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Definitions

• Nanog is a fluorescent protein (GFP) and puromycin-resistant genes into the Nanog locus, forcibly expressed the above four genes in the fibroblasts derived from the transgenic mouse, and selected puromycin-resistant and GFP-positive cells, thereby successfully generated iPS cells (Nanog iPS cells) which are almost equal to embryonic stem (ES) cells in gene expression and epigenetic modification.
• GFP fluorescent protein
• Non-patent literatures 3 and 4 Similar results were also reproduced by other study groups (Non-patent literatures 3 and 4) . Thereafter, it was found that iPS cells can be prepared even using three genes of Oct3/4, Sox2 and Klf4, i.e. the above four genes except for c-Myc gene (Non-patent literature 5) .
• Non-patent literature 6 successfully generated human iPS cells by introducing the four genes similar to those used for generating mouse iPS cells into fibroblasts derived from human skin.
• Yu et al . prepared human iPS cells using Nanog and Lin28 instead of Klf4 and c-Myc.
• Park et al. prepared human iPS cells by using TERT known as a human cell-immortalizing gene and SV40 large T antigen, in
• Non patent literature 1 Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)
• Non patent literature 8 Park, I.H. et al . , Nature, 451: 141-146 (2008)
• Patent literature 2 International Publication No.
• An object of the present invention is to provide a novel method for improving iPS cell generation efficiency and factor for improving iPS cell generation efficiency.
• the present inventors intensively investigated with respect to Myc variants having an effect to improve iPS cell generation efficiency, which is comparable to or greater than that of c-Myc.
• c-Myc the activity to transform NIH3T3 cells
• the activity to induce iPS cells increases as compared with c-Myc
• the present invention relates to the following subject matters.
• a method for improving iPS cell generation efficiency which comprises a step of introducing a Myc variant having the following features (1) and (2).:
• mutation at position 135 in SEQ ID NO: 2 is substitution or deletion of the amino acid.
• reprogramming factors contains one or more factors selected from the- group consisting of members of the Oct family, members of the Sox family, members of the Klf family, members of the Lin28 family and Nanog, as well as nucleic acids encoding them.
• iPS cells are used for regeneration therapy, expression or reactivation of the Myc variant of the present invention or activation of endogenous gene present near the position into which the exogenous nucleic acid of the variant is integrated may increase the risk of tumor formation in the tissues regenerated from the
• differentiated cells derived from the iPS cells differentiated cells derived from the iPS cells.
• nucleic acid is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• a retroviral vector or a lentiviral vector When a retroviral vector or a lentiviral vector is used, silencing of the introduced gene may occur temporarily and then, the gene may be reactivated later. Accordingly, a method can be used preferably in which the nucleic acid encoding the Myc variant of the present invention is excised., for example, using a Cre/loxP system when it becomes unnecessary.
• loxP sequences are located at both ends of the above nucleic acid, a Cre recombinase is allowed to act on the cells using a plasmid or adenoviral vector after induction of iPS cells so that the region flanked with the loxP sequences is excised.
• the procedures may be carried out two or more times, for example, 3 times or 4 times
• a gene introduced may be integrated into the chromosome. Accordingly, it is eventually necessary to verify the absence of a gene insertion into the chromosome by southern blotting or PCR. For this purpose, it may be convenient to use a means in which a gene to be introduced is integrated into the chromosome and the gene is then removed, as in the above Cre-loxP system.
• a gene is integrated into the chromosome using a transposon, a transferase is then allowed to act on the cells using a plasmid or adenoviral vector so that the integrated gene is entirely removed from the chromosome.
• Another preferred non-integral vector includes an episomal vector which is autonomously replicable in
• any of these mutated loxP sequences may be located at any of the 5' and 3' of the above vector element. However, the mutated loxP sequences are needed to be inserted in a direction in which the mutation site is located at outer ends of the loxP
• the verification of removal of the vector element necessary for replication of the introduced gene from the iPS cells can be carried out by using a nucleic acid
• material for preparing iPS cells in the present invention may be any cells derived from a mammal except for germ cells.
• the mammal may be, for example, human, mouse, monkey, cattle, pig, rat or dog. Somatic cells derived from human are particularly preferred. Specifically, the following cells are exemplified: keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal
• Non limited examples of media to be used in this connection include a minimum essential medium (MEM) containing a fetal bovine serum of about 5 to 20%, a Dulbecco's modified
• Sox2 [wherein Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7 or Soxl8, Klf4 with Klfl, Klf2 or Klf5, and c-Myc with T58A (activated mutant) , N-Myc or L-Myc] ;
• Oct3/4 can be replaced with the other member of the Oct family such as OctlA and Oct6.
• Sox2 or Soxl, Sox3., Soxl5, Soxl7, Soxl8 can be replaced with the other member of the Sox family such as Sox7.
• the combination of nuclear reprogramming factors may or may not contain the member of the Myc family.
• remaining factors excepting the members of the Myc family are used as the combination of nuclear reprogramming factors to be used together with the Myc variant of the present invention.
• the combination of factors may be used as the combination of nuclear reprogramming factors together with the Myc variant of the present invention.
• a combination containing any additional factor in addition to the above disclosed nuclear reprogramming factors is also suitably used as the "combination of nuclear reprogramming factors" to be used in combination of the Myc variant of the present invention.
• accession numbers respectively. Those skilled in the art can easily isolate these cDNAs .
• the factor can be prepared by
• these nucleic acids may be carried on separate vectors, or joined in tandem to constitute a polycistronic vector. In the latter case, it is desirable to join a 2A self-cleaving peptide of foot-and-mouth disease virus between the nucleic acids in order to enable an efficient polycistronic expression (see Science, 322, 949-953, 2008 and others) (this document is herein
• the contact of a nuclear reprogramirting factor with somatic cells can be carried out as follows: (a) when the nuclear reprogramming factor is a protein factor, the contact may be carried out in a manner similar to that of the case of the Myc variant of the .present invention as described above, (b) when the nuclear reprogramming factor is a nucleic acid encoding the protein factor of the above (a) , the contact may be carried out in a manner similar to that of the case of a nucleic acid encoding the Myc variant of the present invention as described above, and . (c) when the nuclear reprogramming factor is a low molecular
• Dulbecco's modified Eagle's medium (DMEM) , an RPMI1640 medium, a 199 medium, an F12 medium and the like] suitable for cultivation of the somatic cells isolated from human or other mammals so that the concentration of the compound is in a range which is sufficient for nuclear reprogramming and does not cause cytotoxicity, and culturing the somatic cells in the medium for a certain period of time.
• SV40 large T can also be included in the category of a factor for improving iPS cell generation efficiency from the viewpoint that they are not essential for nuclear reprogramming of somatic cells but are auxiliary factors.
• auxiliary factor other than factors essential for the nuclear reprogramming step is classified as a nuclear reprogramming factor or as a factor for improving iPS cell generation efficiency may be made conveniently.
• a factor for improving iPS cell generation efficiency with somatic cells can be carried out depending on cases in which the factor is (a) a protein factor, (b) a nucleic acid encoding the protein factor, or (c) a low molecular compound, by a method similar to that described above for the Myc variant of the present
• the combination of nuclear reprogramming factors is a combination of nucleic acids encoding the protein factors and the factor for improving iPS cell generation efficiency other than the Myc variant of the invention is a chemical inhibitor
• the former factors require a certain period of time before the protein factors are expressed in large amounts after the nucleic acids are introduced into the cells, while the latter factor can rapidly act on the cells.
• condition means that oxygen concentration in the
• the oxygen concentration in the atmosphere when culturing cells is significantly lower than that in air.
• a condition in which the oxygen concentration is lower than that in the atmosphere of 5-10% CC>2/95-90% air generally used in a conventional cell cultivation is preferably employed, and a condition is included in which the oxygen concentration in atmosphere is not greater than 18%, for example.
• the oxygen concentration in the atmosphere is not greater than 15% (e.g., not greater than 14%, not greater than 13%, not greater than 12% and not greater than 11%), not greater than 10% (e.g., not greater than 9%, not greater than 8%, not greater than 7% and not greater than 6%), or not
• the oxygen is greater than 5% (e.g., not greater than 4%, not greater than 3% and not greater than 2%) . Also, the oxygen
• concentration in atmosphere is preferably not less than 0.1% (e.g., not less than 0.2%, not less than 0.3% and not less than 0.4%), not less than 0.5% (e.g., not less than 0.6%, not less than 0.7%, not less than 0.8% and not less than 0.95%), or not less than 1% (e.g., not less than 1.1%, not less than 1.2%, not less than 1.3% and not less than 1.4%) .
• a procedure generating a hypoxic condition in cell culture environment is not limited to a particular one.
• a method of culturing cells in a C0 2 incubator allowing adjustment of the oxygen concentration may be the easiest and may be employed as a suitable example.
• incubator allowing adjustment of the oxygen concentration is available on the market from various manufacturers (for example, C0 2 incubators for hypoxic cultivation supplied by makers such as Thermo Scientific Co., Ikemoto Scientific Technology Co., Ltd., Juj i Field Inc., and akenyaku Co., Ltd. can be used) .
• the period to culture the cells under the hypoxic condition is not limited to a particular one so far as improvement of iPS cell generation efficiency is not prevented as compared with the case using normal oxygen concentration (20%) .
• the period may be not less than 3 days, not less than 5 days, not less than 7 days or not less than 10 days, and not more than 50 days, not more than 40 days, not more than 35 days or not more than 30 days, but is not limited thereto.
• Preferable period for culturing under the hypoxic condition also varies depending on the oxygen concentration in the atmosphere, and those skilled in the art can suitably adjust the culture period depending on the oxygen
• the cells can be cultured under a condition suitable for cultivation of ES cells, for example.
• ES cells for example.
• a condition suitable for cultivation of ES cells for example.
• mouse cells a
• the selection of candidate colonies of iPS cells can be carried out by using the drug resistance and
• the former method uses, for example, recombinant somatic cells in which a drug
• resistance gene and/or a reporter gene are targeted at a locus of a gene, for example, Fbxl5, Nanog or Oct3/4, and preferably Nanog or Oct3/4, highly expressing specifically in differentiated pluripotent cells are employed, and drug resistance- and/or reporter activity-positive colonies are selected.
• Such recombinant somatic cells include, for example, MEF or TTF derived from a mouse in which geo gene that encodes a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase is knocked into the Fbxl5 locus [Takahashi & Yamanaka, Cell, 126, 663-676 (2006); this document is herein incorporated by reference] , or MEF or TTF derived from a transgenic mouse in which green
• GFP fluorescent protein
• candidate colonies can be selected by visual morphology observation, for example, according to Takahashi et al . , Cell, 131, 861-872 (2007) (this document is herein incorporated by reference) .
• the resulting iPS cells are novel cells different from previously known iPS cells since they contain the exogenous nucleic acid.
• the exogenous nucleic acid is introduced into the somatic cells using a
• the exogenous nucleic acid is usually integrated into the genome of the resulting iPS cells, and therefore, the exogenous nucleic acid is stably retained.
• both c-Myc and L-Myc have an identity near 100% at the amino acid level between human and mouse. Also, even when mouse c-Myc and mouse L-Myc were used, human iPS colonies arose in the same manner as in human c-MYC and human L-MYC were used. Accordingly, mouse genes were used in the experiments as alternatives to the human genes.
• the point mutations in human c-Myc and L-Myc corresponding to those in mouse factors are shown in the following Table 1. The mutated sites of these point mutants on the human amino acid sequence are as shown in Fig. 1.
• Three factors of OCT3/4, KLF4 and SOX2 derived from human are designated as "3F”.
• Retroviral vectors encoding variants cdNl to cdN7 of human c-MYC having N-terminal deletion were prepared.
• fragments were amplified by PCR using human c-MYC cDNA as a template and the following sets of primers.
• HsMyc-AS TCACGCACAAGAGTTCCGTAGCTGTTC AG (SEQ ID NO: 18)
• the retroviral vectors pMXs-cdNl to cdN7 were:
• aHDF-Slc7al a mouse ecotropic virus receptor Slc7al gene
• the aHDF-Slc7al cells were seeded on a 6-well plate in a proportion of 1 x 10 5 cells per well.
• a total of four genes consisting of three genes of OCT3/4, KLF4, and SOX2 that were derived from human and each variant having N-terminal deletion prepared in the above 1) were retrovirally introduced into the cells according to Cell, 131, 861-872 (2007) (in Fig. 3, 3F-cdNl to 3F-cdN7) .
• the aHDF-Slc7al cells were infected with each of the above retroviruses, cultured, recovered after 6 days, and again seeded on MSTO cells (5 x 10 5 cells/100 mm dish) . From the next day, the culture medium was replaced with the medium for culturing primate ES cells (ReproCELL)
• 3F-cdN4 showed very few colonies, and therefore, it was presumed that a region acting positively on the induction of iPS cells is present at positions 65 to 82 in human c-MYC. However, this also suggested a region acting positively on the induction of iPS cells.
• 3FL, 3F-Ms-L, 3F-Ms-cl36 and 3F-Ms- L325 showed a remarkable increase of the number of human iPS colonies (increase of iPS induction activity), and also an increase of the percentage of the number of iPS colonies to the total number of colonies, as compared with the 3Fc.
• Example 2 Investigation of induction of iPS cells with a variant of human C-W135E having N-terminal deletion
• Point mutation of human W135E was introduced by carrying out PCR using pENTR-D-TOPO-cdNl to cdN6
• the retroviral vectors pMXs-cl35dNl to cl35dN6 were individually introduced into Plat-E cells, in the same manner as in Example 1-2) . Then, western blotting was carried out by a conventional method using a lysate of the Plat-E cells. The results are shown in Fig.. 4. From the fact that a band of endogenous c-MYC (an upper band in each lane of cl35dNl to cl35dN6) and also a band having a size corresponding to each variant were detected in each sample, the expression of each variant was verified.
• Example 3 Investigation of induction of iPS cells with variants of L-MYC derivatives having N-terminal deletion
• Retroviral vectors encoding variants LdN2, LdN4, LdN5, LdN6 and LdN7 of human L-MYC having N-terminal deletion were prepared (excepting L-MYC variants corresponding to cdNl and cdN3; see Fig. 1) .
• fragments were amplified by PCR using human L-MYC cDNA as a template and using the following sets of primers.
• the LR reaction was carried out between pENTR-D- TOPO-LdN2, -LdN4, -LdN5, -LdN6 and -LdN7, which were obtained by cloning each of the resulting fragments into pENTR-D-TOPO ( Invitrogen) , and retroviral vector pMXs-gw to prepare pMXs-LdN2, -LdN4 , -LdN5, -LdN6 and -LdN7..
• Example 1 The number of human iPS colonies (ES-like colonies) generated on the thirty-ninth day after the retroviral infection was counted. The results are shown in Fig. 6 . It was found that increasing deletion of the N- terminus of L-MYC1 decreased the number of iPS colonies. Also, 3F-LdN2 and 3F-LdN4 showed an increase of the number of human iPS colonies (increase of iPS induction activity), and also an increase of the percentage of the number of iPS colonies to the total number of colonies, as compared with the 3Fc.
• the present inventors investigated whether the variants prepared have a transformation activity.
• Mouse NIH3T3 cells were seeded on a 6-well plate in a proportion of 1 x 10 5 cells per well. The next day, the following genes derived from human: c-MYC, C-W135E, L- MYC, cdNl to cdN7, and cl35dNl to cl35dN6.; and the following genes derived from human: c-MYC, C-W135E, L- MYC, cdNl to cdN7, and cl35dNl to cl35dN6.; and the following genes derived from human: c-MYC, C-W135E, L- MYC, cdNl to cdN7, and cl35dNl to cl35dN6.; and the following genes derived from human: c-MYC, C-W135E, L- MYC, cdNl to cdN7, and
• cdNl had a transformation activity, but lost the transformation activity by mutating position 135 (cl35dNl in Fig. 9) . Also, any of cdN2 to cdN7 and cl35dN2 to cl35dN6 did not have the transformation activity (Fig. 9) .

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