Induction of pluripotent cells

Abstract

The slow kinetics and low efficiency of reprogramming methods for generating human induced pluripotent stem cells (iPSCs) pose significant limitations to their utility in biomedical applications. We describe here a chemical method that significantly improves (>200-fold) the efficiency of generating iPSCs from human fibroblasts within 7 days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.

Description

[0001] This application is a divisional application of Chinese patent application No. 201610220657.8, filed on October 15, 2010, entitled "Induction of Pluripotent Cells".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 61 / 252,548, filed October 16, 2009, pursuant to 35 USC § 1.119(e), the contents of which are incorporated herein by reference in their entirety. Background Technology

[0004] Recent advances in the production of human induced pluripotent stem cells (iPSCs) (Takahashi, K. et al., Cell 131, 861-72 (2007); Yu, J. et al., Science 318, 1917-20 (2007); Muller, LUW, et al., Mol.Ther. 17, 947-53 (2009)) have raised hopes for their practical applications in biomedical research and clinical settings. However, iPSC production remains a very slow (approximately 4 weeks) and inefficient (<0.01% (Takahashi, K. et al., Cell 131, 861-72 (2007); Yu, J. et al., Science 318, 1917-20 (2007)) process that produces heterogeneous cell populations. Identifying fully reprogrammed iPSCs from such a mixture is tedious and requires specialized skills in human pluripotent cell culture.

[0005] Despite efforts to overcome the risks of genomic insertion of exogenous reprogramming factors, the inefficiency and slow kinetics of reprogramming continue to represent insurmountable problems for the ultimate application of human iPSCs. For example, during reprogramming, the increase of genetic or epigenetic abnormalities can occur, where tumor suppressor factors may be suppressed and oncogenic pathways may be activated. Although recent studies have reported improving the efficiency of reprogramming through genetic manipulation beyond the initial four factors (Feng, B. et al., Cell Stem Cell 4, 301-12 (2009)), such manipulations often further complicate the process and increase the risk of genetic alteration and tumorigenicity. Therefore, there remains a significant need for safer, easier, and more efficient methods for human iPSC production, as well as methods to facilitate the identification and characterization of the underlying mechanisms of reprogramming. Summary of the Invention

[0006] This invention provides mixtures (e.g., those that can be used to induce iPSC). In some embodiments, the mixture comprises:

[0007] mammalian cells;

[0008] TGFβ receptor / ALK5 inhibitor;

[0009] MEK inhibitors; and

[0010] Rho GTPase / ROCK pathway inhibitors.

[0011] In some implementations, at least 99% of the cells are non-pluripotent. In some implementations, all or substantially all of the cells are non-pluripotent.

[0012] In some implementations, the cells are human cells.

[0013] In some embodiments, the TGFβ receptor / ALK5 inhibitor is SB431542.

[0014] In some implementations, the MEK inhibitor is PD0325901.

[0015] In some embodiments, the ROCK inhibitor is a compound having the following formula:

[0016] (I)

[0017] Ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

[0018] Ring B is a substituted or unsubstituted heterocyclic alkyl group, or a substituted or unsubstituted heteroaryl group;

[0019] L 1 It is -C(O)-NR 2 -or-C(O)-NR 2 -;

[0020] L 2 It is a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; and

[0021] R 1 and R 2 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0022] In some implementations, the ROCK inhibitor has the following formula:

[0023] (II)

[0024] Where y is an integer from 0 to 3; z is an integer from 0 to 5; and X is -N=, -CH=, or -CR. 5 =;R 3 R 4 and R 5 CN and S(O) are independent entities. n R 6 NR 7 R 8 C(O)R 9 NR 10 -C(O)R 11 NR 12 -C(O)-OR 13 -C(O)NR 14 R 15 -NR 16 S(O)2R 17 -OR 18 -S(O)2NR 19 substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where if z is greater than 1, then there are 2 R 3 Selectively linked together to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; and R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 and R 19 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0025] In some implementations, the ROCK inhibitor has the following formula:

[0026] ,

[0027] ,

[0028] ,

[0029] ,or

[0030] .

[0031] In some implementations, the ROCK inhibitor is

[0032]

[0033] In some embodiments, when the mixture is subjected to conditions sufficient to induce the conversion of non-pluripotent cells in the mixture into induced pluripotent stem cells, the concentration of the inhibitor is sufficient to increase the efficiency of inducing the cells into induced pluripotent stem cells by at least 10%.

[0034] In some embodiments, the mixture further comprises a GSK3 inhibitor and / or an HDAC inhibitor.

[0035] In some embodiments, the polypeptide is selected from Oct-3 / 4, Sox2, KLF4, and c-Myc. In some embodiments, the cell is selected from human cells, non-human animal cells, mouse cells, non-human primate cells, or other animal cells.

[0036] The present invention also provides a method for inducing non-pluripotent mammalian cells into induced pluripotent stem cells. In some embodiments, the method includes contacting the non-pluripotent cells under conditions sufficient to induce at least some cells to become pluripotent stem cells:

[0037] TGFβ receptor / ALK5 inhibitor;

[0038] MEK inhibitors; and

[0039] ROCK inhibitors.

[0040] In some embodiments, the conditions include introducing at least one exogenous transcription factor into non-pluripotent cells. In some embodiments, the at least one exogenous transcription factor is an Oct peptide, and the cells are further exposed to a histone deacetylase (HDAC) inhibitor.

[0041] In some embodiments, the transcription factor is selected from Oct peptide, Klf peptide, Myc peptide, and Sox peptide.

[0042] In some embodiments, the method includes introducing at least two, three, or four exogenous transcription factors into non-pluripotent cells, wherein the transcription factors are selected from Oct peptide, Klf peptide, Myc peptide, and Sox peptide. In some embodiments, the peptides are selected from Oct-3 / 4, Sox2, KLF4, and c-Myc. In some embodiments, the cells are selected from human cells, non-human animal cells, mouse cells, non-human primate cells, or other animal cells.

[0043] In some embodiments, one or more of the transcription factors are expressed in the cells by introducing a polynucleotide into a non-pluripotent cell, wherein the polynucleotide encodes the at least one exogenous transcription factor.

[0044] In some embodiments, the at least one transcription factor is introduced by contacting a non-pluripotent cell with an exogenous polypeptide, wherein the polypeptide comprises the amino acid sequence of the transcription factor, and the introduction is performed under conditions that allow the polypeptide to be introduced into the cell. In some embodiments, the polypeptide comprises an amino acid sequence that enhances transmembrane transport.

[0045] In some implementations, the cells are human cells.

[0046] In some embodiments, the TGFβ receptor / ALK5 inhibitor is SB431542.

[0047] In some implementations, the MEK inhibitor is PD0325901.

[0048] In some embodiments, the ROCK inhibitor is a compound having the following formula:

[0049] (I)

[0050] Ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

[0051] Ring B is a substituted or unsubstituted heterocyclic alkyl group, or a substituted or unsubstituted heteroaryl group;

[0052] L 1 It is -C(O)-NR 2 -or-C(O)-NR 2 -;

[0053] L 2 It is a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; and

[0054] R 1 and R2 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0055] In some implementations, the ROCK inhibitor has the following formula:

[0056] (II)

[0057] Where y is an integer from 0 to 3; z is an integer from 0 to 5; and X is -N=, -CH=, or -CR. 5 =;R 3 R 4 and R 5 CN and S(O) are independent entities. n R 6 NR 7 R 8 C(O)R 9 NR 10 -C(O)R 11 NR 12 -C(O)-OR 13 -C(O)NR 14 R 15 -NR 16 S(O)2R 17 -OR 18 -S(O)2NR 19 substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where if z is greater than 1, then there are 2 R 3 Selectively linked together to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; and R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 and R 19Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0058] In some implementations, the ROCK inhibitor has the following formula:

[0059] ,

[0060] ,

[0061] ,

[0062] ,or

[0063] .

[0064] In some implementations, the ROCK inhibitor is .

[0065] In some embodiments, when the mixture is subjected to conditions sufficient to induce the conversion of non-pluripotent cells in the mixture into induced pluripotent stem cells, the concentration of the inhibitor is sufficient to increase the efficiency of inducing the cells into induced pluripotent stem cells by at least 10%.

[0066] In some embodiments, the mixture additionally contains a GSK3 inhibitor.

[0067] This invention also provides a kit for inducing pluripotency in non-pluripotent mammalian cells. In some embodiments, the kit comprises,

[0068] TGFβ receptor / ALK5 inhibitor;

[0069] MEK inhibitors; and

[0070] ROCK inhibitors.

[0071] In some embodiments, the TGFβ receptor / ALK5 inhibitor is SB431542.

[0072] In some implementations, the MEK inhibitor is PD0325901.

[0073] In some embodiments, the ROCK inhibitor is a compound having the following formula:

[0074] (I)

[0075] Ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl;

[0076] Ring B is a substituted or unsubstituted heterocyclic alkyl group, or a substituted or unsubstituted heteroaryl group;

[0077] L 1 It is -C(O)-NR 2 -or-C(O)-NR 2 -;

[0078] L 2 It is a bond, a substituted or unsubstituted alkylene group, or a substituted or unsubstituted heteroalkylene group; and

[0079] R 1 and R 2 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0080] In some implementations, the ROCK inhibitor has the following formula:

[0081] (II)

[0082] Where y is an integer from 0 to 3; z is an integer from 0 to 5; and X is -N=, -CH=, or -CR. 5 =;R 3 R 4 and R 5 CN and S(O) are independent entities. n R 6 NR 7 R 8 C(O)R 9 NR 10 -C(O)R 11 NR 12 -C(O)-OR 13 -C(O)NR 14 R 15 -NR 16 S(O)2R 17 -OR 18 -S(O)2NR 19 substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where if z is greater than 1, then there are 2 R 3Selectively linked together to form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; and R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 and R 19 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0083] In some implementations, the ROCK inhibitor has the following formula:

[0084] ,

[0085] ,

[0086] ,

[0087] ,or

[0088] .

[0089] In some implementations, the ROCK inhibitor is

[0090] .

[0091] In some embodiments, the kit further comprises a GSK3 inhibitor and / or a histone deacetylase (HDAC) inhibitor.

[0092] Other implementation methods will become clear from the rest of the content disclosed herein.

[0093] definition

[0094] "Oct polypeptide" refers to any naturally occurring member of the Octomer transcription factor family, or a variant thereof that retains transcription factor activity similar to that of the closest related naturally occurring family member (within at least 50%, 80%, or 90% of the activity range), or a polypeptide that includes at least the DNA-binding domain of a naturally occurring family member, and may further include a transcription activation domain. Exemplary Oct polypeptides include, for example, Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3 / 4 (referred to herein as "Oct4") contains a POU domain, which is a conserved 150-amino acid sequence between Pit-1, Oct-1, Oct-2, and uric-86. See Ryan, AK & Rosenfeld, MGGenes Dev. (Gene Development) 11, 1207-1225 (1997). In some implementations, the variants share at least 85%, 90%, or 95% amino acid sequence identity across their entire sequence compared to naturally occurring Oct polypeptide family members, such as those listed above or those listed in Genbank accessions NP_002692.2 (human Oct4) or NP_038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3 / 4) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins from the same species as the cell species being operated on will be used.

[0095] "Klf polypeptide" refers to any of the following: a naturally occurring member of the Krüppel-like factor (Klfs) family; a zinc finger protein containing an amino acid sequence similar to the Drosophila embryonic model regulator Krüppel; a variant of a naturally occurring member retaining similar transcription factor activity (within at least 50%, 80%, or 90% of the activity range) compared to the most closely related naturally occurring family member; or a polypeptide comprising at least the DNA-binding domain of a naturally occurring family member, and may further comprise a transcriptional activation domain. See, Dang, DT, Pevsner, J. & Yang, VW. Cell Biol. 32, 1103-1121 (2000). Exemplary members of the Klf family include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factors capable of generating iPS cells in mice, and the related genes Klf1 and Klf5 also generated iPS cells in mice, albeit with reduced efficiency. See, Nakagawa, et al., Nature Biotechnology 26: 101–106 (2007). In some embodiments, the variants have at least 85%, 90%, or 95% amino acid sequence identity throughout their entire sequence compared to naturally occurring members of the Klf polypeptide family, such as those listed above or those listed in Genbank accession numbers CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins from the same species as the cell species being operated on will be used. With regard to the Klf polypeptides described herein, they can be replaced with the estrogen-associated receptor β (Essrb) polypeptide. Therefore, it is intended that corresponding embodiments using Essrb instead of the Klf4 polypeptide be described equivalently for the various Klf polypeptide embodiments described herein.

[0096] "Myc polypeptide" refers to any naturally occurring member of the Myc family (see, for example, Adhikary, S. & Eilers, M. Nat. Rev. Mol. Cell Biol. 6:635-645 (2005)), or a variant thereof that retains similar transcription factor activity (within at least 50%, 80%, or 90% of the activity range) to the most closely related naturally occurring family member, or a polypeptide that includes at least the DNA-binding domain of a naturally occurring family member, and may further include a transcription activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity over its entire sequence compared to naturally occurring Myc polypeptide family members, such as those listed above or those listed in Genbank registry number CAA25015 (Human Myc). Myc polypeptides (e.g., c-Myc) may be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins of the same species as the cell species being manipulated will be used.

[0097] "Sox polypeptide" refers to any naturally occurring member of an SRY-associated HMG-box (Sox) transcription factor, characterized by the presence of a high-mobility group (HMG) domain, or a variant thereof retaining transcription factor activity similar to that of the most closely associated naturally occurring family member (within at least 50%, 80%, or 90% of the activity range), or a polypeptide comprising at least the DNA-binding domain of a naturally occurring family member, and may further comprise a transcription activation domain. See, for example, Dang, DT, et al., Int. J. Biochem. Cell Biol. (International Journal of Biochemistry and Cell Biology) 32:1103-1121 (2000). Exemplary Sox peptides include, for example, Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to produce iPS cells with similar efficiency to Sox2, and the genes Sox3, Sox15, and Sox18 have also been shown to produce iPS cells, although with slightly lower efficiency than Sox2. See, Nakagawa, et al., Nature Biotechnology 26: 101–106 (2007). In some embodiments, the variants share at least 85%, 90%, or 95% amino acid sequence identity across their entire sequence compared to naturally occurring members of the Sox polypeptide family, such as those listed above or those listed in Genbank accession number CAA83435 (human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be derived from humans, mice, rats, cattle, pigs, or other animals. Generally, proteins from the same species as the cell species being operated on will be used.

[0098] “H3K9” refers to histone H3 lysine 9. H3K9 modifications related to gene activity include H3K9 acetylation, and H3K9 modifications related to heterochromatin include H3K9 dimethylation or trimethylation. See, for example, Kubicek, et al., Mol. Cell 473-481 (2007).

[0099] The term "pluripotency" or "pluripotency" refers to cells that have the ability to produce progeny cells that, under suitable conditions, differentiate into cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to all embryo-derived tissues in 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 determine the pluripotency of a cell population; however, the identification of different pluripotent stem cell characteristics can also be used to detect pluripotent cells.

[0100] "Pluripotent stem cell characteristics" refers to the cellular characteristics that distinguish pluripotent stem cells from other cells. The ability to produce offspring capable of differentiating under appropriate conditions into cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. The expression or absence of a combination of certain molecular markers is also a pluripotent stem cell characteristic. For example, human pluripotent stem cells express at least some (and in some embodiments, all) 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.

[0101] As used herein, “non-pluripotent cell” refers to mammalian cells that are not pluripotent. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are 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.

[0102] In some embodiments of treating an individual with the resulting pluripotent cells, according to the method of the invention, pluripotent cells are generated using the individual's own non-pluripotent cells.

[0103] The cells may be derived from, for example, humans or non-human mammals. Exemplary non-human mammals include, but are not limited to: mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates (e.g., chimpanzees, macaques, and apes).

[0104] "Recombinant" polynucleotides are polynucleotides that do not exist in their natural state. For example, the polynucleotides include nucleotide sequences that do not exist in nature, or the polynucleotides are in a context different from their natural context, for example, separated from nucleotide sequences that are typically similar to those in nature, or adjacent to (or connected to) nucleotide sequences that are typically dissimilar to those in nature. For example, the sequence under study can be cloned into a vector, or otherwise recombined with one or more other nucleic acids.

[0105] An "expression cassette" refers to a polynucleotide that includes a promoter or other regulatory sequence that is operatively linked to the sequence encoding a protein.

[0106] The terms “promoter” and “expression control sequence” are used herein to refer to an array of nucleic acid control sequences that direct nucleic acid transcription. When used herein, a promoter includes an essential nucleic acid sequence near the transcription start site, such as the TATA element in the case of a polymerase II promoter. A promoter may also optionally include distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. Promoters include constitutive and inducible promoters. A “constitutive” promoter is a promoter active under most environmental and developmental conditions. An “inducible” promoter is a promoter active under environmental or developmental regulation. The term “operable link” refers to a functional link between a nucleic acid expression control sequence (such as a promoter, or a set of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs the transcription of a nucleic acid corresponding to the second sequence.

[0107] When used in this article, "heterologous sequence" or "heterologous nucleic acid" refers to a sequence or nucleic acid derived from a source heterologous to that of a specific host cell, or, if from the same source, modified from its original form. Therefore, a heterologous expression cassette in a cell is not an endogenous expression cassette of a specific host cell, for example, by being linked to a nucleic acid sequence from an expression vector rather than chromosomal DNA, to a heterologous promoter, or to a reporter gene.

[0108] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their polymers. The term includes nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a similar manner to a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methyl phosphonates, chiral methylphosphonates, 2-O-methylribonucleic acid, and peptide-nucleic acid (PNAs).

[0109] Unless otherwise stated, a particular nucleic acid sequence also includes its conserved modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. In particular, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a mixture of bases 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)).

[0110] The terms "inhibitor," "activator," and "regulator" refer to inhibitory, activating, or regulatory molecules, respectively, identified by in vitro and in vivo assays of the expression or activity of the target protein (or encoding a polynucleotide), such as ligands, agonists, antagonists, and their homologues and mimics. The term "regulator" includes both inhibitors and activators. An inhibitor is, for example, an agent that inhibits the expression or binding of the target protein, partially or completely blocks the activity of stimulators or protease inhibitors, reduces, lowers, prevents, delays activation, inactivates, desensitizes, or downregulates the activity of the target protein, such as an antagonist. An activator is, for example, an agent that induces or activates the expression or binding of the target protein, stimulates, increases, opens, activates, promotes, enhances the activation or protease inhibitor activity, sensitizes, or upregulates the activity of the target protein (or encoding a polynucleotide), such as an agonist. Regulators include naturally occurring and synthetic ligands, antagonists, and agonists (e.g., small chemical molecules that function as agonists or antagonists, antibodies, etc.). The assays concerning inhibitors and activators include, for example, applying a proposed modulatory compound to cells expressing the target protein and then determining a functional effect on the activity of the target protein, as described above. The extent of effect is examined by comparing a sample or assay containing the target protein treated with a potential activator, inhibitor, or modulator with a control sample without an inhibitor, activator, or modulator. The control sample (untreated with a modulator) is assigned a relative activity value of 100%. Inhibition of the target protein is achieved when the activity value relative to the control is about 80%, optionally 50%, or 25%, 10%, 5%, or 1%. Activation of the target protein is achieved when the activity value relative to the control is about 110%, optionally 150%, optionally 200%, 300%, 400%, 500%, or 1000-3000% or higher.

[0111] When chemical substituents are specified using their conventional chemical formulas written from left to right, they also include chemically identical substituents produced when the structure is written from right to left, for example, -CH2O- is equivalent to -OCH2-.

[0112] Unless otherwise stated, the term "alkyl" itself, or as part of another substituent, refers to a straight chain (i.e., unbranched) or branched chain, or a combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include divalent and polyvalent groups, and has a specified number of carbon atoms (i.e., C1-C1). 10 This refers to 1-10 carbon atoms. Examples of saturated hydrocarbon groups include, but are not limited to, the following groups: such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, and homologues and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are alkyl groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to: vinyl, 2-propenyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologues and isomers.

[0113] The term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkyl group, examples of which include, but are not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group has 1-24 carbon atoms; those groups having 10 or fewer carbon atoms are examples in this invention. "Lower alkyl" or "lower alkylene" refers to a shorter-chain alkyl or alkylene group, typically having 8 or fewer carbon atoms.

[0114] Unless otherwise stated, the term "heteroalkyl" on its own or in combination with another term refers to a stable straight-chain, branched, or cyclic hydrocarbon group or a combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, P, S, and Si may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to other parts of the molecule. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms can be adjacent, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" itself, or as part of another substituent, refers to a divalent group derived from a heteroalkyl group, examples of which include, but are not limited to, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatom can also occupy any one or both chain ends (e.g., alkeneoxy, alkenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the direction in which the general formula of the linking group is written does not imply the orientation of the linking group. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As mentioned above, the heteroalkyl groups used herein include those groups that are linked to other parts of the molecule via heteroatoms, such as -C(O)R', -C(O)NR', ​​and -NR'R. '' -OR', -SR ' And / or -SO2R'. The list of "heteroalkyl" is followed by a list of specific heteroalkyl groups (such as -NR'R). '' In cases such as (etc.), it should be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are listed for clarity. Therefore, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R. '' wait.

[0115] Unless otherwise stated, the terms "cycloalkyl" and "heterocycloalkyl" on their own or in combination with other terms represent cyclic forms of "alkyl" and "heteroalkyl," respectively. Additionally, for heterocycloalkyl groups, the heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. Examples of heterocycloalkyl groups include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc. "Cycloalkylene" and "heterocycloalkylene" refer to divalent groups derived from cycloalkyl and heterocycloalkyl groups, respectively.

[0116] Unless otherwise stated, the term "halogenated" or "halogen" itself, or as part of another substituent, refers to a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "halogenated alkyl" are intended to include both monohalogenated and polyhalogenated alkyl groups. For example, the term "halogenated (C1-C4) alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0117] Unless otherwise stated, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1-3 rings) fused together or covalently linked. The term "heteroaryl" refers to an aryl group (or ring) containing 1-4 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Heteroaryl groups can be linked to other parts of the molecule via carbon or heteroatoms. Non-limiting examples of aryl and heteroaryl groups include: phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrole, 2-pyrrole, 3-pyrrole, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- azole group, 4- azole, 2-phenyl-4- azole, 5- azole group, 3-iso azole group, 4-iso azole group, 5-iso Azolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinel, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl, and 6-quinolinyl. Substituents in each of the aryl and heteroaryl ring systems indicated above are selected from the acceptable substituents listed below. "Arylidene" and "heteroarylidene" refer to divalent groups derived from aryl and heteroaryl groups, respectively.

[0118] For the sake of brevity, when used in combination with other terms (e.g., aryloxy, arylthoxy, arylalkyl), the term "aryl" includes the aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include groups in which the aryl group is linked to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), said alkyl group including those in which a carbon atom (e.g., methylene) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0119] The term "oxo" as used in this article refers to oxygen that forms a double bond with a carbon atom.

[0120] As used herein, the term "alkylsulfonyl" refers to a portion having the formula -S(O2)-R', where R' is an alkyl group as defined above. R' may have a specified number of carbons (e.g., "C1-C4 alkylsulfonyl").

[0121] Each of the above terms (e.g., "alkyl", "heteroalkyl", "aryl", and "heteroaryl") is intended to include both substituted and unsubstituted forms of the indicated group. Exemplary substituents for each class of group are provided below.

[0122] Substituents in alkyl and heteroalkyl groups (including those often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, ynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be selected from, but not limited to, one or more of the following groups: -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R"', -OC( R', R"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and -NO2, the number of which is 0 to (2m'+1), where m' is the total number of carbon atoms in such groups. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or arylalkyl. When the compounds of the present invention comprise more than one R group, for example, each R group is independently selected as R', R", R'" and R"" (when more than one such group is present). When R' and R" are attached to the same nitrogen atom, they can combine with said nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups that contain a carbon atom attached to a group other than a hydrogen group, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0123] Similar to the substituents described for alkyl groups, the substituents for aryl and heteroaryl groups are diverse and selected from: for example, halogens, -OR', -NR'R", -SR', -halogens, -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R"', -NR"C(O)R',

[0124] -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'R"R'")=NR"",

[0125] -NR-C(NR'R")=NR'", -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -CN and –NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy and fluoro(C1-C4)alkyl, the number of which ranges from 0 to the total number of open valences on the aromatic ring system; and wherein R', R", R"' and R"" are preferably independently selected from: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When the compounds of the present invention comprise more than one R group, for example, each R group is independently selected as each R', R", R'" and R"" group (when more than one such group is present).

[0126] The two substituents on adjacent atoms of the aryl or heteroaryl ring can optionally form the formula -TC(O)-(CRR'). q A -U- ring, where T and U are independently -NR-, -O-, -CRR'-, or single bonds, and q is an integer from 0 to 3. Alternatively, the two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -A-(CH2). r The substituents of -B- are replaced, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or single bonds, and r is an integer from 1 to 4. One of the single bonds in this new ring can optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced by the formula -(CRR'). s -X'-(C''R''') d - Substituent substitution, wherein s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. Substituents R, R', R" and R'" are preferably independently selected from: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[0127] The term “heteroatom” or “cyclic heteroatom” as used in this article is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0128] As used in this article, "substituent" refers to a group selected from the following:

[0129] (A)-OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0130] (B) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, which are substituted by at least one substituent selected from the following:

[0131] (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0132] (ii) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl groups, which are substituted by at least one substituent selected from the following:

[0133] (a) Oxygenated, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogens, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

[0134] (b) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, which is substituted by at least one of the following substituents: oxo, -OH, -NH2, -SH, -CN, -CF3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl and unsubstituted heteroaryl.

[0135] As used herein, "size-restricted substituent" or "size-restricted substituent group" refers to a group selected from all the substituents described above regarding "substituent," wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C2 group. 20 Alkyl groups, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2-20-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C4-C8 cycloalkyl group, and each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 4-8-membered heterocycloalkyl group.

[0136] As used herein, “lower substituent” or “lower substituent group” refers to a group selected from all the substituents described above regarding “substituent”, wherein each substituted or unsubstituted alkyl group is a substituted or unsubstituted C1-C8 alkyl group, each substituted or unsubstituted heteroalkyl group is a substituted or unsubstituted 2-8-membered heteroalkyl group, each substituted or unsubstituted cycloalkyl group is a substituted or unsubstituted C5-C7 cycloalkyl group, and each substituted or unsubstituted heterocycloalkyl group is a substituted or unsubstituted 5-7-membered heterocycloalkyl group.

[0137] The term "pharmaceutically acceptable salt" is intended to include salts of active compounds prepared with relatively nontoxic acids or bases, depending on the specific substituents present on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting a neutral form of such a compound in a pure compound or in a suitable inert solvent with a sufficient amount of the desired base. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting a neutral form of such a compound in a pure compound or in a suitable inert solvent with a sufficient amount of the desired acid. Examples of pharmaceutically acceptable acid addition salts include: salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrophosphoric acid, dihydrophosphoric acid, sulfuric acid, monohydrosulfuric acid, hydroiodic acid, or phosphorous acid, etc.; and salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, succinic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are: salts of amino acids such as arginine salts; and salts of organic acids (such as glucuronic acid or galacturonic acid), etc. (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain both basic and acidic functional groups, which allow the compounds to be converted into basic or acidic addition salts.

[0138] Therefore, the compounds of the present invention can exist as salts formed with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobroms, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof, including racemic mixtures), succinates, benzoates, and salts formed with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art.

[0139] Preferably, the neutral form of the compound is regenerated as follows: the salt is contacted with a base or acid, and the parent compound is separated in a conventional manner. The parent form of the compound differs from the different salt forms in certain physical properties, such as solubility in polar solvents.

[0140] In addition to the salt form, the present invention also provides compounds in prodrug form. The prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in an in vitro environment. For example, when placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents, prodrugs can be slowly converted into the compounds of the present invention.

[0141] Some compounds of the present invention may exist in both non-solventized and solvated forms (including hydrated forms). Generally, solvated and non-solventized forms are equivalent and both are included within the scope of the present invention. Some compounds of the present invention may exist in various crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended uses of the present invention and are intended to fall within the scope of the present invention.

[0142] Some compounds of this invention have asymmetric carbon atoms (optical centers) or double bonds; within the scope of this invention, they include racemic derivatives, diastereomers, tautomers, geometric isomers, and monomeric derivatives. The compounds of this invention do not include those known in the art that are too unstable to be synthesized and / or isolated.

[0143] The compounds of the present invention may also contain atomic isotopes in non-natural proportions at one or more atoms constituting such compounds. For example, radioactive isotopes (e.g., tritium) can be used. 3 H), Iodine-125 ( 125 I) or carbon-14 ( 14 C) Radiolabeling of the compounds. All isotopic variants (radioactive or non-radioactive) of the compounds of the present invention are included within the scope of the present invention. Attached Figure Description

[0144] Figure 1. Seven days of compound treatment is sufficient to induce pluripotent stem cells from human fibroblasts transduced with four reprogramming factors. (A) Timeline of human iPSC induction using the combination of SB431542 and PD0325901 along with 4TF. Treatment began with cell reseeding on day 7 after 4TF transduction and was maintained for 7 days. (B) ALPs appearing in untreated culture (left) or culture treated with the two compounds (right) within 7 days. +Colony staining. (C) RT-PCR showing elevated endogenous mRNA expression of pluripotency markers OCT4 and NANOG in cultures treated with the two compounds. (D) TRA-1-81 staining on day 14 with or without (left) the two compounds. (E) Plotting of NANOG on day 14 under different treatment conditions. + Number of colonies. (F) Typical staining of hESC-specific markers (NANOG and SSEA4) exhibited by iPSCs on day 14. Scale bar, 50 µm (d and f).

[0145] Figure 2. Prolonged compound treatment and cell passage dramatically increased the number of reprogrammed colonies. (A) Timeline of human iPSC induction using SB431542, PD0325901, and thiazovivin. (B) iPSC expression of pluripotency markers NANOG, SSEA4, and TRA-1-81 on day 30. Scale bar, 50 µm. (C) ALP staining of cultures on day 30 with (top) or without (bottom) the three compound treatments. The boxed area in the left image is magnified in the right image. Scale bar, 200 µm. (D) NANOG on day 30 under different treatment conditions without splitting. + Number of colonies. (E) NANOG of cultures treated with trypsin digestion of three compounds as directed on day 30. + Number of colonies. (F) RT-PCR of iPSC colonies treated with the three compounds showed reactivated expression of endogenous pluripotency markers. HDF: Human dermal fibroblasts.

[0146] Figure 3. In vitro and in vivo differentiation of iPSCs generated by treatment with three compounds. (A) Photomicrographs showing embryoids (EB) generated from iPSCs and cell types differentiating in vitro into ectoderm (βIII tubulin), mesoderm (BRACHYURY), and endoderm (PDX1). Scale bar, EB: 100 µm; others 10 µm. (B) RT-PCR showing the expression of representative lineage markers and the absence of OCT4 mRNA expression in differentiated cells. U - undifferentiated, D - differentiated. (C) Teratomas generated from iPSCs in nude mice (testing 3 independent colonies) consist of tissues from all 3 germ layers. Left: 1- muscle, 2- neuroepithelium; Middle: 1- skin, 2- intestinal epithelium; Right: 1- bone, 2- cartilage. Scale bar, 20 µm.

[0147] Figure 4. Compound treatment enhances iPS cell production in a dose-dependent manner.

[0148] Figure 5 Stable, expanding iPS cell colonies generated by compound treatment exhibited a normal karyotype.

[0149] Figure 6. Transgenic expression and silencing are independent of compound treatment.

[0150] Figure 7 The chemical structure of thiazovivin.

[0151] Figure 8. Human induced pluripotent stem cell (iPSC) generation from primary keratinocytes via single gene, OCT4, and small molecules. (A) Treatment with 0.5 μM PD0325901 (PD) and 0.5 μM A-83-01 (A83) significantly improved iPSC generation from primary human keratinocytes transduced with 4 TF (4F, OKSM) or 3 TF (3F, OKS). NHEK cells were seeded at a density of 100,000 transduced cells / 10 cm dish. (B) Further chemical screening identified PS48, NaB, and combinations thereof, which substantially enhanced the reprogramming of primary human keratinocytes transduced with 2 TF (OK). NHEK cells were seeded at a density of 100,000 transduced cells / 10 cm dish. (C) Experimental protocol for generating human iPSCs from primary human keratinocytes transduced with a single reprogramming gene, OCT4. KCM, keratinocyte culture medium; hESCM, human ESC culture medium. (D) TRA-1-81 live immunostaining of iPSC colonies generated from primary human keratinocytes transduced with 2TF / OK or 1TF / OCT4 prior to colony picking. (E) Established human iPSC-OK and iPSC-O cells expressing typical pluripotency markers, including ALP (alkaline phosphatase), OCT4, SOX2, NANOG, SSEA-4, and TRA-1-81. Nuclear staining was performed using DAPI.

[0152] Figure 9. In-depth characterization of human iPSC-OK and iPSC-O cells. (A) RT-PCR expression analysis of endogenous pluripotency genes and exogenous OCT4 and KLF4. GAPDH was used as an input control. (B) Methylation analysis of the OCT4 and NANOG promoters by bisulfate genome sequencing. Hollow and solid circles indicate unmethylated and methylated CpG in the promoter regions, respectively. (C) Scatter plot comparing the total gene expression patterns between iPSC-O cells and NHEK and hESC. Arrows indicate the locations of the pluripotency genes OCT4, NANOG, and SOX2. Black lines indicate linear equivalence and fold change in gene expression levels between samples. (D) Using the EB method, human iPSC-OK and iPSC-O cells can be efficiently differentiated into cells in vitro in three germ layers: neuroectodermal cells (βIII tubulin), mesodermal cells (SMA), and endoderm cells (AFP). (E) Quantitative PCR detection of three germ layer markers from differentiated human iPSCs using the EB method: ectoderm (PAX6, βIII tubulin), mesoderm (FOXF1, HAND1), and endoderm (AFP, GATA6). Data indicate fold change in GAPDH-normalized values ​​compared to undifferentiated parental human iPSCs. (F) Human iPSC-OK and iPSC-O efficiently generate complete teratomas containing differentiated cells from all three germ layers in SCID mice.

[0153] Figure 10. Generation and characterization of human induced pluripotent stem cells (iPSCs) from human umbilical vein endothelial cells using single genes, OCT4, and small molecules. (A) Experimental protocol for generating human iPSCs from OCT4-transduced HUVECs. HCM, HUVEC medium; hESCM, human ESC medium. (B) hiPSC-O cells generated from HUVECs expressing typical pluripotency markers, including NANOG and SSEA-4. Nuclear staining with DAPI. (C) Expression analysis by RT-PCR of endogenous pluripotency genes. GAPDH was used as an input control. (D) Methylation analysis of the OCT4 and NANOG promoters by bisulfate genome sequencing. Hollow and solid circles indicate unmethylated and methylated CpG in the promoter regions, respectively. (E) hiPSC-O cells from HUVECs can efficiently differentiate into cells in vitro in three germ layers using the EB method: neuroectodermal cells (βIII tubulin), mesodermal cells (SMA), and endoderm cells (AFP). (F) hiPSC-O cells can effectively generate complete teratomas containing differentiated cells from three germ layers in SCID mice.

[0154] Figure 11. Characterization of human iPSC-O cells from AHEK. (A) HiPSC-O cells derived from adult keratinocytes express typical pluripotency markers, including NANOG, SOX2, and SSEA-4. Nuclear staining was performed using DAPI. (B) Using the EB method, these hiPSC-O cells can be efficiently differentiated in vitro into cells from three germ layers: neuroectodermal cells (βIII tubulin), mesodermal cells (SMA), and endoderm cells (AFP).

[0155] Figure 12. Characterization of human iPSC-O cells from AFDC. (A) HiPSC-O cells derived from amniotic fluid expressed typical pluripotency markers, including NANOG, SOX2, and SSEA-4. Nuclear staining was performed using DAPI. (B) Using the EB method, these hiPSC-O cells could be efficiently differentiated in vitro into cells from three germ layers: neuroectodermal cells (βIII tubulin), mesodermal cells (SMA), and endoderm cells (AFP).

[0156] Figure 13 Other hiPSC cell lines express typical pluripotency markers. Other established hiPSC-O cell lines express typical pluripotency markers, including NANOG and SSEA-4. Nuclear staining was performed using DAPI.

[0157] Figure 14 Feeder-free cultures of hiPSC cell lines. HiPSCs were aliquoted into previously reported hESC medium with known chemical composition on Matrigel / ECM-coated plates. These hiPSCs could be maintained and expanded in a feeder-free environment. ICC was used to demonstrate the expression of the pluripotency markers OCT4 and SSEA4. Nuclear staining was performed using DAPI.

[0158] Figure 15. Genotyping of hiPSCs. RT-PCR analysis using genomic DNA showed that only the OCT4 transgene was integrated into the genome of the hiPSC-O lines (hiPSC-O#1, hiPSC-O#3, hiPSC-O#21, hiPSC-O#26, and hiPSC-O#31). NHEK (a) and HUVEC (b) were used as negative controls, while the vector was used as a positive control.

[0159] Figure 16 Integration of the OCT4 transgene into hiPSCs. Genomic DNA (10 μg) was digested with EcoRI and hybridized with the OCT4 cDNA probe (EcoRI / SpeI fragment of pSin-EF2-OCT4-Pur). Multiple transgene integrations were detected.

[0160] Figure 17. Karyotype analysis of hiPSC cell lines. Metaphase spreads of hiPSC-O#1 (a) and hiPSC-O#21 (b) show normal karyotypes after the 15th generation. Detailed Implementation

[0161] I. Introduction

[0162] This invention is based on the surprising discovery that a combination of ALK5 inhibitors, MEK inhibitors, and ROCK inhibitors significantly improves the efficiency of pluripotency induction in non-pluripotent mammalian cells transformed with these four transcription factors. Therefore, this invention provides a method for inducing pluripotency in non-pluripotent mammalian cells, wherein the method comprises: exposing the non-pluripotent cells to at least a TGFβ receptor / ALK5 inhibitor, preferably in combination with a MEK / ERK pathway inhibitor, and in a specific embodiment, in combination with a Rho GTPase / ROCK inhibitor.

[0163] II. TGFβ receptor / ALK5 inhibitors

[0164] Activin receptor-like kinase 5 (ALK-5) is the major TGFβ receptor mediating the cellular response to TGF-β (Massague J. Annu Rev Biochem 67:753–791 (1998); Massague J, Chen YG. GenesDev 14:627–644 (2000); Franzen P, et al. Cell 75:681–692 (1993)). Upon ligand binding, constitutively active TβRII kinases phosphorylate ALK-5, which in turn activates downstream signal transduction cascades. ALK-5-activated Smad2 and Smad3 phosphorylation is the most prominent pathway (Massague J, Chen YG. Genes Dev 14:627-644 (2000)). Once activated, Smad2 / 3 bind to Smad4 and translocate to the nucleus, where the complex transcribes and regulates the expression of target genes.

[0165] TGFβ receptor (i.e., ALK5) inhibitors may include antibodies against TGFβ receptor (e.g., ALK5), dominant inactivating mutants of TGFβ receptor (e.g., ALK5), and siRNA, microRNA, antisense nucleic acid, and other polynucleotides that inhibit the expression of TGFβ receptor (e.g., ALK5). Exemplary TGFβ receptor / ALK5 inhibitors include, but are not limited to: SB431542 (see, for example, 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-thioamide (see, for example, Tojo, et al., Cancer Science 96(11):791-800 (2005), and are commercially available, for example, from Toicris Bioscience);2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-diazanaphthalene, Wnt3a / BIO (see, e.g., Dalton, et al., WO2008 / 094597, incorporated herein by reference), BMP4 (see, Dalton, ibid.), 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-(quinoxolin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, for example, Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, for example, de Gouville, et al., DrugNews Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]m-dioxacyclopenten-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, for example, DaCosta, et al., Molecular) Pharmacology (Molecular Pharmacology) 65(3):744-752 (2004)) and pyrimidine derivatives (see, for example, those listed in Stiefl, et al., WO2008 / 006583, which are incorporated herein by reference). Furthermore, while “ALK5 inhibitor” is not intended to include nonspecific kinase inhibitors, “ALK5 inhibitor” should be understood to include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as, for example, SB-431542 (see, for example, Inman, et al., J Mol. Phamacol. (Molecular Pharmacology) 62(1): 65-74 (2002).

[0166] Given the data shown in this document regarding the effect of inhibiting ALK5, it is believed that inhibition of the TGFβ / activin pathway will have a similar effect. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or as an alternative to the ALK5 inhibitor described in each paragraph herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to: TGFβ receptor inhibitors, SMAD 2 / 3 phosphorylation inhibitors, SMAD 2 / 3 and SMAD 4 interaction inhibitors, and activators / agonists of SMAD 6 and SMAD 7. Furthermore, the classifications described below are for organizational purposes only, and those skilled in the art will recognize that compounds can affect one or more points in the pathway, and therefore compounds can function in more than one defined classification.

[0167] TGFβ receptor inhibitors can include antibodies against the TGFβ receptor, dominant inactivating mutants of the TGFβ receptor, and siRNAs or antisense nucleic acids that target the TGFβ receptor. Specific examples of inhibitors include, but are not limited to: SU5416; 2-(5-benzo[1,3]dioxane-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; LY2109761; 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 antisense transfected tumor cells targeting the TGFβ receptor. (See, for example, Wrzesinski, et al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiers in Bioscience 12:4393-4401 (2007)). Furthermore, the inventors have discovered that TGFβ inhibitors BMP-4 and BMP-7 have similar cellular reprogramming effects to the ALK5 inhibitors described in the examples, thus providing further evidence that TGFβ inhibitors can be used for reprogramming (e.g., in combination with MEK / ERK pathway inhibitors and Rho GTPase / ROCK inhibitors). Exemplary human BMP-4 and BMP-7 protein sequences are described, for example, in U.S. Patent No. 7,405,192.

[0168] Inhibitors of SMAD 2 / 3 phosphorylation can include antibodies against SMAD2 or SMAD3, dominant-inactivating mutants 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, ibid.; Kaminska, ibid.; Shimanuki, et al., Oncogene 26:3311-3320 (2007); and Kataoka, et al., EP1992360, which are incorporated herein by reference.)

[0169] Inhibitors of the interaction between SMAD 2 / 3 and smad4 may include antibodies against SMAD2, SMAD3, and / or smad4, dominant inactivating mutants of SMAD2, SMAD3, and / or smad4, and antisense nucleic acids targeting SMAD2, SMAD3, and / or smad4. Specific examples of inhibitors of the interaction between SMAD 2 / 3 and SMAD4 include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1 (see, for example, Cui, et al., Oncogene 24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell, 17:3819-3831 (2006)).

[0170] Activators / agonists of SMAD 6 and SMAD 7 include, but are not limited to, antibodies against SMAD 6 or SMAD 7, dominant-inactivating mutants 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, for example, Miyazono, et al., US6534476, and Steinbrecher, et al., US2005119203, both of which are incorporated herein by reference.

[0171] Technicians will understand that the concentration of TGFβ receptor / ALK5 inhibitors will depend on the specific inhibitor used. Typically, the concentration of TGFβ receptor / ALK5 inhibitors in cell cultures is in the IC20-IC100 range (i.e., the concentration required to achieve 20% to 100% inhibition of cells). For example, SB432542 can be used at 0.5-10 μM, optimally at about 1-5 μM. In some embodiments, a combination of two or more different TGFβ receptor / ALK5 inhibitors may be used.

[0172] III. MEK / ERK pathway inhibitors

[0173] The MEK / ERK pathway refers to the MEK and ERK serine / threonine kinases that constitute part of the signal transduction pathway. Normally, activated Ras activates the protein kinase activity of RAF kinase. RAF kinase phosphorylates and activates MEK, which in turn phosphorylates and activates mitogen-activated protein kinase (MAPK). MAPK was originally called "extracellular signal-regulated kinase" (ERK) and microtubule-associated protein kinase (MAPK). Therefore, "ERK" and "MAPK" are used synonymously.

[0174] MEK / ERK pathway inhibitors refer to inhibitors of MEK or ERK that are part of the Raf / MEK / ERK pathway. Because the inventors have found that MEK inhibitors can effectively improve iPSC induction, and because MEK directly controls ERK activity, it is believed that the MEK inhibitors described herein can be replaced by ERK inhibitors as needed.

[0175] Inhibitors of MEK (i.e., MEK1 (also known as mitogen-activated protein kinase kinase 1) and / or MEK2 (also known as mitogen-activated protein kinase kinase 2)) may include antibodies against MEK, dominant-inactivating mutants of MEK, and siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides that inhibit MEK expression. Specific examples of MEK inhibitors include, but are not limited to: PD0325901 (see, e.g., Rinehart, et al., Journal of Clinical Oncology 22: 4456-4462 (2004)), PD98059 (e.g., available from Cell Signaling Technology), U0126 (e.g., available from Cell Signaling Technology), SL 327 (e.g., available from Sigma-Aldrich), ARRY-162 (e.g., available 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 Medicine). Therapeutics (Journal of Pharmacology and Experimental Therapeutics) 316:456–465 (2006)), sunitinib (see, for example, Voss, et al., US2008004287, which is incorporated herein by reference), sorafenib (see, Voss ibid.), vandetanib (see, Voss ibid.), pazopanib (see, for example, Voss ibid.), axitinib (see, Voss ibid.), and PTK787 (see, Voss ibid.).

[0176] Currently, several MEK inhibitors are undergoing clinical trials for evaluation. CI-1040 has been evaluated in phase I and phase II cancer clinical trials (see, for example, Rinehart, et al., Journal of Clinical Oncology 22(22):4456-4462 (2004)). Other MEK inhibitors evaluated in clinical trials include PD184352 (see, for example, English, et al., Trends in Pharmaceutical Sciences 23(1):40-45 (2002)), BAY 43-9006 (see, for example, Chow, et al., Cytometry (Communications in Clinical Cytometry) 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, for example, information from the National Institutes of Health at www.clinicaltrials.gov and information from the National Cancer Institute at www.cancer.gov / clinicaltrials.)

[0177] Exemplary ERK (i.e., ERK1 (also known as MAPK3) and / or ERK2 (also known as MAPK1)) inhibitors include PD98059 (see, for example, Zhu, et al., Oncogene 23:4984-4992 (2004)), U0126 (see, Zhu, as previously stated), FR180204 (see, for example, Ohori, Drug News Perspective 21(5):245-250 (2008)), sunitinib (see, for example, US2008004287, incorporated herein by reference), sorafenib, vandetanib, pazopanib, axitinib, and PTK787.

[0178] Technicians will understand that the concentration of MEK / ERK pathway inhibitors will depend on the specific inhibitor used. In specific implementation schemes, combinations of two or more different MEK / ERK pathway inhibitors can be used.

[0179] IV. Rho GTPase / ROCK Inhibitor

[0180] This invention provides the use of inhibitors of the Rho-GTPase / ROCK pathway and compositions comprising said inhibitors. The pathway includes the downstream protein myosin II, which is further downstream of ROCK (Rho-ROCK-myosin II forms this pathway / axis). Therefore, the effects described herein can be achieved using any or all of a Rho GTPase inhibitor, a ROCK inhibitor, or a myosin II inhibitor. Those skilled in the art will understand that the concentration of the Rho-GTPase / ROCK pathway inhibitor will depend on which specific inhibitor is used. In other embodiments, a combination of two or more different Rho-GTPase / ROCK pathway inhibitors can be used.

[0181] Any Rho GTPase will be effective in the methods and compositions of the present invention. Inhibitors of Rho GTPases may include antibodies that bind to Rho GTPases, dominant-inactivating mutants of Rho GTPases, and siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides that target Rho GTPases. An exemplary Rho GTPase inhibitor is Clostridium botulinum C3 toxin.

[0182] Any myosin II inhibitor will be effective in the methods and compositions of the present invention. Inhibitors of myosin II may include antibodies that bind to myosin II, dominant-inactivating mutants of myosin II, and siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides that target myosin II. An exemplary myosin II inhibitor is blebbistatin. The inventors have found that blebbistatin can replace SB431542 (an ALK5 inhibitor) in the mixtures and methods described in the Examples section, although with reduced efficacy. Other inhibitors include, but are not limited to, those described in U.S. Patent No. 7,585,844.

[0183] In this article, “ROCK” refers to a serine / threonine kinase that acts downstream of Rho. Both ROCK I (also known as ROKβ or p160ROCK) and ROCK II (also known as ROKα or Rho kinase) are regulated by RhoA. See, for example, Riento, K. and Ridley, AJ, Nat. Rev. Mol. Cell. Biol., 4, 446-456 (2003). “ROCK inhibitor” refers to an agent that inhibits two or more types of ROCK. ROCK inhibitors can include antibodies that bind to ROCK, dominant-inactivating mutants of ROCK, and siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides that target ROCK. Some exemplary ROCK inhibitors include, but are not limited to, those described in the following publications: International Publication Nos. WO98 / 06433, WO00 / 78351, WO01 / 17562, WO02 / 076976, WO02 / 076977, WO2003 / 062227, WO2003 / 059913, WO2003 / 062225, WO2002 / 076976 WO2004 / 039796, WO03 / 082808, WO05 / 035506, WO05 / 074643 and U.S. Patent Application Nos. 2005 / 0209261, 2005 / 0192304, 2004 / 0014755, 2004 / 0002508, 2004 / 0002507, 2003 / 0125344 and 2003 / 0087919. ROCK inhibitors include, for example, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride or Wf536; 4-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzamide monohydrochloride or fasudil; 5-(hexahydro-1H-1,4-diaza... -1-ylsulfonyl)isoquinoline hydrochloride or compound 1; 4-[(trans-4-aminocyclohexyl)amino]-2,5-difluorobenzamide or compound 2; 4-[(trans-4-aminocyclohexyl)amino]-5-chloro-2-fluorobenzamide or compound 3; 2-[4-(1H-indazol-5-yl)phenyl]-2-propylamine dihydrochloride or compound 4; N-(3-methoxybenzyl)-4-(4-pyridyl)benzamide, Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil (also known as HA1077) (see, for example, Uenata et al., Nature 389: 990-994 (1997)), sc-3536 (see, for example, Darenfed, H., et al. Cell Motil. Cytoskeleton. 64: 97-109, 2007), H-1152 (see, for example, Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (see, for example, Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), Y-30141 (described in U.S. Patent No. 5,478,838) and their derivatives, as well as antisense nucleic acids of ROCK, nucleic acids that induce RNA interference (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, their dominant inactivating mutants and expression vectors.

[0184] As needed, the above compounds can be formulated into acid addition salts using pharmaceutically acceptable inorganic or organic acids. Examples of acid addition salts include: salts containing inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.); salts containing organic carboxylic acids (such as formic acid, acetic acid, fumaric acid, maleic acid, oxalic acid, citric acid, malic acid, tartaric acid, aspartic acid, and glutamic acid); and salts containing sulfonic acids (such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydroxybenzenesulfonic acid, dihydroxybenzenesulfonic acid, etc.).

[0185] The compound and its acid addition salt can be its acid anhydride, hydrate or solvate.

[0186] For the purpose of practicing this invention, ROCK inhibitors are generally suitable, but not limited to, as long as the inhibitor can inhibit the function of Rho-kinase (ROCK). Suitable inhibitors include Y-27632 (e.g., see Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), sc-3536 (see, e.g., Darenfed, H., et al. Cell Motil. Cytoskeleton. 64: 97-109, 2007), fasudil (also known as HA1077) (e.g., see Uenata et al., Nature 389: 990-994 (1997)), H-1152 (e.g., see Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), and Wf-536. (For example, see Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)), Y-30141 (described in U.S. Patent No. 5,478,838) and their derivatives and ROCK antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, their dominant inactivating mutants and expression vectors.) Furthermore, since other low-molecular-weight compounds are known to be ROCK inhibitors, such compounds or their derivatives can also be used in this invention (e.g., see U.S. Patent Applications Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, and 20030087919 and International Patent Publications Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). In this invention, combinations of one, two, or more ROCK inhibitors can also be used.

[0187] Other ROCK inhibitors include, for example, HA1100, 3-(4-pyridyl)-1H-indole and N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, each of which is commercially available (e.g. from Alexis Biochemicals (PlymouthMeeting, PA)).

[0188] In some implementations, ROCK inhibitors have the following formula:

[0189] (I).

[0190] In formula (I), ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. Ring B is a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

[0191] L 1 It is -C(O)-NR 2 -or-NR 2 -C(O)-。 L 2 It is a bonded, substituted or unsubstituted alkylene, or a substituted or unsubstituted heteroalkylene.

[0192] R 1 and R 2 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0193] In some embodiments, ring A is a substituted or unsubstituted aryl group. Ring A may also be a substituted or unsubstituted phenyl group.

[0194] In other embodiments, ring B is a substituted or unsubstituted heterocyclic alkyl group, or a substituted or unsubstituted heteroaryl group. Ring B may also be a substituted or unsubstituted heteroaryl group. In other embodiments, ring B is a substituted or unsubstituted pyrazolyl group, a substituted or unsubstituted furanyl group, a substituted or unsubstituted imidazolyl group, or a substituted or unsubstituted isopropyl group. Azolium group, substituted or unsubstituted Diazole group, substituted or unsubstituted Azolyl, substituted or unsubstituted pyrroloyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted thiophene, substituted or unsubstituted dihydrothiophene-pyrazolyl, substituted or unsubstituted thioindyl, substituted or unsubstituted carbazole, substituted or unsubstituted benzothiophene, substituted or unsubstituted benzofuranyl, substituted or unsubstituted indole, substituted or unsubstituted quinolinyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted benzo[] Azolium, substituted or unsubstituted benzimidazolium, substituted or unsubstituted isoquinolinium, substituted or unsubstituted isoydinol, substituted or unsubstituted acridineyl, substituted or unsubstituted benzimidazolyl benzoisazolyl, or substituted or unsubstituted dimethylhydantoin.

[0195] L 2 It can be substituted or unsubstituted C1-C 10 Alkyl group. In some embodiments, L 2 It is unsubstituted C1-C 10 Alkyl group. L 2 It can also be a substituted or unsubstituted methylene group (e.g., an unsubstituted methylene group).

[0196] R 2 It could be hydrogen. R 1 It can be hydrogen or unsubstituted C1-C 10 Alkyl group. In some embodiments, R 1 Simply put, it's hydrogen.

[0197] In some embodiments of formula (I), ring A is a substituted or unsubstituted aryl group, ring B is a substituted or unsubstituted heteroaryl group, and R... 1 It is hydrogen, and L 2 It is unsubstituted C1-C 10 alkyl.

[0198] In another embodiment, the ROCK inhibitor has the following formula:

[0199] (II).

[0200] In equation (II), y is an integer from 0 to 3, and z is an integer from 0 to 5. X is -N=, -CH=, or -CR. 4 =. R 1 and L 2 As defined above in the definition of equation (I).

[0201] R 3 R 4 and R 5 Independently, they are -CN and -S(O). n R 6 -NR 7 R 8 -C(O)R 9 -NR 10 -C(O)R 11 -NR 12 -C(O)-OR 13 -C(O)NR 14 R 15 -NR 16 S(O)2R 17 -OR 18 -S(O)2NR19 substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2, and where if z is greater than 1, then there are 2 R 3 Selectively linked together to form substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0202] R 6 R 7 R 8 R 9 R 10 R 11 R 12 R 13 R 14 R 15 R 16 R 17 R 18 and R 19 Independently, it is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0203] In some implementations, L 2 Is it substituted or unsubstituted C1-C? 10 Alkyl group. L 2 It can also be unsubstituted C1-C 10 Alkyl. Or, L 2 It is a substituted or unsubstituted methylene group (e.g., an unsubstituted methylene group).

[0204] In other implementations, X is -N= or -CH=. The symbol z can be 2. In other implementations, the two R's at adjacent vertices... 3 Partially linked together to form substituted or unsubstituted heterocyclic alkyl groups. The symbol z can also be 1. The symbol y can be 0 or 1.

[0205] R 3 It can be -OR 18 R 18 It can be hydrogen or unsubstituted C1-C 10 alkyl.

[0206] In some implementations, L 2 It is a substituted or unsubstituted methylene group (e.g., a substituted methylene group), X is -N= or -CH=, R 1 It is hydrogen, and y and z are 0.

[0207] In other embodiments, the compound has the following formula:

[0208] ,

[0209] ,

[0210] (Sometimes called Thiazovivin or "Tzv")

[0211] ,or

[0212] .

[0213] V.GSK3 inhibitors

[0214] In various embodiments, the methods, mixtures, and kits of the present invention may include one or more GSK3 inhibitors. The inventors have discovered that GSK3 inhibitors, preferably in combination with MEK / ERK pathway inhibitors, and in specific embodiments with Rho GTPase / ROCK inhibitors, may be included. GSK3 inhibitors may include antibodies that bind to GSK3, dominant-inactivating mutants of GSK3, and GSK3-targeting siRNAs, microRNAs, antisense nucleic acids, and other polynucleotides. Specific examples of GSK3 inhibitors include, but are not limited to: Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418 (see, for example, Gould, et al., The International Journal of Neuropsychopharmacology 7:387-390 (2004)), CT 99021 (see, for example, Wagman, Current Pharmaceutical Design 10: 1105-1137 (2004)), CT 20026 (see, Wagman, ibid.), SB216763 (see, for example, Martin, et al., Nature Immunology 6:777-784 (2005)), AR-A014418 (see, for example, Noble, et al., PNAS 102:6990-6995). (2005)), lithium (see, for example, Gould, et al., Pharmacological Research 48: 49-53 (2003)), SB 415286 (see, for example, Frame, et al., Biochemical Journal 359:1-16 (2001)) and TDZD-8 (see, for example, Chin, et al., Molecular Brain Research, 137(1-2): 193-201 (2005)).Other exemplary GSK3 inhibitors available from Calbiochem (see, for example, Dalton, et al., WO2008 / 094597, which is incorporated herein by reference) include, but are not limited to: BIO(2'Z,3'E)-6-bromoindorubin-3'-oxime (GSK3 inhibitor IX); BIO-acetone oxime(2'Z,3'E)-6-bromoindorubin-3'-acetone oxime (GSK3 inhibitor X); (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazoline-4-yl)amine (GSK3 inhibitor XIII); pyridocarbazole-cyclopentadiene ruthenium complex (GSK3 inhibitor XV); TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazole-3,5-dione (GSK3β inhibitor I); 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-. Diazole (GSK 3β inhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK 3β inhibitor III); α-4-dibromoacetylphenyl (GSK 3β inhibitor VII); AR-AO 14418 N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazo-2-yl)urea (GSK 3β inhibitor VIII); 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrolo-2,5-dione (GSK 3β inhibitor XI); TWSl 19 Pyrrolopyrimidine compound (GSK 3β inhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its myristylated form (GSK 3β inhibitor XIII); 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-acetone (GSK3β inhibitor VI); AR-AO144-18; SB216763; and SB415286. GSK3β residues interacting with the inhibitors have been identified. See, for example, Bertrand et al., J. Mol Biol. (Journal of Molecular Biology) 333(2): 393-407 (2003). GSK3 inhibitors can activate, for example, the Wnt / β-catenin pathway. Many downstream β-catenin genes co-regulate pluripotency gene networks. For example, GSK inhibitors activate cMyc expression, as well as enhance its protein stability and transcriptional activity. Thus, in some embodiments, GSK3 inhibitors can be used to stimulate the expression of endogenous MYC peptides in cells, thereby eliminating the need for MYC expression to induce pluripotency.

[0215] Technicians will understand that the concentration of the GSK3 inhibitor will depend on which specific inhibitor is used. In some implementations, a combination of two or more different GSK3 inhibitors may be used.

[0216] VI. Methods for Inducing Pluripotency

[0217] To date, numerous different methods and protocols have been established for inducing non-pluripotent mammalian cells into induced pluripotent stem cells (iPSCs). It is believed that the reagents described herein can be combined with virtually any protocol for generating iPSCs, thereby improving the efficiency of said protocols. Therefore, this invention provides incubation of non-pluripotent cells with at least a TGFβ receptor / ALK5 inhibitor, preferably in combination with a MEK / ERK pathway inhibitor, and in a specific embodiment, a RhoGTPase / ROCK inhibitor in combination with any protocol for generating iPSCs. The choice of protocol is described below, and it is believed that each protocol can be combined with the reagents of this invention to improve the efficiency of said protocol.

[0218] The improvement in efficiency of the iPSC production protocol will depend on the protocol and the reagents of the present invention used. In some embodiments, the efficiency is improved by at least 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the same protocol without the reagents of the present invention (i.e., TGFβ receptor / ALK5 inhibitors, MEK / ERK pathway inhibitors, and Rho GTPase / ROCK inhibitors). Efficiency is measured in relation to the improvement in the number of iPSCs produced or the rate of iPSC production within a specific time frame.

[0219] Studies have confirmed that retroviral transduction of mouse fibroblasts with four transcription factors (Oct-3 / 4, Sox2, KLF4, and c-Myc) that are highly expressed in ESCs produces induced pluripotent stem (iPS) cells. See Takahashi, K. & Yamanaka, S. Cell 126, 663-676 (2006); Okita, K., Ichisaka, T. & Yamanaka, S. Nature 448, 313-317 (2007); Wernig, M. et al. Nature 448, 318-324 (2007); Maherali, N. et al. Cell Stem Cell 1, 55-70 (2007); Meissner, A., Wernig, M. & Jaenisch, R. Nature Biotechnol. 25, 1177-1181 (2007); Takahashi, K. et al. Cell 131, 861-872 (2007); Yu, J. et al. Science 318, 1917-1920 (2007); Nakagawa, M. et al. Nature Biotechnol. 26, 101-106 (2007); Wernig, M., Meissner, A., Cassady, JP & Jaenisch, R. Cell Stem Cell. 2, 10-12 (2008). Based on teratoma formation and chimeric contribution, iPS cells are similar to ESCs in morphology, proliferation, and pluripotency.

[0220] As noted above, although the initial protocols involved introducing four transcription factors into non-pluripotent cells, it has been more recently discovered that some transcription factors can be omitted. Therefore, in some embodiments, the protocol involves introducing one, two, or three of the following peptides—Oct, Klf, Myc, and Sox—into non-pluripotent cells, under conditions that allow the non-pluripotent cells to become iPSCs. For example, Maherali and Konrad Hochedlinger, “Tgfβ Signal Inhibition Cooperates in the Induction of iPSCs and Replaces Sox2 and cMyc” Current Biology (2009) and WO / 2009 / 117439 each describe protocols that do not require all four transcription factors to induce pluripotency. Furthermore, the inventors have discovered that iPSCs can be generated by introducing Oct4 alone into cells and incubating the cells with a TGFβ receptor / ALK5 inhibitor, a MEK / ERK pathway inhibitor, a Rho GTPase / ROCK inhibitor, and a histone deacetylase (HDAC) inhibitor. For example, exogenous Oct4 was successfully introduced into mammalian cells in the presence of sufficient amounts of SB431542, PD0325901, Tzv and valproic acid or sodium butyrate, resulting in the generation of iPSC cells.

[0221] Exemplary HDAC inhibitors may include antibodies that bind to one or more HDACs, dominant-inactivating mutants of one or more HDACs, and siRNAs and antisense nucleic acids that target one or more HDACs. HDAC inhibitors include, but are not limited to: TSA (trichostatin A) (see, for example, Adcock, British Journal of Pharmacology 150:829-831 (2007)), VPA (valproic acid) (see, for example, Munster et al., Journal of Clinical Oncology 25: 18S (2007):1065), sodium butyrate (NaBu) (see, for example, Han et al., Immunology Letters 108:143-150 (2007)), SAHA (saturated aniline hydroxamic acid or vorinostat) (see, for example, Kelly et al., Nature Clinical Practice Oncology 2:150-157 (2005)), sodium phenylbutyrate (see, for example, Gore et al., Cancer) Research (Cancer Research) 66:6361-6369 (2006)), depsipeptide (FR901228, FK228) (see, for example, Zhu, et al., Current Medicinal Chemistry 3(3):187-199 (2003)), trapoxin (TPX) (see, for example, Furumai, et al., PNAS 98(1):87-92 (2001)), cyclic hydroxamic acid-containing peptide 1 (CHAP1) (see, Furumai, ibid.), MS-275 (see, for example, Carninci, et al., WO2008 / 126932, incorporated herein by reference)), LBH589 (see, for example, Goh, et al., WO2008 / 108741, incorporated herein by reference) and PXD101 (see, Goh, ibid.). Generally, at the whole-cell level, pluripotent cells exhibit more histone acetylation, while differentiated cells exhibit less. Histone acetylation is also involved in the regulation of histone and DNA methylation. In some implementations, HDAC inhibitors promote the activation of silenced pluripotency genes.

[0222] To address the safety concerns arising from target cell genomes carrying integrated foreign sequences, numerous improved genetic protocols have been developed. These protocols generate iPS cells with potentially reduced risk and include non-integrating adenoviruses for delivering reprogrammed genes (Stadtfeld, M., et al. (2008) Science 322, 945-949), transient transfection of reprogrammed plasmids (Okita, K., et al. (2008) Science 322, 949-953), the piggyBac transposon system (Woltjen, K., et al. (2009) Nature 458, 766-770, Kaji, K., et al. (2009) Nature 458, 771-775), Cre-cleavable viruses (Soldner, F., et al. (2009) Cell 136, 964-977), and oriP / EBNA1-based addotype expression systems (Yu, J., et al. (2009) Science DOI: 10.1126). Furthermore, strategies utilizing endogenous gene expression in certain cell types also allow for easier reprogramming and / or less need for exogenous genes (Shi, Y., et al. (2008b). Cell Stem Cell 2, 525-528; Aasen, T., et al. (2008) NatBiotechnol 26, 1276-1284; Kim, JB, et al. (2008). Nature 454, 646-650). In addition, small molecules that enhance reprogramming efficiency and replace certain reprogramming factors have been identified (Shi, Y., et al. (2008) CellStem Cell 2, 525-528; Shi, Y., et al. (2008) Cell Stem Cell 3, 568-574; Li, W., et al. (2009) Cell Stem Cell 4, 16-19; Huangfu, D., et al. (2008) Nat Biotechnol 26, 1269-1275; Huangfu, D., et al. (2008) Nat Biotechnol 26, 795-797).

[0223] Furthermore, it has recently been demonstrated that transcription factors can be delivered as exogenous proteins to non-pluripotent cells to generate iPSCs. See, for example, WO / 2009 / 117439; Zhou et al., Cell Stem Cell 4:381-384 (2009). Exogenous peptides (i.e., proteins provided from outside the cell and / or not produced by the cell) can be introduced into cells via a variety of different methods that do not involve the introduction of polynucleotides encoding peptides. Thus, in some embodiments, non-pluripotent cells are exposed to a TGFβ receptor / ALK5 inhibitor, preferably in combination with a MEK / ERK pathway inhibitor, and in specific embodiments, in combination with a Rho GTPase / ROCK inhibitor and one or more exogenous transcription factor proteins (e.g., one, two, three, or all four of Oct peptide, Klf peptide, Myc peptide, and Sox peptide).

[0224] Various methods for introducing relevant protein factors into target cells have been described. In one embodiment, introducing a peptide into a cell may include: introducing a polynucleotide containing one or more expression cassettes into the cell and inducing expression, thereby introducing the peptide into the cell through transcription and translation of the expression cassettes.

[0225] Alternatively, one or more proteins can be simply cultured in the presence of target cells, provided that the protein can be introduced into the cells. See, for example, Zhou H et al., Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell. 2009 May 8;4(5):381-4. In some embodiments, the exogenous protein comprises a target transcription factor polypeptide linked to a polypeptide that enhances the ability of the transcription factor to enter the cell (and in some embodiments, the nucleus) (e.g., linked as a fusion protein, or otherwise covalently or non-covalently linked).

[0226] Examples of polypeptide sequences that enhance transmembrane transport include, but are not limited to: the Drosophila homeotype protein Anthrapodal 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); and the HIV-1 transcription 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. Patent No. 6,730,293 (including, but not limited to, peptide sequences comprising at least 7-25 consecutive arginine residues); and commercially available Penetratin. TM One peptide, and Diatos peptide carriers (“DPVs”) available from the Vectocell® platform of Daitos SA, Paris, France. See also WO / 2005 / 084158 and WO / 2007 / 123667 and other transporters described therein. Not only can these proteins cross the plasma membrane, but the attachment of other proteins, such as transcription factors described herein, is sufficient to stimulate cellular uptake of these complexes.

[0227] In some embodiments, the transcription factor peptides described herein are exogenously introduced as part of a liposome or liquid mixture, such as commercially available Fugene6 and Lipofectamine. Alternatively, the transcription factor protein may be directly introduced into target cells via microinjection or other methods.

[0228] As discussed in the examples of WO / 2009 / 117439, prolonged incubation of cells with the transcription factor peptides of the present invention is toxic to the cells. Therefore, the present invention uses one or more of the following peptides—Klf, Oct, Myc, and / or Sox—to intermittently incubate non-pluripotent mammalian cells, with the one or more peptides absent during the intervals between cell incubations. In some embodiments, the incubation cycles with and without the peptides can be repeated 2, 3, 4, 5, 6, or more times for a sufficiently long period (i.e., incubation with and without the protein) to achieve pluripotent cell formation. Various reagents (e.g., MEK / ERK pathway inhibitors and / or GSK3 inhibitors and / or TGFβ / ALK5 inhibitors and / or RhoGTPase / ROCK pathway inhibitors) may be included to improve the efficiency of this method.

[0229] Different inhibitors (e.g., TGFβ receptor / ALK5 inhibitors, MEK / ERK pathway inhibitors, and in specific embodiments, in combination with Rho GTPase / ROCK inhibitors and / or GSK3 inhibitors) can be contacted with non-pluripotent cells before, simultaneously with, or after the delivery of programmed transcription factors (e.g., via expression cassettes or as protein delivery). For convenience, the day of delivery of the reprogrammed transcription factor is designated "Day 1". In some embodiments, the inhibitors are contacted with the aggregated (i.e., as a "mixture") cells around Day 3–7 and continued for 7–14 days. Alternatively, in some embodiments, the mixture is contacted with the cells on Day 0 (i.e., the day before the preprogrammed factor) and incubated for approximately 14–30 days.

[0230] In other embodiments, different inhibitors are added at different times. In some embodiments, cells are exposed to a combination of a TGFβ receptor / ALK5 inhibitor (e.g., SB431542) and a ROCK inhibitor for 1-8 days 1-7 days after delivery of the reprogrammed factor, followed by exposure to a TGFβ receptor / ALK5 inhibitor, a ROCK inhibitor, and a MEK / ERK pathway inhibitor (e.g., PD0325901) for 1-8 days. This may optionally be followed by exposure to a TGFβ receptor / ALK5 inhibitor and a MEK / ERK pathway inhibitor (but not necessarily a ROCK inhibitor) for 1-4 days, followed by exposure to a MEK / ERK pathway inhibitor (but not a TGFβ receptor / ALK5 inhibitor or a ROCK inhibitor), and optionally finally exposure to a basal (e.g., basal human) ES medium without inhibitors for 1-4 days. Other combinations may also be used.

[0231] IV. Transformation

[0232] This invention employs conventional techniques in the field of recombinant genetics. Basic textbooks disclosing the general methods used in this invention include Sambrook et al., *Molecular Cloning, A Laboratory Manual* (3rd edition, 2001); Kriegler, *Gene Transfer and Expression: A Laboratory Manual* (1990); and *Current Protocols in Molecular Biology* (Ausubel et al., eds., 1994).

[0233] In some implementations, the cell species and the protein species to be expressed are the same. For example, if mouse cells are used, a mouse ortholog is introduced into the mouse. If human cells are used, a human ortholog is introduced into the cells.

[0234] It should be understood that when expressing two or more proteins in a cell, one or more expression cassettes can be used. For example, when an expression cassette is used to express multiple peptides, a polycistronic expression cassette can be used.

[0235] A. Plasmid vector

[0236] In some implementations, plasmid vectors are considered for transforming host cells. Typically, plasmid vectors containing replicons and control sequences derived from species compatible with the host cells can be used together with these hosts. The vector may carry a replication site, as well as marker sequences capable of providing phenotypic selection in the transformed cells.

[0237] B. Viral vector

[0238] The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis, and to integrate into the host cell genome and stably and efficiently express viral genes, has made them attractive candidates for transferring exogenous nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver the nucleic acids of the present invention are described below.

[0239] i. Adenovirus vector

[0240] One specific method for delivering nucleic acids involves the use of adenoviral expression vectors. Although adenoviral vectors are known to have a low ability to integrate into genomic DNA, this characteristic is compensated by the high gene transfer efficiency provided by these vectors. “Adenoviral expression vector” is intended to include those constructs containing an adenoviral sequence sufficient to (a) support the packaging of the construct and (b) ultimately express tissue- or cell-specific constructs cloned therein. Knowledge of genetic tissues or adenoviruses (i.e., a ~36 kb linear double-stranded DNA virus) allows for the replacement of large fragments of adenoviral DNA with exogenous sequences of up to 7 kb (Grunhaus et al., Seminar in Virology, 200(2):535-546, 1992).

[0241] ii. AAV carrier

[0242] Nucleic acids can be introduced into cells using adenovirus-assisted transfection. Increased transfection efficiency has been reported in cell systems utilizing 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 Immunology, 13(2-3):141-64, 1994). Adeno-associated viruses (AAVs) are attractive vector systems due to their high integration frequency and ability to infect undivided cells, thus making them effective for delivering genes into mammalian cells, for example, in tissue culture (Muzyczka, CurrTop Microbiol Immunol, 158:97-129, 1992) or in vivo. Detailed information regarding the production and use of rAAV vectors is set forth in U.S. Patent Nos. 5,139,941 and 4,797,368, which are incorporated herein by reference.

[0243] iii. Retroviral vectors

[0244] Retroviruses hold promise as gene delivery vectors due to their ability to integrate their genes into the host genome, transfer large amounts of exogenous genetic material, infect a wide range of species and cell types, and be packaged in specific cell lines (Miller et al., Am. J. Clin. Oncol, 15(3):216-221, 1992).

[0245] To construct retroviral vectors, nucleic acids (e.g., nucleic acids encoding the target gene) are inserted into the viral genome to replace certain viral sequences, thereby generating replication-defective viruses. To generate viral particles, packaging cell lines containing the gag, pol, and env genes but lacking LTRs and packaging elements are constructed (Mann et al., Cell, 33:153-159, 1983). When a recombinant plasmid containing cDNA, along with a retroviral LTR and packaging sequence, is introduced into a specific cell line (e.g., via calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged within viral particles, which are then secreted into the culture medium (Nicolas and Rubinstein, in: Vectors: A survey of molecular cloning vectors and their uses, eds. Rodriguez and Denhardt, Stoneham: Butterworth, pp. 494–513, 1988; Temin, in: GeneTransfer, Kucherlapati, ed., New York: Plenum Press, pp. 149–188, 1986; Mann et al., Cell, 33:153–159, 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression typically involve host cell division (Paskind et al., Virology, 67:242-248, 1975).

[0246] Lentivirals are complex retroviruses that contain not only the common retroviral genes gag, pol, and env, but also other genes with regulatory or structural functions. Lentiviral vectors are well known in the field (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; US Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been produced by significantly weakening HIV virulence genes, for example, by deleting genes env, vif, vpr, vpu, and nef, to make the vectors biologically safe.

[0247] Recombinant lentiviral vectors can infect undivided cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting undivided cells, wherein suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat, are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. Recombinant viruses can be targeted by linking envelope proteins to antibodies or specific ligands that target receptors on specific cell types. By inserting the target sequence (including the regulatory region) and, for example, another gene encoding a ligand for a receptor on a specific target cell into a viral vector, the vector becomes target-specific.

[0248] iv. Delivery using modified viruses

[0249] The nucleic acid to be delivered can reside within an infectious virus that has been engineered to express a specific binding ligand. The viral particle should therefore specifically bind to the relevant receptor on the target cell and deliver its contents into the cell. Based on retroviral chemical modifications by adding lactose residues to the viral envelope, a novel method has been developed to allow for the specific targeting of retroviral vectors. This modification allows for the specific infection of hepatocytes via the sialic acid glycoprotein receptor.

[0250] Another method for designing targeted recombinant retroviruses involves using biotinylated antibodies against retroviral envelope proteins and antibodies against specific cell receptors. These antibodies are conjugated via a biotinylate assembly using strepto-biotin (Roux et al., Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989). Antibodies against major histocompatibility complex class I and II antigens have demonstrated the in vitro infection of various human cells with those surface antigens by troponin-dependent viruses (Roux et al., 1989).

[0251] C. Vector delivery and cell transformation

[0252] The concept of suitable methods for nucleic acid delivery to transform cells, tissues, or organisms for use in this invention substantially includes any such method by which nucleic acids (e.g., DNA) are introduced into cells, tissues, or organisms, as described herein or as should be known to those skilled in the art. The methods include, but are not limited to: direct delivery of DNA, such as by in vitro-to-in vivo transfection (Wilson et al., Science, 244:1344-1346, 1989; Nabel and Baltimore, Nature, 326:711-713, 1987), wherein Fugene6 (Roche) or lipoferrin (Invitrogen) is optionally used, by injection (US Patent 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 incorporated herein by reference), including microinjection (Harland and Weintraub, J. Cell). Biol. (Journal of Cell Biology), 101: 1094-1099, 1985; US Patent No. 5,789,215, incorporated herein by reference); by electroporation (US Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. CellBiol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); by 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. (Molecular Cell Biology), 10:689-695, 1990); by using DEAE-dextran and subsequently polyethylene glycol (Gopal, Mol. Cell Biol. (Molecular Cell Biology), 5:1188-1190, 1985); by direct sonic loading (Fechheimer et al., Proc. Nat'l Acad. Sci.).USA (Proceedings of the National Academy of Sciences of the United States of America), 84:8463-8467, 1987); Transfection mediated by liposomes (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, 1991) and receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of the methods described, each incorporated herein by reference.

[0253] VII. Mixtures

[0254] This invention provides mixtures that improve the production efficiency of iPSCs. For example, the invention provides mixtures of TGFβ receptor / ALK5 inhibitors, MEK / ERK pathway inhibitors, and Rho GTPase / ROCK inhibitors, and in a particular embodiment, mixtures of these with mammalian cells. For example, said mixtures may be included in cell culture media containing or without cells. The contents of the cell culture media are generally known in the art. Exemplary cell culture media are described in detail in the examples. Typically, cell cultures containing mammalian cells and the reagents of this invention (TGFβ receptor / ALK5 inhibitors, MEK / ERK pathway inhibitors, and Rho GTPase / ROCK inhibitors) initially contain all or substantially all of the non-pluripotent cells. However, over time, particularly under the conditions of the protocols described herein, a subset of the cells will become pluripotent (i.e., iPSCs).

[0255] Cells to be induced to be pluripotent can be cultured according to any method known in the art. General guidelines for culture conditions for generating iPSCs can be found, for example, Maherali, et al., Cell Stem Cell 3:595-605 (2008).

[0256] In some embodiments, the cells are cultured by contacting feeder cells. Exemplary feeder cells include, but are not limited to, fibroblasts, such as mouse embryonic fibroblast (MEF) cells. Methods for culturing cells on feeder cells are known in the art.

[0257] In some embodiments, cells are cultured under conditions lacking feeder cells. Cells, for example, can be directly attached to the surface of a solid culture (e.g., a culture plate) via molecular adhesion. The inventors have discovered that when cells are directly attached to a solid culture surface, the efficiency of induced pluripotency in culturing cells is much higher (i.e., a larger proportion of cells acquire pluripotency) compared to the efficiency of culturing otherwise identically treated cells on feeder cells. Exemplary molecular adhesives include, but are not limited to: Matrix Gel®, extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen. However, those skilled in the art will recognize that this is a non-limiting list and other molecules can be used to attach cells to a solid surface. Methods for initially attaching the adhesive to a solid surface are known in the art.

[0258] As described herein, in some embodiments, the mixtures of the present invention may or may not include mammalian cells (including pluripotent or non-pluripotent cells) and one or more of the following substances: HDAC inhibitors, GSK3 inhibitors or L-type calcium channel agonists; cAMP pathway activators; DNA methyltransferase (DNMT) inhibitors; nuclear receptor ligands, for example, as described in PCT WO / 2009 / 117439.

[0259] VIII. Reagent Kit

[0260] This invention also provides kits, for example, for generating induced pluripotent stem cells. Such kits may contain any or all of the reagents described herein, including, but not limited to: TGFβ receptor / ALK5 inhibitors, MEK / ERK pathway inhibitors, and / or Rho GTPase / ROCK inhibitors described herein. These three reagents, or subsets thereof, may be present in separate vials within the kit or together as a mixture. The kits of this invention may also include one or more of the following: HDAC inhibitors, GSK3 inhibitors, or L-type calcium channel agonists; cAMP pathway activators; DNA methyltransferase (DNMT) inhibitors; and nuclear receptor ligands.

[0261] In one embodiment, the kit of the present invention comprises one or more types of mammalian cells (e.g., human, mouse, rat, etc.) and / or cell culture medium.

[0262] In one specific embodiment, the kit of the present invention comprises one or more polynucleotides containing an expression cassette for expressing one or more of the Oct peptide, Klf peptide, Myc peptide, and Sox peptide. Additionally or alternatively, the kit may contain one or more isolated transcription factor proteins, for example, 1, 2, 3, or all four of the Oct peptide, Klf peptide, Myc peptide, and Sox peptide. In another specific embodiment, the transcription factor protein may be fused to a polypeptide sequence for enhancing transmembrane transport of the transcription factor protein.

[0263] VI. Applications of pluripotent cells

[0264] This invention allows for further research and development of stem cell technology, including but not limited to preventative or therapeutic applications. For example, in some embodiments, the cells of this invention (pluripotent cells or cells induced to differentiate along a desired cell fate) are introduced into individuals in need, including but not limited to individuals requiring regeneration of organs, tissues, or cell types. In some embodiments, the cells are initially obtained from the individual via biopsy; pluripotency is induced as described herein, optionally differentiation is induced (e.g., differentiation into a particularly desirable progenitor cell), and then transplanted back into the individual. In some embodiments, the cells are genetically modified prior to their introduction into the individual.

[0265] In some embodiments, the pluripotent cells generated according to the method of the present invention are subsequently induced to form, for example, hematopoietic (stem / progenitor) cells, neural (stem / progenitor) cells (and optionally more differentiated cells, such as subtype-specific neurons, oligodendrocytes, etc.), pancreatic cells (e.g., endocrine progenitor cells or pancreatic hormone-expressing cells), hepatocytes, cardiovascular (stem / progenitor) cells (e.g., cardiomyocytes, endothelial cells, smooth muscle cells), retinal cells, etc.

[0266] Various methods for inducing pluripotent stem cells to differentiate into desired cell types are known. A non-limiting list of recent patent publications describing methods for inducing stem cell differentiation into different cell fates is as follows: US Patent Publications 2007 / 0281355; 2007 / 0269412; 2007 / 0264709; 2007 / 0259423; 2007 / 0254359; 2007 / 0196919; 2007 / 0172946; 2007 / 0141703; 2007 / 0134215.

[0267] By introducing the pluripotent cells of the present invention and optionally targeting specific injured tissues, many diseases can be improved. Examples of diseases caused by tissue damage include, but are not limited to: neurodegenerative diseases, cerebral infarction, obstructive vascular disease, myocardial infarction, heart failure, chronic obstructive lung disease, pulmonary emphysema, bronchitis, interstitial pulmonary disease, asthma, hepatitis B (liver damage), hepatitis C (liver damage), alcoholic hepatitis (liver damage), hepatic cirrhosis (liver damage), hepatic insufficiency (liver damage), pancreatitis, diabetes mellitus, Crohn's disease, and inflammatory colitis. Colitis, IgA glomerulonephritis, glomerulonephritis, renal insufficiency, decubitus ulcers, burns, suture wounds, lacerations, cuts, bite wounds, dermatitis, cicatricial keloids, keloids, diabetic ulcers, arterial ulcers, and venous ulcers.

[0268] In one implementation, iPSCs can be used in different experiments and screened to identify molecules that regulate their function (including, but not limited to, promoting iPSC survival and / or differentiation).

[0269] Example

[0270] The following embodiments are provided to illustrate, but are not intended to limit, the invention.

[0271] Example 1: A chemical platform for enhancing the induction of human iPSCs

[0272] Mesenchymal fibroblasts reprogrammed with “four factors” (OCT4, SOX2, KLF4, and c-MYC; hereinafter referred to as 4TF) undergo dramatic morphological changes that produce iPSCs with unique cell polarity, boundaries, and cell-cell interactions. The reprogrammed cells express E-cadherin, a marker of epithelial cells (Hay, ED, Acta Anat. (Basel) 154, 8-20 (1995)), which is also highly expressed in human embryonic stem cells (hESCs). We hypothesize that factors promoting mesenchymal-to-epithelial transition (MET), such as TGFβ pathway antagonists, have a direct influence on the reprogramming process. Furthermore, previous studies have shown that MEK-ERK pathway inhibition plays a crucial role in different steps of the reprogramming process (Chen, S. et al., Proc. Natl. Acad. Sci. USA 104, 10482-87 (2007); Shi, Y. et al., Cell StemCell 2, 525-8 (2008)). In addition, factors that promote cell survival are also beneficial in improving the efficiency of the reprogramming process. Therefore, we focused on small molecules that can regulate these three processes and pathways because small molecules offer many advantages in biological research (Feng, B. et al., Cell Stem Cell 4, 301-12 (2009); Shi, Y. et al., CellStem Cell 2, 525-8 (2008); Xu, Y. et al., Nature 453, 338-44 (2008)) and are a safer option than genetic manipulation. Here we describe a simple chemical platform that substantially increases the production of completely reprogrammed human iPSCs from fibroblasts through a far faster and more efficient process.

[0273] We are in 1x10 4 The effects of known inhibitors of TGFβ receptor and MEK on reprogramming kinetics and efficiency were tested on human primary fibroblasts (CRL2097 or BJ) transduced with 4TF retrovirus (Feng, B. et al., Cell Stem Cell 4, 301-12 (2009)). (See details in [link to article]). Figure 1A On day 7 post-infection (D7), add the compound alone or in combination, and examine the iPSCs of the culture over the next 1–3 weeks.

[0274] On day 7 post-treatment (D14), we observed the strongest effect in cultures treated with a combination of the ALK5 inhibitor SB431542 (2 µM) and the MEK inhibitor PD0325901 (0.5 µM), which produced approximately 45 large ALPs with characteristic hESC-like morphology. + Colony ( Figure 1B (Of which more than 24 settlements are TRA-1-81) + ( Figure 1D Approximately 6-10 colonies were positive for SSEA4 and NANOG (mature pluripotent factors without ectopic introduction). Figure 1E and 1F Furthermore, the treated cultures exhibited high levels of endogenous mRNA expression of pluripotency genes. Figure 1C In contrast, in the untreated control culture ( Figure 1E and Figure 4A ), or in cultures treated with PD0325901 alone, NANOG was not observed. + Colony ( Figure 4A However, in cultures treated with only SB431542, we still observed 1-2 ALPs. + hESC-sample set ( Figure 4A Importantly, the combined effect of the two inhibitors ( Figure 4B and 4C The effects of SB431542 alone are dose-dependent.

[0275] When we maintained the SB431542 + PD0325901 treated culture for 30 days without splitting, we obtained approximately 135 iPSC colonies / well. Figure 2D This is >100 times more efficient than conventional methods. Consistent with previous reports (Takahashi, K. et al., Cell 131, 861-72 (2007)), we observed 1-2 iPSC colonies and several granular colonies in the untreated control carrying 4 TF. Figure 2CIt has been suggested that these granular structures are colonies of partially reprogrammed cells (Takahashi, K. et al., Cell 131, 861-72 (2007)). We also observed granular colonies in cultures treated with SB431542, which were several times more numerous than the rare hESC-like colonies. Interestingly, in the combined treatment with SB431542 and PD0325901, the number of granular colonies decreased sharply, which led to a corresponding increase in the number of hESC-like colonies. This suggests that the combined inhibition of ALK5 and MEK may guide partially reprogrammed colonies to a fully reprogrammed state, thereby improving the overall reprogramming process. Furthermore, the fact that we observed the induction of improved iPSCs as early as 7 days after treatment suggests that treatment with these small molecules not only improves the efficiency of the reprogramming process but may also have accelerated its kinetics. Figure 1A Further experiments are needed to determine whether the reprogrammed cells truly become completely independent of exogenous reprogramming factors at this stage earlier than in untreated cultures.

[0276] Despite the selection and amplification of iPSC colonies, culture fragmentation via trypsin digestion, as in hESC cultures, resulted in poor survival. From recent screenings conducted in our lab, we identified a novel small molecule, Thiazovin (… Figure 7 Thiazovivin significantly improved hESC survival after trypsin digestion. The addition of thiazovivin to our mixture of SB431542 and PD0325901 also greatly improved iPSC survival after fragmentation via trypsin digestion. Figure 2A ), and obtained a large number of reprogrammed colonies. From the initial 10,000 cells, a single 1:4 split on day 14 produced approximately 1,000 hESC-like colonies on day 30. Figure 2E The two-round split (on day 14 and day 21 (1:10)) produced approximately 11,000 hESC-sampled colonies on day 30. Figure 2C and 2E These colonies exhibited high levels of endogenous mRNA, a marker of pluripotency. Figure 2F ) and protein expression ( Figure 2B and 2C ), while the expression of the four transgenic genes was almost undetectable ( Figure 2F In contrast, no iPSC colonies were obtained from untreated samples digested with trypsin or from samples treated with two compounds (Table 1).

[0277] Table 1: Comparison of the number of iPSC colonies observed on day 30 in untreated, 2-compound-treated, and 3-compound-treated cultures.

[0278]

[0279] To examine whether the positive effect of Thiazovin was solely due to colony survival after splitting, or whether it also enhanced the reprogramming effect of the combined SB431542 and PD0325901 treatments, we tested a mixture of the three compounds on unsplit 4TF-transduced cells. In these cultures, approximately 25 large colonies expressing Nanog were observed before day 14. Figure 1E Before day 30, we observed approximately 205 very large nanogs. + Colony ( Figure 2D They are also TRA-1-81 + and SSEA4 + (Data not shown) Compared to no compound treatment, they show an efficiency increase of more than 200 times, and compared to two compound treatments, the efficiency increased by 2 times.

[0280] When reprogramming factors were introduced using a lentiviral system instead of a retroviral system, both compound treatments also produced a greater number of alkaline phosphatase-positive colonies compared to the untreated control. Figure 6A Furthermore, the mixture of the three compounds did not show any effect on the expression of reprogramming factors derived from retroviral vectors. Figure 6B -F).

[0281] iPSC colonies generated using a mixture of three compounds can be easily and stably expanded long-term (over 20 generations) under conventional hESC culture conditions. They are very similar to hESCs in morphology, expression of typical pluripotency markers, and differentiation potential. They exhibit a normal karyotype ( Figure 5 ), and can be performed outside the body ( Figure 3A and 3B ) and in the body ( Figure 3C It differentiates into derivatives of all three germ layers. These results also suggest that there are no short-term side effects associated with the far more convenient trypsin digestion procedure.

[0282] The inhibition of TGFβ and MEK-ERK pathways modulates fibroblast reprogramming, suggesting the crucial roles of these two signaling pathways and the MET mechanism in the process. Consistently, the addition of TGFβ has an inhibitory effect on the reprogramming of fibroblasts mediated by four factors (data not shown). TGFβ and its family members play important contextual roles in ESC self-renewal and differentiation (Watabe, T. and Miyazono, K., CellRes. 19, 103-15 (2009)). Furthermore, TGFβ is a proto-cytokine for inducing epithelial-mesenchymal transition (EMT) and maintaining mesenchymal state (Willis, BC and Borok, Z., Am. J. Physiol. Lung Cell Mol. Physiol. 293, L525-34 (2007)). In this context, the primary endpoint of this signaling pathway is the deregulation of E-cadherin (Thiery, JP and Sleeman, JP, Nat. Rev. Mol. Cell Biol., 7, 131-42 (2006)). E-cadherin has been shown to be important for maintaining the pluripotency of ESCs and has recently been suggested as a regulator of NANOG expression (Chou, YF et al., Cell 135, 449-61 (2008)). Therefore, inhibition of TGFβ signaling (which leads to deinhibition of epithelial fate) can benefit the reprogramming process in multiple pathways. ERK signaling also promotes EMT (Thiery, JP and Sleeman, JP, Nat. Rev. Mol. Cell Biol. 7, 131-42 (2006)), and in this process is downstream of TGFβ (Chou, YF et al., Cell 135, 449-61 (2008)). We have previously demonstrated that the action of reversine (a small molecule that can reprogram the pluripotent state of myoblasts) is mediated in part through the inhibition of MEK-ERK (Chen, S. et al., Proc. Natl. Acad. Sci. USA 104, 10482-87 (2007)). This could explain the role observed in the reprogramming when it is combined with TGFβ inhibition.

[0283] The chemical platform described here is unique because it modulates upstream signaling pathways and can fundamentally improve reprogramming for common cell types such as fibroblasts. The chemical conditions described here provide a foundational platform for more efficient and safer reprogramming methods based on non-viral and non-DNA (Zhou, H. et al., Cell Stem Cell 4, 381-84, (2009)), which can generate an unlimited supply of safe human iPSCs for various applications.

[0284] method

[0285] Cell culture

[0286] Primary skin fibroblasts CRL2097 and BJ (neonatal foreskin) were purchased from ATCC. All cell culture media and reagents were purchased from Invitrogen Corporation, CA. Cells were maintained in DMEM (10313-021) containing 10% FBS (10439-024), 1X MEM non-essential amino acids (11140-050), 1X Glutamax (35050-061), 10 mM Hepes (15630-080), and 0.11 mM 2-mercaptoethanol (21985-023). Cells were passaged at a 1:5 ratio using 0.05% (1X) trypsin-EDTA (25300-054).

[0287] plasmid

[0288] From ADDGENE, pMX vectors encoding human cDNAs of OCT4, SOX2, c-MYC, and KLF4, as described above (Takahashi, K. et al., Cell 131, 861-72 (2007)), were obtained. As previously described (Takahashi, K. et al., Cell 131, 861-72 (2007)), mouse Slc7a1 ORF was cloned into pWPXLD (Addgene).

[0289] Retroviral infection and iPS cell production

[0290] As previously described (Yu, J. et al., Science 318, 1917-20 (2007)), lentiviruses carrying OCT4, NANOG, SOX2, and LIN28 are produced. For retrovirus production, at a rate of 1 x 102... 6Cells / wells: PLAT-E packaged cells were seeded into 6-well plates. After 24 hours, cells were transfected with pMXs vectors carrying OCT4, SOX2, c-MYC, and KLF4 cDNA using Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions. 24 hours post-transfection, the medium was replaced with fresh medium, and the plates were transferred to 32 °C for retroviral production. Viruses were collected at 48 and 72 hours, filtered through a 0.45 µm filter, and then transduced.

[0291] On day 1, with 1x10 5 Cells / wells: Human fibroblasts expressing Slc7a1 were seeded in 6-well plates. On day 2, 0.25 ml of supernatant for each retrovirus was added to the cells in the presence of 6 µg / ml 1,5-dimethyl-1,5-diazadecylmethylene polybromide. On day 3, a second round of transduction was performed. Infection efficiency was estimated on cells transduced in parallel with retroviruses carrying either the GFP or RFP gene by fluorescence microscopy. Seven days after initial transduction, fibroblasts were harvested by trypsin digestion and cultured at 1 x 10⁻⁶ cells / well. 4 Cells / wells were re-seeded in 6-well plates coated with matrix gel (1:50 dilution, catalog number 354234, BD Biosciences). For compound treatment, cells were cultured in human reprogrammed medium (DMEM / F12, 20% knockout serum substitute, 1x MEM non-essential amino acids, 1x glutamax, 0.11 mM 2-mercaptoethanol, 20 ng / ml bFGF, and 1,000 U / ml LIF) and treated with 2 µM SB431542 (Stemgent), 0.5 µM MPD0325901 (Stemgent), 0.5 µM Thiazovivin, or a combination of the compounds. The medium was changed every 2–3 days depending on cell density. Seven days after compound treatment, the plates were either fixed and stained for alkaline phosphatase (ALP) activity or protein marker staining, or cultured until day 30 with or without indicated splitting by trypsin digestion. For split cultures, cells were split (1:4) and reseeded into irradiated CF-1 MEF feeder layers (2.5 x 10⁶ cells / well) in each well of a 6-well plate. 5Cells were split on a 1:10 scale and then split again on day 21. The cells were maintained in the same culture medium and compound mixture described above, except for the concentrations of PD0325901 (0.5 µM on day 14 and 1 µM on day 21) and SB431542 (0.5–1 µM after day 14). Subsequently, iPSC colonies were maintained in standard hESC medium in the absence of the aforementioned compounds.

[0292] Alkaline phosphatase staining and immunocytochemistry

[0293] According to the product instructions, alkaline phosphatase staining was performed using the ALP detection kit (catalog number: SCR004, Chemicon). For immunocytochemistry, cells were fixed in 4% paraformaldehyde (10 min, RT), washed twice with PBS, blocked with 5% normal donkey serum (Chemicon) and 0.1% Triton X-100 (15 min, RT), and then treated overnight at 4°C with the primary antibody. The primary antibodies used were: anti-NANOG (catalog number: AB9220, Chemicon, 1:1,000); anti-OCT4 (catalog number: sc-5279, Santa Cruz biotech, 1:200); anti-SSEA 4 (catalog number: mab4304, Chemicon, 1:500); anti-Tra-1-81 (catalog number 560123, BD Biosciences, 1:100); anti-Tra-1-81 (mAb 4381, Chemicon, 1:500); anti-βIII tubulin (catalog number: MMS-435P, Covance Research Products Inc, 1:1000); anti-PDX 1 (1:500) (a gift from Dr. C. Wright); and anti-BRACHYURY (catalog number: AF2085, R&D). The final concentration was 0.2 µg / ml. Cells were washed twice with PBS and then treated with a secondary antibody at room temperature for 1 hour. The secondary antibodies used were Alexa fluor 488 donkey anti-rabbit or anti-mouse IgG (Invitrogen, 1:1,000) and Alexa fluor 555 donkey anti-rabbit or anti-mouse IgG (Invitrogen, 1:1,000). Cell nuclei were stained with 0.5 µg / ml DAPI (Sigma). Images were captured using a Nikon Eclipse TE2000-U / X-cite 120 EXFO microscope with a CoolSnap HQ2 camera for photometric determination.

[0294] In vitro differentiation and teratoma assay

[0295] As described elsewhere, embryoid formation and in vitro differentiation were performed (Takahashi, K. et al., Cell 131, 861-72 (2007)). For teratoma assays, 3-5 million cells were injected subrenally into SCID mice. After 31 days, the tumors were excised, fixed in 4% paraformaldehyde, and histologically analyzed at the TSRI Histology Core facility. The use of SCID mice was approved by the UCSD Animal Research Committee.

[0296] RT-PCR

[0297] Total RNA was extracted from cells using the RNeasy mini kit (Qiagen). cDNA was synthesized using the Superscript III first-strand synthesis kit (Invitrogen) according to the product instructions. Two μL of reaction product was used for 24–28 PCR cycles using various primers. Primer sequences are as described elsewhere (Takahashi, K. et al., Cell 131, 861–72 (2007)).

[0298] Flow cytometry

[0299] For flow cytometry analysis, cultures were gently digested with trypsin and harvested from 6-well plates. Cells were washed, resuspended in FACS buffer (PBS, 2 mM EDTA, 2 mM HEPES, 1% FBS), and analyzed using the CellQuest program on a FACS Calibur cell counter (Becton Dickinson, San Jose, CA).

[0300] Example 2: Synthesis of N-(cyclopropylmethyl)-4-(4-(6-hydroxy-3,4-dihydroquinoline-1(2H)-yl)pyrimidin-2-ylamino)benzenesulfonamide (Thiazovivin)

[0301] A reaction flask containing 2,4-dichloropyrimidine (372 mg, 2.5 mmol), 6-methoxy-1,2,3,4-tetrahydroquinoline (489 mg, 3 mmol), and diisopropylethylamine (0.52 mL, 3 mmol) in n-butanol (10 mL) was heated overnight at 40 °C. The solvent was evaporated, and the residue was purified by rapid column chromatography to give 2-chloro-4-(6-methoxy-3,4-dihydroquinoline-1(2H)-yl)pyrimidine (551 mg, 80%). This intermediate (250 mg, 0.91 mmol) was then dissolved in dichloromethane and treated with BBr3 (1 M, in dichloromethane) (1 mL, 1 mmol) at -78 °C. The reaction mixture was slowly warmed to room temperature and stirred for 1 hour, then poured into water and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated. The residue was purified by rapid column chromatography to give 2-chloro-4-(6-hydroxy-3,4-dihydroquinoline-1(2H)-yl)pyrimidine (154 mg, 65%). To a stirred solution of 2-chloro-4-(6-hydroxy-3,4-dihydroquinoline-1(2H)-yl)pyrimidine (29 mg, 0.11 mmol) and 4-amino-N-(cyclopropylmethyl)benzenesulfonamide (27 mg, 0.12 mmol) in DMF (0.5 mL), p-toluenesulfonic acid (2 M, in diethyltoluenesulfonic acid solution) was added. (in alkane) (55 µL, 0.11 mmol). The reaction mixture was stirred overnight at 90 °C and then purified by HPLC to give the title compound (27 mg, 56%).

[0302]

[0303] Example 3: Reprogramming of human primary somatic cells using OCT4 and compounds

[0304] We report here a novel small molecule mixture that can reprogram human primary somatic cells into iPSCs that express only exogenous OCT4.

[0305] Among several readily available primary human somatic cell types, keratinocytes, which can be easily isolated from human skin or hair follicles, represent an attractive cellular source for reprogramming because they endogenously express KLF4 and cMYC, and have been reported to be more effectively reprogrammed using conventional four TFs or three TFs (without MYC) (Aasen, T. et al., Nat Biotechnol 26:1276-1284 (2008); Maherali, N. et al., Cell Stem Cell 3,340-345 (2008)). More recently, we reported that dual inhibition of the TGFβ and MAPK / ERK pathways using small molecules (i.e., SB431542 and PD0325901, respectively) provided conditions for significantly enhanced reprogramming of human fibroblasts by four exogenous TFs (i.e., OSKM) (Lin, T. et al., Nat Methods 6:805-808 (2009)). Furthermore, we have demonstrated that dual-pathway inhibition using two small molecules, parnate (an inhibitor of lysine-specific demethylase 1) and CHIR99021 (a GSK3 inhibitor), can also enhance the reprogramming of human keratinocytes by two exogenous TFs (i.e., OK) (Li, W. et al., Stem Cells 27:2992-3000 (2009)). However, such 2-TF reprogramming processes are highly inefficient and complex (e.g., involving two exogenous TFs and four chemical reagents), and even reprogramming at least one TF appears frustrating. To reprogram OCT4-only, we developed a stepwise strategy in improving reprogramming conditions and identifying novel reprogramming chemical entities. We first attempted to further optimize the reprogramming process in neonatal human epidermal keratinocytes (NHEK) under four or three TF conditions (i.e., OSKM or OSK), where various inhibitors of different concentrations of the TGFβ and MAPK pathways were tested using previously reported human iPSC characterization methods (Lin, T. et al., Nat Methods 6:805-808 (2009)). Encouragingly, we found that the combination of 0.5 μM PD0325901 and 0.5 μM A-83-01 (a more effective and selective TGFβ receptor inhibitor) more effectively enhanced the reprogramming process in human keratinocytes transduced with OSKM or OSK. Figure 8ANotably, when we further reduced viral transduction to only 2 factors / OK, we still generated iPSCs from NHEK, albeit at a lower efficiency, when treated with 0.5 μM PD0325901 and 0.5 μM A-83-01. We then began screening other small molecules from a collection of previously reported concentrations of known bioactive compounds. Among the many compounds tested to date, we were surprised to find that PS48 (5 μM), a small molecule activator of PDK1 (3'-phosphoinositol-dependent kinase-1) (which had never been reported in reprogramming), significantly increased reprogramming efficiency by approximately 15-fold. Interestingly, we also found that 0.25 mM sodium butyrate (NaB, a histone deacetylase inhibitor) proved far more reliable and effective than the previously reported 0.5 mM VPA for iPSC generation under OK conditions. Figure 8B Subsequent studies confirmed that the combination of 5 μM PS48 and 0.25 mM NaB can further enhance the efficiency of reprogramming by more than 25 times. Figure 8B (and Table 4). Leveraging this unprecedented efficiency in reprogramming NHEK under only two TFs, we further investigated the possibility of generating iPSCs using OCT4 alone by improving the combinations of those small molecules in different processing windows. Primary NHEK was transduced with OCT4 and treated with chemical reagents ( Figure 8C Under different conditions, small iPSC colonies (4-6 colonies per 1,000,000 seeded cells) similar to hESCs appeared in OCT4-infected NHEK cells treated for the first 4 weeks with 0.25 mM NaB, 5 μM PS48, and 0.5 μM A-83-01, followed by another 4 weeks of treatment with 0.25 mM NaB, 5 μM PS48, 0.5 μM A-83-01, and 0.5 μM PD0325901. Figure 8C Such TRA-1-81 positive iPSC colonies ( Figure 8D iPSC clones grow larger in standard hESC medium and can be continuously passaged to produce stable iPSC clones that can be further characterized. Figure 8E(and 9). More notably, iPSCs with only OCT4 could also be generated from human adult keratinocytes by adding 2 μM tranylcypromine sulfate and 3 μM CHIR99021 (which have been shown to improve reprogramming of NHEK under OK conditions). After reliably reprogramming primary keratinocytes into iPSCs with OCT4 and small molecules, we further applied the conditions to other human primary cell types, including HUVECs (differentiated mesodermal cells) and AFDCs (cells obtained from amniotic fluid). Similarly, TRA-1-81 positive iPSC colonies appeared in HUVECs and AFDCs infected with OCT4 treated with chemical reagents. Notably, under OCT4 and small molecule conditions, reprogramming of HUVECs and AFDCs appeared to be more efficient and faster than reprogramming of NHEK (Table 4). Two clones of iPSCs from each cell type were extended for more than 20 passages under standard hESC culture conditions and further characterized (Table 5).

[0306] These stably expanding hiPSC-OK and hiPSC-O cells are morphologically indistinguishable from hESCs and can be cultured on ECM-coated surfaces under conditions without feeder cells and with known chemical composition. Figure 8E and Figure 13 They are alkaline phosphatase (ALP) positive and express typical pluripotency markers, including OCT4, SOX2, NANOG, TRA-1-81, and SSEA4, which can be detected by immunocytochemistry / ICC. Figure 8E , 10B (Figures 11-12). Furthermore, RT-PCR analysis confirmed the expression of endogenous human OCT4, SOX2, NANOOG, REX1, UTF1, TDGF2, and FGF4 genes and the silencing of exogenous OCT4 and KLF4. Figure 9A and 10C Furthermore, bisulfite sequencing analysis revealed that the OCT4 and NANOG promoters in hiPSC-OK and hiPSC-O cells were largely demethylated. Figure 9B and 10D This result provides further evidence for the reactivation of pluripotent transcriptional programs in hiPSC-OK and hiPSC-O cells. Total gene expression analysis of hiPSC-O cells, NHEK, and hESC confirmed that hiPSC-O cells differed from NHEK (Pearson correlation: 0.87) and were most similar to hESC (Pearson correlation: 0.98). Figure 9CGenotyping analysis confirmed that hiPSC-O cells contained only the OCT4 transgene and were not contaminated with the KLF4 or SOX2 transgenes (Figure 15). DNA blot analysis confirmed the presence of multiple distinct integration sites of the OCT4 transgene in different clones. Figure 16 Furthermore, karyotype analysis confirmed that hiPSC-O maintained a normal karyotype throughout the reprogramming and expansion process (Figure 17). In addition, DNA fingerprinting experiments ruled out the possibility that these hiPSCs originated from laboratory-acquired hESC contamination (Table 6). To examine the developmental potential of these hiPSC-O cells, they were differentiated in vitro using standard embryoid (EB) differentiation methods. ICC analysis confirmed that they could efficiently differentiate into βIII-tubulin. + Characteristic neurons (ectoderm), SMA + Mesodermal cells and AFP + Endoderm cells ( Figure 9D and 10E Quantitative PCR analysis further confirmed the expression of these and other lineage-specific marker genes, including those from ectoderm cells (βIII-tubulin and nestin), mesodermal cells (MSX1 and MLC2a), and endoderm cells (FOXA2 and AFP). Figure 9E Following the EB protocol, these hiPSC-OK and hiPSC-O cells can also generate rhythmically beating cardiomyocytes. To test their in vivo pluripotency, they were transplanted into SCID mice. After 4–6 weeks, these hiPSC-O cells effectively generated typical teratomas containing derivatives of all three germ layers. Figure 9F and 10F These in vitro and in vivo characterizations collectively confirm that a single transcription factor, OCT4, combined with a defined mixture of small molecules, is sufficient to reprogram several human primary somatic cells into iPSCs, which are morphologically, molecularly, and functionally similar to pluripotent hESCs.

[0307] The above research has several important implications: (1) Although fetal NSCs have been confirmed to be reprogrammed into iPSCs by ectopic expression of Oct4 alone, significant skepticism remains regarding whether exogenous Oct4 alone is sufficient to reprogram other, more practical human somatic cells that do not endogenously express Sox2 (one of the two dominant pluripotent genes in reprogramming), are at a later developmental stage (e.g., early embryo / fetus vs. newborn / adult), and can be obtained without significant harm to the individual. To the best of our knowledge, our study is the first to demonstrate in practice that iPSCs can be derived in practice from readily available primary human somatic cells (e.g., keratinocytes) transduced by a single exogenous reprogramming gene, Oct4. Keratinocytes are more readily available than neural stem cells from the brain and can be easily obtained from newborn individuals with less invasive procedures. This further reinforces the strategy of developing different, practically available human somatic cells for iPSC production (with safer protocols and / or better quality). Therefore, this new method and its further development will significantly facilitate the production of patient-specific pluripotent stem cells for various applications. (2) Although small molecules and combinations thereof have been identified to replace only one or two reprogramming TFs, the challenge of generating iPSCs increases exponentially when more exogenous reprogramming TFs are omitted together. The identification of this new small molecule mixture (which functionally replaces all three major transcription factors (i.e., Sox2, Klf4, and cMyc) in the production of iPSCs using Oct4 alone) represents another major step in the final reprogramming process using only small molecules and further confirms and solidifies the chemical scheme for generating iPSCs. (3) This confirms the important use of single-gene conditions in protein-induced pluripotent stem cell (piPSC) technology. The practical challenge of piPSC technology is the large-scale and reliable production of four transducible reprogramming proteins, each of which performs differently in production (e.g., their expression, folding, stability, etc.). Clearly, combining this mixture of small molecules with a single transducible protein would significantly simplify piPSC technology and facilitate its application. (4) More notably, we identified a novel small molecule, PS48, which has a novel target / mechanism in enhancing reprogramming. PS48 is an allosteric small molecule activator of PDK1, which is an important upstream kinase for several AGC kinases, including Akt / PKB (Alessi et al., Curr Biol 7, 261-269 (1997)).Its reprogramming-enhancing effect can be partly attributed to the activation of Akt / PKB, which promotes cell proliferation and survival (Manning, BD, Cantley, LC, Cell 129, 1261-1274 (2007)). Further in-depth characterization of how the mechanisms involving PDK1 are precisely regulated during reprogramming will provide further insights into reprogramming and pluripotency. In addition, because there may be even greater hidden risks arising from the inefficiency and slow kinetics of reprogramming (e.g., more subtle genetic and / or post-natal abnormalities may be generated or selected during reprogramming), the identification of new small molecules for enhancing reprogramming is always valuable for safer, easier, and more efficient human iPSC preparation procedures (as illustrated again in this study). (5) Finally, this new and efficient mixture of small molecules for reprogramming will be validated against the stepwise chemical optimization and screening strategy presented here for a final pure chemical induced pluripotent stem cell production protocol. Furthermore, the finding that different small molecules targeting the same target / mechanism can have significantly different effects on reprogramming in different contexts (exemplified by the better reprogramming-enhancing activity of A-83-01 and NaB in human keratinocytes) suggests the importance of “personalized” optimization and treatment of different schemes for specific reprogramming contexts.

[0308] Cell culture

[0309] Normal human epidermal keratinocytes (Lonza) were maintained in keratinocyte culture medium (KCM, Lonza). Human umbilical vein endothelial cells (HUVEC, Millipore) were maintained in EndoGRO-VEGF complete medium (HCM, CHEMICON). Human ESCs and hiPSCs were cultured on MEF feeder cells in standard human ESC medium (hESCM: DMEM / F12, 15% knockout serum substitute, 1% Glutamax, 1% non-essential amino acids, 1% penicillin / streptomycin, 0.1 mM β-mercaptoethanol and 10 ng / ml bFGF). Unless otherwise specified, all cell culture products were derived from Invitrogen / Gibco BRL.

[0310] Lentiviral production

[0311] As previously described (Yu, J. et al., Science 318:1917-1920 (2007)), lentiviral supernatants are produced and harvested. Plasmids used for lentiviral production include pSin-EF2-Puro-hOCT4, pSin2-EF2-Puro-hSOX2, pLove-mKlf4, pLove-mMyc, packaging plasmid psPAX2, and membrane-encoding plasmid pMD2.G (Yu, J. et al., Science 318:1917-1920 (2007) and Li, W. et al., Stem Cells 27:2992-3000 (2009)).

[0312] NHEK recompilation program

[0313] NHEK cells were cultured in 100 mm tissue culture dishes and transduced three times with freshly prepared lentiviral supernatant (3-4 hours each time). 1,000,000 transduced NHEK cells were seeded onto irradiated X-ray-inactivated CF1 MEF feeder cells in 100 mm dishes and cultured in KCM. The cells were treated with 5 μM PS48, 0.25 mM NaB (Stemgent), and 0.5 μM A-83-01 (Stemgent) for 2 weeks. Half the culture medium was then replaced with hESCM, and 5 μM PS48, 0.25 mM NaB, and 0.5 μM A-83-01 were added for another 2 weeks. Then, the cell culture medium was replaced with hESCM, and 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01, and 0.5 μM PD0325901 (Stemgent) were added for another 4 weeks. Keratinocytes infected with the same OCT4 and cultured in a chemical-free medium were used as controls. Cultures were split with Accutase (Millipore) and treated with 1 μM Thiazovivin (Stemgent) on day 1 post-split. iPSC colonies stained positive for Alexa Fluor 555 mouse anti-human TRA-1-81 antibody (BD Pharmingen) were selected for expansion on feeder cells within hESCM and cultured routinely.

[0314] HUVEC reprogramming

[0315] HUVECs were cultured in 100 mm tissue culture dishes and transduced twice with freshly prepared lentiviral supernatant (4-6 hours each time). 200,000 transduced HUVECs were seeded onto gelatin-coated 100 mm dishes and cultured in HCM. The culture medium was treated with 5 μM PS48, 0.25 mM NaB, and 0.5 μM A-83-01 for 2 weeks. Half the volume of medium was then replaced with hESCM, and 5 μM PS48, 0.25 mM NaB, and 0.5 μM A-83-01 were added for another 2 weeks. Then, the cell culture medium was replaced with hESCM, and 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01, and 0.5 μM PD0325901 were added for another 1-2 weeks. iPSC colonies stained positive for Alexa Fluor 555 mouse anti-human TRA-1-81 antibody were selected for expansion on feeder cells within hESCM and cultured routinely. Cultures were split with Accutase and treated with 1 μM Thiazovivin on day 1 post-splitting.

[0316] In vitro differentiation

[0317] In vitro differentiation of hiPSCs was performed using the standard embryoid (EB) method. Briefly, hiPSCs were detached with Accutase (Millipore), cultured for 8 days in ultra-low adhesion 6-well plates, and then transferred to differentiation medium in a Substrate-coated 6-well plate. The cells were fixed for immunocytochemical analysis or harvested after 8 days for RT-PCR experiments. Differentiation medium: DMEM / F12, 10% FBS, 1% Glutamax, 1% non-essential amino acids, 1% penicillin / streptomycin, 0.1 mM β-mercaptoethanol.

[0318] Alkaline phosphatase staining and immunocytochemical assay

[0319] Alkaline phosphatase staining was performed using an alkaline phosphatase assay kit (Stemgent) according to the manufacturer's protocol. Standard immunocytochemical assays were performed as previously reported (Li, W. et al., Stem Cells 27:2992-3000 (2009)). The primary antibody used is shown in Table 3. The secondary antibody was Alexa Fluor 488 donkey anti-mouse or anti-rabbit IgG (1:1000) (Invitrogen). Cell nuclei were visualized by DAPI (Sigma-Aldrich) staining. Images were captured using a Nikon Eclipse TE2000-U microscope.

[0320] Gene expression analysis using RT-PCR and qRT-PCR

[0321] For RT-PCR and qRT-PCR analysis, total RNA was extracted from human iPSCs using the RNeasy Plus mini kit (Qiagen) in combination with QIAshredder. iScript was used. TM The cDNA synthesis kit (BioRad) was used to perform first-strand reverse transcription using 2 μg of RNA. The expression of pluripotency markers was analyzed by RT-PCR using Platinum PCR SuperMix (Invitrogen). The expression of lineage-specific markers after differentiation was analyzed by qRT-PCR using iQ SYBR Green Supermix (Bio-Rad). Primers are listed in Table 2.

[0322] Microarray analysis

[0323] Microarray hybridization was performed using human Ref-8_v3 expression Beadchip (Illumina, CA, USA) to examine overall gene expression in NHEK, hiPSC, and hES cells. Biotin-16-UTP-labeled cRNA was synthesized from 500 ng of total RNA using the Illumina TotalPrep RNA Amplification Kit (Ambion AMIL1791, Foster City, CA, USA). Hybridization mixtures containing 750 ng of labeled amplified cRNA were prepared according to the Illumina BeadStation 500X System Manual (Illumina, San Diego, CA, USA) using the provided reagents and GE Healthcare's streptavidin-Cy3 staining solution. Hybridization with Illumina human Ref-8_v3 expression Beadchip was performed on a BeadChip HybWheel at 55 °C for 18 h. The array was scanned using an Illumina BeadArray reader. All samples were prepared in duplicate biological replicates. The microarray data were processed and analyzed using Illumina BeadStudio software. The data were background subtracted and normalized using the rank invariant option.

[0324] Bisulfate genome sequencing

[0325] Genomic DNA was isolated using a Non-Organic DNA Isolation Kit (Millipore) and then treated with an EZ DNA Methylation-Gold Kit (Zymo Research Corp., Orange, CA). The treated DNA was then used as a template to amplify the target sequence. Primers used for amplification of the OCT4 and NANOG promoter fragments are shown in Table 2. The obtained fragments were cloned using a TOPO TA Cloning Kit (Invitrogen) for sequencing and then sequenced.

[0326] hiPSC genotyping

[0327] Genotyping of hiPSC lines was performed using RT-PCR with specific primers and genomic DNA (Table 2; Yu, J. et al., Science 318:1917-1920 (2007) and Li, W. et al., Stem Cells 27:2992-3000 (2009)).

[0328] Teratoma formation

[0329] The hiPSC line was harvested using 0.05% trypsin-EDTA. Five million cells were injected subrenally into the kidney capsules of SCID mice (n=3). After 4–6 weeks, well-developed teratomas were harvested, fixed, and then subjected to histological analysis at the TSRI Histology Core facility.

[0330] Table 2 Primers used

[0331]

[0332] Table 3 shows the first antibody used.

[0333]

[0334] Table 4 Summary of the reprogramming experiment

[0335]

[0336] NHEK, neonatal human epidermal keratinocytes; HUVEC, human umbilical vein endothelial cells; AHEK, adult epidermal keratinocytes; AFDC, cells obtained from amniotic fluid. Chemical reagent concentrations used: PD, 0.5 μM PD0325901; A83, 0.5 μM A-83-01; PS48, 5 μM PS48; VPA, 0.5 mM valproic acid; NaB, 0.25 mM sodium butyrate; Par, 2 μM transphenylcyclopropane sulfate; CHIR, 3 μM CHIR99021. For reprogramming with four or three factors, NHEK cells were seeded at a density of 100,000 transduced cells / 10 cm dish, and positive colonies were counted after 4 weeks. For reprogramming with two factors, NHEK cells were seeded at a density of 100,000 transduced cells / 10 cm dish, and positive colonies were counted after 6 weeks. For reprogramming with one factor, NHEK and AHEK cells were seeded at a density of 1,000,000 transduced cells / 10 cm dish, and positive colonies were counted after 8 weeks. HUVECs and AFDCs were seeded at a density of 200,000 transduced cells / 10 cm dish, and positive colonies were counted after 6 weeks.

[0337] Table 5 Characterization of the established human iPSC cell lines

[0338]

[0339] Under standard hESC culture conditions, the characterized cell lines were proliferated for more than 20 generations, and their biomarker expression and pluripotency were further characterized; other established cell lines were stored at the 5th or 6th generation. (Blank entries indicate no assays were performed.)

[0340] Table 6. DNA fingerprinting analysis of Oct4-induced iPSCs and parental cell lines.

[0341]

[0342] We studied 15 polymorphic short tandem repeat (STR) DNA loci and the sex chromosome marker amelioprotein.

[0343] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art will propose different modifications or variations upon consideration of them. Such modifications or variations will be included within the spirit and scope of this application and within the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A reagent kit comprising: ALK5 inhibitors; MAP / ERK kinase (MEK) inhibitors; Rho kinase (ROCK) inhibitors, The ALK5 inhibitor, MEK inhibitor, and ROCK inhibitor are present in amounts that effectively increase the efficiency of reprogramming non-pluripotent mammalian cells into pluripotent cells, compared to non-pluripotent mammalian cells treated with ALK5 inhibitors and MEK inhibitors but without ROCK inhibitors. The ROCK inhibitor described herein has the following structure: (II) in, L 2 It is unsubstituted C1-C 10 Alkylene; y is an integer between 0 and 1; z is an integer between 0 and 1; X is either -N= or -CH=; R 1 It is hydrogen or an unsubstituted C1-C6 alkyl group; R 3 Yes - OR 18 , or substituted or unsubstituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is substituted with a halogen; R 4 Yes - OR 18 ;and R 18 It is hydrogen or an unsubstituted C1-C6 alkyl group; Or its pharmaceutically acceptable salt.

2. The kit according to claim 1, wherein the MEK inhibitor is PD0325901.

3. The kit according to claim 1, wherein the ALK5 inhibitor is A-83-01 or SB431542.

4. The kit according to claim 1, further comprising: (i) Glycogen synthase kinase 3 (GSK3) inhibitors; or (ii) Histone deacetylase (HDAC) inhibitors.

5. The kit according to claim 1, further comprising one or more non-pluripotent mammalian cells, wherein the non-pluripotent mammalian cells contain one or more exogenous transcription factors selected from the group consisting of Oct-3 / 4, Klf2 / 4, Sox2 and c-Myc.

6. An in vitro method for inducing non-pluripotent mammalian cells into induced pluripotent stem cells, the method comprising: Under conditions sufficient to induce pluripotent stem cells, The exogenous transcription factor containing Oct-3 / 4 was introduced into the non-pluripotent mammalian cells; and The non-pluripotent mammalian cells were induced using the kit described in any one of claims 1-4.

7. The method of claim 6, wherein introducing the exogenous transcription factor into the non-pluripotent mammalian cell comprises: One or more polynucleotides encoding the exogenous transcription factor are introduced into the non-pluripotent mammalian cells.

8. The method of claim 6, wherein introducing the exogenous transcription factor into the non-pluripotent mammalian cell comprises: The non-pluripotent mammalian cells are exposed to a polypeptide, wherein the polypeptide contains the amino acid sequence of the exogenous transcription factor.

9. A mixture comprising: One or more mammalian cells; ALK5 inhibitors; MAP / ERK kinase (MEK) inhibitors; GSK3 inhibitors; and Rho kinase (ROCK) inhibitors, The ROCK inhibitor described herein has the following structure: (II) in, L 2 It is unsubstituted C1-C 10 Alkylene; y is an integer between 0 and 1; z is an integer between 0 and 1; X is either -N= or -CH=; R 1 It is hydrogen or an unsubstituted C1-C6 alkyl group; R 3 Yes - OR 18 , or substituted or unsubstituted C1-C6 alkyl, wherein the substituted C1-C6 alkyl is substituted with a halogen; R 4 Yes - OR 18 ;and R 18 It is hydrogen or an unsubstituted C1-C6 alkyl group; Or its pharmaceutically acceptable salt; The mammalian cells mentioned therein include non-pluripotent cells.

10. The mixture according to claim 9, wherein (i) L 2 It is methylene; X is N= or CH=; R 1 It is hydrogen; or y and z are 0; or (ii) L 2 It is methylene; X is N= or CH=; R 1 It is hydrogen; and y and z are 0; or (iii) The structure is: , , or 。 11. The mixture according to claim 9, further comprising a histone deacetylase (HDAC) inhibitor.

12. The kit according to claim 1, wherein L2 is methylene; X is N= or CH=; R1 is hydrogen; or y and z are 0.

13. The kit according to claim 1, wherein the ROCK inhibitor has the following structure: ， ,or 。

Images