Oct4, Nanog, and SSEA1, and differentiate into cells of the three germ layers in vitro. It is noteworthy that reprogramming efficiency in the absence of Oct4 expression is relatively low.

[0182] Finally, we observed that application of PD0325901, a specific small molecule inhibitor of MEK, to later stages of reprogramming (e.g., after Oct4-GFP activation) can serve as a good selection strategy to generate iPS cells. Due to the very low efficiency of reprogramming, iPS cells are typically selected by using reporters (e.g., Neo / Puro or GFP) under the control of regulatory elements of pluripotency markers using genetically modified cell lines, or manually picked out based on cell morphology. The latter method, applicable to genetically unmodified cells, is more suitable for the final clinical use of iPS cells, but is a much more tedious and unreliable technique that typically requires picking and growing many colonies for several generations, of which only a small fraction effectively becomes true iPS cells. This is in part because the majority of similar-looking colonies may be rapidly growing partially reprogrammed cells and / or simply transformed cells, and may interfere with the growth and reprogramming of iPS cells. It would therefore be highly desirable to have an alternative selection strategy for genetically unmodified cells. We found that PD0325901 efficiently promoted proliferation and stable reprogramming of iPS cells while inhibiting proliferation of non-iPS cells, resulting in larger and more homogeneous colonies of iPS cells. This observation may be due in part to the mechanism that mESCs lack such proliferation restrictions and that inhibition of MEK also inhibits differentiation of mESCs (contributing to further stabilization of the iPS cell state), while MEK activity is required for cell cycle progression of somatic cells.

[0183] The results presented herein have a number of important implications. (1) Lower endogenous expression levels (overexpression) of key genes required for reprogramming by (tissue-specific precursor) somatic cells may be sufficient to substitute for corresponding exogenous gene expression via viral transduction to generate iPS cells. This suggests an alternative strategy to generate iPS cells from somatic cells with less genetic manipulation by utilizing practically accessible cells that endogenously express certain relevant reprogramming genes by endogenous tissue specificity and / or via ex vivo culture manipulation. (2) This is a proof-of-principle demonstration that rationally designed cell-based screens can identify small molecules that functionally replace viral transduction of certain transcription factors, improve reprogramming efficiency, or act as selection conditions in generating iPS cells. Such pharmacological approaches to substitute for specific genetic manipulations may not only substantially reduce the risks associated with the insertion and insertional mutation of cancer genes (e.g., cMyc and Oct4), but also open up the possibility of precisely controlled and highly efficient reprogramming processes with defined small molecules. This is especially important for studying the molecular mechanisms of reprogramming, which is currently largely difficult due to very low efficiency and slow kinetics. (3) In contrast to gain-of-function approaches in generating iPS cells, the highly effective use of their specific small molecule inhibitors suggests that loss-of-function of specific genes may be at least equally important and effective in generating iPS cells. More importantly, the function of BIX01294 defines a specific epigenetic mechanism / target in generating iPS cells, namely, inhibition of G9a-mediated H3K9me2. This is consistent with previous findings that repressive H3K9 methylation is associated with Oct4 inactivation during differentiation (Feldman, N. et al., Nature Cell Biology 8, 188-194 (2006)) and that histone lysine methylation, although robust, is dynamic and regulated by HMTases and lysine demethylases.BIX01294 may function to promote the transition of epigenetic balance from the silenced state of Oct4 to active transcription. (4) Exploiting the difference between somatic cells and ESCs by small molecules, exemplified by using MEK inhibitors for easy selection of iPS cells, represents an alternative / attractive strategy for selecting iPS cells. Finally, it is considered that the strategies and small molecules reported herein can be further explored for improved methods and better mechanistic understanding of the stage of generating iPS cells, and can be combined with additional small molecules (which can replace the function of remaining introduced transcription factors and improve reprogramming) and other non-genetic methods (e.g., protein introduction) that ultimately allow the generation of highly efficient iPS cells in completely chemically defined conditions without any genetic modification.

[0184] method Neural progenitor cell culture Neural progenitor cells were derived from mESCs or mouse fetal brains according to the procedure reported by Conti et al. (Conti, L. et al., PLoS Biol. 3, e283 (2005)). Briefly, mESCs were plated at 1 × 10 4 cells / cm 2Neurospheres were then plated in Neural Induction Medium (50% DMEM / F12 Basal Medium, 50% Neurobasal Medium, 0.5x N2, 0.5x B27, 1x Glutamax, 50ug / ml BSA) at 4°C and allowed to differentiate for 7-8 days. The formed neural rosettes were then trypsinized to single cells and replated in Neural Progenitor Expansion Medium (DMEM / F12, 1x N2, 10ng / ml bFGF, 10ng / ml EGF, 50ug / ml BSA) in Ultra-Low Attachment dishes (Corning) to form neurospheres. After 3 days in suspension, neurospheres were allowed to reattach to gelatin-coated dishes and they were further passaged as single cells and allowed to differentiate further for 4-6 days before being expanded in monolayers on gelatin-coated dishes in Neural Progenitor Expansion Medium for >5-6 passages.

[0185] Neurospheres from 12.5-16.5 dpc ROSA26 / OG2 heterozygous fetal brains were generated as previously described (Do, JT et al., Stem Cells 25, 1013-1020 (2007)). Briefly, cortices were dissected, enzymatically dissociated, and passed through a 70 μm nylon mesh (Falcon; Franklin Lakes, NJ). Neurons were further purified by centrifugation at 750 g for 10 min in 0.9 M sucrose in 0.5× HBSS and at 200 g for 7 min in 4% BSA in EBSS solution. Such cells were further expanded in suspension to form neurospheres and then serially passaged in monolayers and on gelatin-coated dishes in Neural Progenitor Expansion Medium as described above. Animal experiments were approved and performed in accordance with the Animal Protection Guidelines of the Government of Max Planck Society, Germany.

[0186] Retroviral transduction Mouse cDNAs for Oct4, Klf4, Sox2, and c-Myc were cloned into the pMSCV retroviral vector and verified by sequencing. pMX-based retroviral vectors were obtained from Addgene. Virus production and transduction were performed as described in 2–3.

[0187] Induction of iPS cells from neural progenitor cells mESC-derived or primary OG2 mouse neural progenitor cells were plated at 3.5 × 10 in 6-well plates coated with Matrigel (1:50, BD Biosciences). 4 Cells / well were plated in neural progenitor expansion medium. One day later, the cells were transduced with retrovirus overnight and the medium was changed to mESC growth medium [DMEM, 5% FBS, 10% KSR, 1× non-essential amino acids (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), and 10 mM ethanol (Gibco)] with or without BIX01294 (0.5–1 μM). 3 The medium was then replaced with 1000 μg / ml LIF (Chemicon). GFP-positive iPS cell colonies appeared after 9–14 days and were selected and expanded on MEF feeder cells using mESC proliferation medium.

[0188] Characterization assays ALP staining was performed as directed by the Alkaline Phosphatase Detection Kit (Chemicon). Cells were fixed in 4% paraformaldehyde, washed three times with PBS, and then incubated in PBS containing 0.3% TritonX-100 (Sigma) and 10% normal donkey serum (Jackson ImmunoResearch) for 30 min at room temperature. Cells were then incubated overnight at 4°C with the following primary antibodies: mouse anti-Oct4, mouse anti-SSEA1 (1:200, Santa Cruz), rabbit anti-Sox2 (1:200, Chemicon), rabbit anti-Nanog (AbCam), rabbit anti-Pdx1 (1:200, from Dr. C. Wright), mouse anti-βIII-tubulin (1:500, Covance), mouse anti-cardiac troponin T (1:200, DSHB), and rabbit anti-albumin (DAKO). After washing, cells were further incubated with secondary antibodies: Alexa Fluro555 donkey anti-mouse IgG or Alexa Fluro555 donkey anti-rabbit IgG (1:500, Invitrogen) for 30 min at RT. Nuclei were detected by DAPI (Sigma) staining. Images were captured by Nikon TE2000-U.

[0189] Aggregation of iPS cells with zona-free embryos To obtain aggregate chimeras, iPS cells were aggregated with exposed compacted 8-cell embryos. 8-cell embryos (B6C3F1) flushed from females at 2.5 dpc were cultured in microdroplets of KSOM medium (10% FCS) under mineral oil. Clumps of iPS cells (10–20 cells) were selected after brief trypsinization and transferred into microdroplets containing zona-removed 8-cell embryos. The aggregated 8-cell embryos with iPS cells were cultured overnight at 37°C and 5% CO2. Aggregated blastocysts developed from the 8-cell stage were transferred into one uterine horn of pseudopregnant recipients at 2.5 dpc.

[0190] Example 2 Somatic cells can be induced into pluripotent stem cells (iPSCs) using a four-transcription factor combination, Oct4 / Sox2 / Klf4 / c-Myc or Oct4 / Sox2 / Nanog / LIN28. This provides a platform that allows obtaining patient-specific cells for various therapeutic and research applications. However, several issues remain for this approach to be therapeutically relevant due to the efficiency and drawbacks associated with viral genomic integration. As explained above, Oct4 / Klf4-transduced neural progenitor cells (NPCs) can be reprogrammed into iPSCs. However, NPCs endogenously express Sox2, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small molecule combination, BIX-01294 and BayK8644, that allows reprogramming of Oct4 / Klf4-transduced mouse embryonic fibroblasts that do not endogenously express factors essential for reprogramming. This study demonstrates that small molecules identified by phenotypic screening can compensate for viral transfer of key factors such as Sox2 and improve reprogramming efficiency.

[0191] This example aims to evaluate whether small molecules can replace specific viral transduction to obtain iPSCs from common cell lineages that do not express any of the TFs considered essential for reprogramming: Oct4, Sox2, and Klf4. To this end, mouse embryonic fibroblasts (MEFs) were used. Finding small molecules that can replace one of these TFs in inducing MEF reprogramming could lead to the identification of common pathways involved in this process. Such chemical strategies may be more suitable for therapeutic applications. Therefore, we screened a collection of known drugs to identify small molecules that can enable the generation of iPSCs from OK-transduced MEFs, thus compensating for the lack of Sox2 overexpression. Through various screens performed, we identified that the combination of L-channel calcium agonist Bayk8644 (BayK) (Schramm, M. et al., Nature, 303:535-537 (1983)) and BIX was the most effective one. Bayk was of interest because it exerts its effects upstream in cell signaling pathways and does not directly cause epigenetic modifications. This type of molecule, such as BayK or activators of the Wnt signaling pathway (Marson, A. et al., Cell Stem Cell, 3:132-135 (2008)), could likely be exploited to induce reprogramming in a more specific manner than molecules that act directly at the epigenetic level to cause DNA or histone modifications. Some of these epigenetic modifiers, such as BIX (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)), valproic acid (Huangfu, D. et al., Nat Biotechnol, 26:795-797 (2008)), and 5'azacytidine (Mikkelsen, T. et al., Nature, 454:49-55 (2008)), have already been shown to promote the reprogramming process.

[0192] This study demonstrates that small molecules identified by phenotypic screening can be used to effectively compensate for viral transduction of Sox2, another important iPSC TF that is not endogenously expressed in fibroblasts, and further highlights the important contribution that small molecule screening may ultimately make to discover new molecular targets and mechanisms involved in complex biological processes such as reprogramming.

[0193] result Phenotypic screening leads to the discovery of small molecules that enable reprogramming of MEFs when only two TFs are introduced. Unmodified MEFs derived from E13–14 embryos of 129 mice were used for the initial screen. MEFs were plated on Matrigel at 3.5 × 10 per well in a 6-well plate. 4Cells were plated with 1000 ng / ml GFP-10 ... BIX appeared to have the strongest effect on reproducibly inducing more than one or two compact ESC-like colonies with high ALP expression. We observed that when MEFs were treated with BIX after OK virus transduction, compact colonies with strong ALP expression could be easily detected within approximately 14-21 days. These cells were also positive for Nanog, Oct4, and SSEA-1 expression. The present results, obtained with a more common cell type that does not endogenously express any of the three essential reprogramming genes, further confirm our previous observations that BIX has strong reprogramming-inducing activity and that G9a HMTase can promote reprogramming (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)). However, the reprogramming efficiency in OK-transduced and BIX-treated MEFs was still low compared to four-factor-induced reprogramming of MEFs or OK / BIX NPC reprogramming, at approximately 2 colonies / 3.5 × 10 4cells (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)). Therefore, we performed a second screening using a similar procedure but adding BIX to the basal medium after OK virus transduction. This provided a more permissive platform to identify new small molecules that could further improve reprogramming efficiency. More importantly, this second screening could facilitate the discovery of small molecules that affect reprogramming in a more specific manner, for example, by acting on signaling pathways rather than on histones or DNA-modifying enzymes. In this second screening, we again tested a library of approximately 2000 known small molecules (see Experimental Procedures) and identified two compounds that could act in a synergistic manner with BIX to improve reprogramming based on the screening criteria. One example is RG108, a DNA methyltransferase (DMNT) inhibitor (Brueckner, B. et al., Cancer Res, 65:6305-6311 (2005)), which enhanced reprogramming of OK-transduced MEFs in the presence of BIX (Figure 3). However, like BIX, RG108 is known to affect cells at a general epigenetic level, and another DNA methyltransferase inhibitor, 5-azacytidine, has already been shown to enhance reprogramming (Mikkelsen, TS et al., Nature, 454:49-55 (2008)). Therefore, RG108 was not pursued further for this study. Instead, we focused phenotypic and functional characterization on another small molecule identified in the second screen, the L-calcium channel agonist BayK. This small molecule, which showed the strongest effect in the screen besides known DNA / histone modifiers, was studied further.Because in the absence of BIX, it has no observable reprogramming activity on OK-transduced MEFs and is known not to directly affect cells at the epigenetic level but rather at the cell signaling level. Therefore, BayK may play a more specific role in the reprogramming process. When 129MEFs were transduced with empty retrovirus (negative control); no colonies were observed. When 129MEFs were transduced with OK without small molecules; only a few small flat colonies with weak ALP expression were present. After 14-21 days of transducing 129MEFs with OK and treating them with BIX / BayK, ESC-like iPSC colonies were observed; these ESC-like colonies showed strong ALP expression. When OK-transduced MEFs were treated with BIX in combination with BayK, the ALP expression was more similar to that of mESCs compared to OK-transduced MEFs treated with BIX alone (~2 colonies). + A significant increase in the number of colonies could be observed (~7 colonies). Further characterization of these primary iPSC colonies showed that they were positive for typical pluripotency markers such as Oct4, Sox2, Nanog, and SSEA1, as measured by immunofluorescence.

[0194] iPSCs derived from OK-transduced and BIX / BayK-treated MEFs have pluripotent properties characteristic of mESCs. To further confirm and characterize that OK transduction and BIX / BayK treatment can induce MEFs to become iPSCs, we cultured OG2 cells containing an Oct4-GFP reporter. + / - / ROSA26 + / -Primary MEFs derived from (OG2) transgenic mice were used (Do, JT and Scholer, HR, Stem Cells, 22:941-949 (2004)). Once reprogrammed, these cells can then be used to conveniently assess chimeras and germline competence. Similar to 129 MEFs, OG2 MEFs transduced with OK were able to generate iPSCs when treated with the BayK / BIX combination (OK2B iPSCs) (Figure 3). GFP + iPSC colonies could first be detected 14–21 days after viral transduction and compound treatment. When OG2 MEFs were transduced with OK and not treated with any compound, 3.5 × 10 4 Only a few small colonies appeared, with an average of 0.5 ± 0.7 colonies per cell. These colonies were difficult to passage and therefore were not studied further. Treatment of OK-transfected OG2 MEFs with BIX alone resulted in 3.5 × 10 4 Compared to OK alone, OK-transduced OG2 MEFs were easily and reproducibly reprogrammed with 2.5±0.7 colonies per cell. There was a further significant improvement in reprogramming efficiency when OK-transduced OG2 MEFs were treated with a combination of BIX (2 μM) and BayK (2 μM); we obtained 3.5×10 4 We observed 7.7±1.5 colonies per cell (Figure 3). Treatment of OK-transduced OG2 MEFs with BayK alone in the absence of BIX did not increase the reprogramming efficiency of the OK-transduced MEF controls described above (data not shown).

[0195] OK2B colonies were picked and serially expanded on irradiated MEF feeder cells for more than 20 passages in the absence of small molecules under normal mESC growth conditions. +OK2B iPSCs were shown to express typical pluripotency markers, including ALP, Nanog, Sox2, Oct4, SSEA1, c-Myc, eRas, Esg1, Ecat1, and Fgf4. RT-PCR assay also demonstrated that OK2B iPSCs expressed endogenous Oct4 and Klf4 (Figure 4A). Bisulfite genomic sequencing analysis of the Nanog promoter revealed that the Nanog promoter in MEFs was highly methylated, but demethylated in OK2B iPSCs, similar to the mESC control (R1) (Figure 4B). This result further suggests the reactivation of stem cell transcriptional programs in these OK2B iPSCs. In addition, transcriptome analysis showed that the expression profile of OK2B iPSCs was significantly different from that of MEFs, as illustrated in the clustering analysis, with a Pearson correlation value of 0.84, but highly similar to that of mESCs, with a Pearson correlation value of 0.96.

[0196] Transcriptome analysis was performed to compare the OK2B transcriptome with mES and MEF cells. RNA was extracted from OK2B iPSCs at passage 13 using the Qiagen RNAeasy Mini Kit. RNA expression data for OK2B iPSCs was generated from polyA RNA using GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix). Expression data for MEF and mES cells were obtained from the Gene Expression Omnibus (GEO) website http: / / www.ncbi.nlm.nih.gov / geo / . mES cell data accession numbers: GSM198062, GSM198063, and GSM198064. MEF cell data accession numbers: GSM198070 and GSM198072. Preprocessing, normalization (GC-RMA), and hierarchical clustering were performed using dChip (http: / / biosun1.harvard.edu / complab / dchip / ; (Distance metric: correlation (Pearson); Linkage method: centroid; Gene order: by cluster tightness). p-value for OK2B iPSC vs. MEF cells: 0.84; p-value for OK2B iPSC vs. mES cells: 0.96. p-values ​​were obtained using the Pearson correlation test.

[0197] OK2B iPSCs differentiate into cells derived from all three germ layers and contribute to germline transmission. OK2B iPSCs could efficiently form embryoid bodies (EBs) in suspension, which could differentiate into derivatives of the three primary germ layers: endodermal cells (albumin and Pdx1), mesodermal cells / cardiomyocytes (CT3), and ectodermal cells / neurons (βIII-tubulin, Tuj1). In addition, OK2B iPSCs could efficiently incorporate into the inner cell mass of blastocysts following aggregation with 8-cell embryos, leading to chimerism with germline contribution in vivo after implantation of aggregated embryos into pseudopregnant mice. Furthermore, mating of one adult male progeny obtained from these blastocysts with female CD1 wild-type mice resulted in LacZ+ This resulted in the production of progeny, three of which were Oct4-GFP, further confirming that these iPSCs were able to contribute to germline transmission. + These in vitro and in vivo characterizations confirmed that retroviral transduction of the two genes alone, in combination with BIX / BayK treatment, was sufficient to reprogram MEFs into iPSCs that were phenotypically and functionally similar to classical mESCs.

[0198] Consideration The work presented herein provides proof-of-principle demonstration that small molecules can be identified from rationally designed phenotypic screens to functionally replace viral transfer of certain TFs and improve reprogramming efficiency in generating iPSCs from common cell types such as MEFs. Such chemical approaches to generate iPSCs, which provide more precise and temporal control of targets / processes, are considered to be advantageous over genetic manipulation with oncogenes, which may also introduce deleterious and undetectable insertional genomic changes. Similar strategies are being used to find additional small molecules that may ultimately enable reprogramming of lineage-restricted cells to pluripotent or multipotent states in fully chemically defined conditions. BIX was originally identified and characterized as a specific inhibitor of G9a HMTase (Kubicek, S. et a., Mol Cell, 25:473-481 (2007)). It has been shown to reduce H3K9me2 levels at G9a target genes (Feldman, N. et al., Nat Cell Biol, 8:188-194 (2006)). Interestingly, G9a-mediated histone H3K9 methylation and heterochromatinization represent a highly specific mechanism for epigenetic silencing of embryonic genes such as Oct4 and Rex1 (Feldman, N. et al., Nat Cell Biol, 8:188-194 (2006)). Furthermore, it has been demonstrated that knockdown of G9a can assist in fusion-based reprogramming of adult neural cells (Ma, DK et al., Stem Cells, 26:2131-2141 (2008)). It is therefore pertinent that we previously observed that BIX can promote the generation of iPSCs from NPCs transduced with either OK or Klf4 / Sox2 / c-Myc (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)), suggesting that it can compensate for endogenous expression of Sox2 or Oct4.However, NPCs already expressed significant levels of Sox2, which may have rendered these cells more susceptible to reprogramming in the conditions described above. This study aimed to identify small molecules that could enable reprogramming of MEFs that do not express any of the TFs thought to be required for reprogramming. Our identification of BIX in our screens of both NPCs and MEFs was unexpected, further confirming that this molecule has a role in enabling and improving the generation of iPSCs from somatic cells. Given the characterized mechanism of action of BIX, our study potentially identified a molecular target whose loss of function via pharmacological inhibition is sufficient to compensate for the gain of function of essential iPSC reprogramming genes. It further mechanistically links the inhibition of G9a-mediated H3K9me2, a specific epigenetic process, to iPSC generation. BIX may function to promote the transition of the epigenetic balance from a silenced state of pluripotency genes to an active transcriptional state. Apparently, the combination of BIX with other chromatin-modifying small molecules with different targets and mechanisms of action, such as RG108, could be exploited for better reprogramming. On the other hand, our observation that BayK, which has activity characterized as a specific L-type calcium channel agonist (Schramm, M. et al., Nature, 303:535-537 (1983)), improves reprogramming efficiency is intriguing. L-type calcium channels are known to mediate intracellular processes in various tissues, such as blood pressure regulation, smooth muscle contraction, insulin secretion, cardiac development, etc. (Tosti, E., Reprod Biol Endocrinol, 4:26(2006)). Furthermore, activation of L-type calcium channels by various agonists, including BayK, has been shown to mediate CREB activation, sarcoplasmic reticulum Ca. 2+It has been shown that BayK induces intracellular signals by the release of calcium and changes in cAMP activity. More importantly, some reports suggest that calcium may play a role in controlling the proliferation of mES cells (Heo, JS et al., Am J Physiol Cell Physiol, 290:C123-133 (2006)). However, in our hands, treatment of mES cells with 2 μM BayK alone or in combination with 1 μM BIX does not result in changes in proliferation (Figure 5). Furthermore, treatment of OG2 MEFs with 2 μM BayK alone or in combination with 1 μM BIX does not induce SOX2 expression (Figure 6). Needless to say, more work needs to be done to dissect the exact mechanism by which BayK affects the reprogramming process. However, it is intriguing to find that small molecules with activity in signal pathways not previously associated with reprogramming can significantly enhance its efficiency. So far, it is the first small molecule of a type other than Wnt3 protein (Marson, A. et al., Cell Stem Cell, 3:132-135 (2008)) that shows an effect on reprogramming without directly acting on chromatin modifiers, since most of the other small molecules found to affect reprogramming to date appear to directly modify the epigenetic state of cells: namely, BIX (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)), valproic acid (Huangfu, D. et al., Nat Biotechnol, 26:795-797 (2008)), and 5'azacytidine (Mikkelsen, TS et al., Nature, 454:49-55 (2008)). Importantly, BayK appears to possess several key characteristics that may ultimately be desirable for a therapeutically relevant molecule for in vivo reprogramming and / or regeneration.The fact that it does not act / reprogram by itself but requires the presence of BIX to exert its effect suggests that cells that have already undergone a form of reprogramming, possibly caused by injury, may be more susceptible to its effects, which may allow it to ultimately reprogram target cells in a more specific manner without affecting healthy cells systemically, as direct epigenetic modifiers do.

[0199] In summary, we have identified defined small molecule conditions, namely, BIX, and combinations of BIX / BayK or BIX / RG108, that can enable and improve the reprogramming of fibroblasts to iPSCs with the introduction of only two TFs: Oct4 and Klf4. This study further confirms the usefulness of phenotypic screening approaches in identifying small molecules that can effectively compensate for viral introduction of essential iPSC TFs, such as Sox2 in this study, or Oct4 as previously reported (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)). Ultimately, phenotypic small molecule screening may lead to the identification of small molecules that are likely to be powerful tools that provide new insights into the reprogramming process, and may ultimately be useful in in vivo stem cell biology and therapy.

[0200] Experimental procedure Origin of MEF 129S2 / SvPasCrlf or ROSA26 + / - / OG2 + / - MEFs were obtained according to the procedure reported on the WiCell Research Institute website: “Introduction to human embryonic stem cell culture methods.” Animal experiments were performed in accordance with the Animal Protection Guidelines of the Max Planck Institute for Biomolecular Research, Germany.

[0201] Retroviral transduction and compounds pMX-based retroviral vectors for mouse Oct4, Klf4, c-Myc, and Sox2 were obtained from Addgene (Cambridge, MA). Virus production and transduction processes were performed as described (Takahashi, K. et al., Cell, 131:861-872 (2007)). Synthesis and full characterization of compound BIX-01294 was as previously described (Kubicek, S. et al., Mol Cell, 25:473-481 (2007)), and Bayk8644 was purchased from EMD / Calbiochem Biochemical (San Diego, CA).

[0202] Screening for iPSC generation from MEFs For primary and secondary screening, a collection of known compounds was used, consisting of approximately 2000 known commercially available bioactive molecules, including FDA-approved drugs, known inhibitors and activators of characterized enzymes (including the LOPAC collection from Sigma-Aldrich (St. Louis, MO), the Known Bioactive Library from BIOMOL (Plymouth Meeting, PA), and non-redundant known compounds from EMD Calbiochem (San Diego, CA)).

[0203] Original 129S2 / SvPasCrlf (first screening) or ROSA26 + / - / OG2 + / - (Secondary screening) MEFs were plated on Matrigel (1:50; BD Biosciences, Bedford, MA)-coated dishes at 3.5 × 10 per well in a 6-well plate. 4Cells were plated at a density of 100x100 / mL. After 24 hours, these cells were transduced with the defined retrovirus overnight at 37°C and 5% CO2. After 12–14 hours, the medium on the transduced cells was replaced with mESC medium [Knockout DMEM, 10% ES-qualified FBS, 10% Knockout serum replacement, 1% Glutamax, 1% Non-essential amino acids, penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 1% EmbryoMax ESC Qualified Nucleosides (Millipore, Temecula, CA), and 10% ethanol. 3 U / ml LIF (Millipore)] (all products are from Invitrogen, Carlsbad, CA, unless otherwise noted). On that same day, individual small molecules from our known drug collection were added to the cells in the range of 0.5-2 μM. Compound treatment continued for 10-14 days; cells were fixed and stained using a standard ALP detection kit (Millipore) on days 14-21. For the second round of screening, 1 μM BIX was added to the mESC medium 1 day after transduction. Five days later, individual small molecules from the known drug collection were added to each well in the range of 0.5-2 μM in addition to 1 μM BIX. Mouse ESC medium containing the defined small molecules was refreshed every 3 days until colonies with morphology similar to mESCs were observed, which was usually between 14-21 days after transduction. In addition to the confirmed compounds as presented in the text, primary hits from a second synergist screen that were not pursued further also included PD173074, riversine, 5'azacytidine, pluripotin, and dexamethasone. Further characterization studies and iterations were performed on the primary 129S2 / SvPasCrlf or ROSA26 + / - / OG2 + / - The test was performed on either MEF or ROSA26 + / - / OG2 + / -When MEFs were used, iPSC colonies could also be identified by GFP expression as a marker for Oct4 expression. Once iPSC colonies were identified, they were picked onto MEF feeder cells in mESC medium for expansion. Some colonies were expanded in the presence of the MEK inhibitor PD0325901 at concentrations of 0.5-2 μM to further confirm their pluripotency.

[0204] Immunocytochemistry and immunofluorescence assays ALP staining was performed using an Alkaline Phosphatase Detection Kit (Millipore) according to the manufacturer's instructions. For immunofluorescence assays, cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT) and washed with PBS. They were then incubated in blocking buffer (BB) [0.3% Triton X-100 (Sigma-Aldrich), 10% normal donkey serum (Jackson ImmunoResearch Laboratories Inc) in PBS (Invitrogen)] for 30 min at RT. They were then incubated with primary antibodies overnight in BB at 4°C. Cells were then washed with PBS and incubated with secondary antibodies in BB for 45–60 min at RT. Primary antibodies were: mouse anti-Oct4 (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-SSEA1 (1:200) (Santa Cruz Biotechnology Inc.), rabbit anti-Nanog (1:500) (Abcam Inc., Cambridge, MA), mouse anti-Sox2 (1:200) (Millipore), rabbit anti-Pdx1 (1:200) (kind gift from Dr. C. Wright), mouse anti-βIII-tubulin (Tuj1) (1:500) (Covance Research Products Inc., Denver, PA), mouse anti-cardiac troponin T (CT3) (1:200) (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA), and rabbit anti-albumin (DAKO). Secondary antibodies were Alexa Fluor555 donkey anti-mouse or rabbit IgG (1:500) (Invitrogen). Nuclei were detected by DAPI (Sigma-Aldrich) staining. Images were captured using a Nikon Eclipse TE2000-U / X-cite 120 EXFO microscope with a photometric CoolSnap HQ2 camera.

[0205] RT-PCR assay RNA was extracted from iPSCs and control cell lines using the RNeasy Plus Mini Kit in combination with QIAshredder. RNA was converted to cDNA using the iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA). Amplification of specific genes was performed on a Mastercycler ep gradient PCR machine (Eppendorf) using previously published primers (Takahashi, K. et al., Cell, 131:861-872 (2007); Takahashi, K. and Yamanaka, S., Cell, 126:663-676 (2006)) and Platinum PCR SuperMix (Invitrogen).

[0206] Methylation Assays DNA was isolated from R1, OG2 MEF, and OK iPSC (passage 10) cells using the Non Organic DNA Isolation Kit (Millipore). The DNA was then treated with EZ DNA Methylation-Gold Kit™ (Zymo Research Corp., Orange, CA) for bisulfite sequencing. The treated DNA was then used to amplify sequences of interest. The primers used for promoter fragment amplification were as previously published (Blelloch, R. et al., Stem Cells, 24:2007-2013 (2006)). The resulting fragments were cloned and sequenced using the TOPO TA cloning Kit for sequencing (Invitrogen).

[0207] Aggregation of iPSCs with zona-removed embryos To obtain aggregate chimeras, iPSCs were aggregated with exposed compacted 8-cell embryos. 8-cell embryos (B6C3F1) were flushed from females at 2.5 dpc and cultured in microdroplets of KSOM medium (10% FCS) under mineral oil. Clumps of iPSCs (10–20 cells) were selected after brief trypsinization and transferred into microdroplets containing zona-removed 8-cell embryos. The aggregated 8-cell embryos with iPSCs were cultured overnight at 37°C and 5% CO2. Aggregated blastocysts developed from the 8-cell stage were implanted into one uterine horn of a pseudopregnant recipient at 2.5 dpc. One adult male chimera was mated with a female CD1 wild-type mouse. X-gal staining showed that six F1 embryos obtained from this natural mating of chimeric and wild-type mice were generated by germline transmission.

[0208] statistical analysis Bar graphs and statistical analysis were performed using standard t-tests in Excel.

[0209] Microarray analysis OK2B iPSCs were grown for 2 days on gelatin (Millipore, Temecula, CA) in complete mES cell medium [Knockout DMEM, 10% ES-qualified FBS, 10% Knockout serum replacement, 1% Glutamax, 1% Non-essential amino acids, penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 1% EmbryoMax ESC Qualified Nucleosides (Millipore), and 10 3U / ml LIF (Millipore)] (all products are from Invitrogen, Carlsbad, CA, unless otherwise noted). RNA from duplicate wells was then isolated using the RNAeasy Mini Kit (Qiagen, Valencia, CA). Total RNA samples were amplified and labeled using the MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, TX). Amplified and labeled samples were then hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix) and analyzed using hierarchical clustering (Pearson, log-transformed, row-centered values) using GenePattern (World Wide Web: broad.mit.edu / cancer / software / ).

[0210] Proliferation Assay mES R1 cells were plated on gelatin-coated 6-well plates at 2 × 10 5 Cells were plated in complete mES cell medium at a density of 1000 cells / well. Concurrent with cell attachment, approximately 12 hours, cells were treated in triplicate with either DMSO, 1 μM BIX, 2 μM BayK, or a combination of both. At 15, 24, and 48 hours, cells were detached with trypsin and counted using a hemocytometer. Trypan blue (Sigma-Aldrich, St. Louis, MO) was used to remove dead cells.

[0211] Assessment of SOX2 expression after compound treatment OG2 + / - / ROSA26 + / - MEFs were plated on a 6-well plate at 3.4 × 10 per well. 4Cells were plated at a density of 100x100 / well. The next day, cells were treated with DMSO, 1 μM BIX, 2 μM BayK, and a combination of both in triplicate for 6 days. The medium was refreshed on day 3. RNA from each well was then isolated using an RNAeasy Mini Kit (Quiagen). Reverse transcription of RNA was performed using an iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA). Amplification of endogenous Sox2 was performed using previously published primers (Takahashi, K., Okita, K., Nakagawa, M., and Yamanaka, S. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2, 3081-3089; Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676) with Platinum PCR SuperMix (Invitrogen) in a Mastercycler® ep gradient PCR machine (Eppendorf).

[0212] Example 3 This example demonstrates that incubation of mammalian cells with transcription factor proteins is sufficient to induce pluripotency.

[0213] Genetic constructs: To obtain high-level protein expression in E. coli, the codon regions of all four human TF genes were first optimized (GA Gutman and GW Hatfield (1989). PNAS. vol. 86.pp:3699-3703) and the full-lengths were synthesized using DNA oligo-based / PCR gene construction techniques (Danilo R Casimiro, Peter E Wright and H Jane Dyson. (1997). Structure. Vol.5. pp: 1407- 1412.). Polyarginine tag: TIFF2025015628000003.tif4128 was added to the C-terminus of each protein in the design (Gump JM, Dowdy SF. (2007) Trends Mol Med. 2007 Oct;13(10):443-8). The final DNA fragment was flanked by NdeI and XhoI sites and inserted into the NdeI-XhoI sites of the pET41 expression vector for protein expression. Each plasmid was DNA sequence verified and then transformed into BL21 starting competent cells overnight with autoinducing medium for recombinant protein production (Studier FW, (2005) Protein Expr Purif. 41(1). Pp: 207-234.).

[0214] Protein preparation Escherichia coli BL21(DE3) cells were transformed with pET-Oct4-PTD ("PTD" refers to protein transduction domain), pET-Soc2-PTD, pET-Klf4-PTD, and pET-c-Myc-PTD separately, and protein expression was performed using the autoinduction method (Studier FW, Protein Expression and Purification, 41 (2005) 207-234.). Inclusion bodies were solubilized and proteins were refolded as described (LaFevre BM, Wu S. and Lin X. Molecular Cancer Therapeutics 7, 1420-1429, June 1, 2008. doi: 10.1158 / 1535-7163;Medynski D., Tuan M., Liu, W., Wu, S. and Lin, X. Protein Expression and Purification Vol. 52, 395-402, April 2007;Hou W., Medynski D., Wu, S., Lin, X. and Li, LY. Clinical Cancer Research Vol. 11, 5595-5602, August 1, 2005).

[0215] Briefly, E. coli containing the expression plasmid was inoculated into 1.0 L of Luria-Bertani Broth containing kanamycin, induced with 500 umol / L IPTG at A600 nm = 0.6, and stirred at 37 °C for 3 h. Cells were harvested by centrifugation, and the pellet was subjected to freeze-thaw cycles. Released inclusion bodies were washed extensively with a buffer containing 20 mmol / L Tris, 100 mmol / L NaCl, 1% TritonX-100 (pH 8.0), and dissolved at A280 nm = 2.0 in a buffer containing 8 mol / L urea, 0.1 mol / L Tris, 1 mmol / L glycine, 1 mmol / L EDTA, 10 mmol / L b-mercaptoethanol, 10 mmol / L DTT, 1 mmol / L reduced glutathione, and 0.1 mmol / L oxidized glutathione (pH 10). The solubilized inclusion bodies were refolded using the rapid dilution method as described (Lin XL, Lin YZ, Tang J., Methods Enzymol 1994. 241. 195-224; Lin X, Koelsh G., Wu.S, Downs D, Dashti A. Tang J. Proc Natl Acad Sci USA. 2000; 97. 1556-1560; Kim YT. Downs D. Wu S, et al. Eur J Biochem 2002. 269: 5669-77; Michelle LaFevre-Bernt, Shili Wu, and Xinli Lin. (2008). Molecular Cancer Therapeutics. 7: pp:1420-1429). The refolded proteins were concentrated by N2 ultrafiltration and purified by molecular sieve chromatography using Sephacryl S 300. The endotoxin concentration in each of the protein preparations was less than 100 EU / mg. Most of the refolded protein samples have a solubility of at least >1.5 mg / ml.

[0216] The refolded protein was concentrated using tangential flow filtration, purified using molecular sieve chromatography on a Superdex-200 column (XK26x850-mm, GE, Piscataway, NJ), and confirmed using SDS-PAGE.

[0217] Mouse fibroblasts were grown for 6-8 hours in mESC medium supplemented with either Oct4 / Sox2 / Klf4 or Oct4 / Sox2 / Klf4 / Myc (all proteins contain poly-Arg as described above) at 8 μg / ml, washed, and incubated for 2-3 days in mESC medium without the transcription factors listed above. This was repeated several times (1, 2, 3, 4, or more) (4-12 hours with, 1-3 days without), after which the cells were cultured in mESC for 2 weeks. At the end of this period, the cultures were determined to contain pluripotent cells by colony morphology and marker expression (data not shown). Strikingly, constant incubation of the cells with transcription factors (i.e., without a period of 1-3 days without proteins) was found to be toxic to the cells. Although not required, in some cases cells were incubated with a MEK inhibitor (PD0325901) and / or a GSK inhibitor (CHIR99021) and / or a TGFβ inhibitor (SB431542), and the presence of these agents improved the efficiency and rate of pluripotent cell generation.

[0218] The above examples are provided to illustrate the present invention, but are not provided to limit its scope.Other variations of the present invention will be readily apparent to those skilled in the art and are encompassed by the appended claims.All publications, databases, Genbank sequences, patents, and patent applications cited herein are incorporated herein by reference.

[0219] Sequence information SEQUENCE LISTING <110> THE SCRIPPS RESEARCH INSTITUTE <120> COMBINED CHEMICAL AND GENETIC APPROACHES FOR GENERATION OF INDUCED PLURIPOTENT STEM CELLS <150> US 61 / 069,956 <151> 2008-03-17 <150> US 61 / 197,986 <151> 2008-10-31 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Poly-arginine tag <400> 1 Glu Ser Gly Gly Gly Gly Ser Pro Gly Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20

Claims

1. 1. A method for producing induced pluripotent stem cells from mammalian non-pluripotent cells, comprising: (a) introducing into the mammalian non-pluripotent cell one or more expression cassettes comprising an Oct4 polynucleotide, a Sox2 polynucleotide, and a Klf4 polynucleotide; and (b) contacting the cells of step (a) with a TGFβ receptor / ALK5 inhibitor and a GSK-3 inhibitor, thereby generating induced pluripotent stem cells from the mammalian non-pluripotent cells; A method comprising:

2. The method of claim 1, further comprising contacting the cells with an Erk inhibitor.

3. The method of claim 1, further comprising the step of contacting the mammalian non-pluripotent cell with a histone deacetylase (HDAC) inhibitor.

4. The method of claim 1, wherein the TGFβ receptor / ALK5 inhibitor is selected from the group consisting of A-83-01 and SB431542.

5. The method of claim 1, wherein the GSK-3 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, and 6-bromoindirubin-3'-oxime (BIO).

6. The method of claim 1, further comprising contacting the mammalian non-pluripotent cell with at least one of an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, an L-type Ca channel agonist, an activator of the cAMP pathway, a DNA methyltransferase (DNMT) inhibitor, or a nuclear receptor ligand.

7. The method described in claim 1, wherein the one or more expression cassettes further contain a c-Myc polynucleotide.

8. The method of claim 1 , wherein the non-pluripotent cell is a somatic cell.

9. The method of claim 8, wherein the somatic cells are fibroblasts.

10. The method of claim 1, wherein the non-pluripotent cells are progenitor cells.

11. The method of claim 10, wherein the progenitor cells are neural progenitor cells, skin progenitor cells, or hair follicle progenitor cells.

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