WO2014181682A1 - Composition pour la production de cellules souches pluripotentes induites et méthode de production de cellules souches pluripotentes induites - Google Patents

Composition pour la production de cellules souches pluripotentes induites et méthode de production de cellules souches pluripotentes induites Download PDF

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WO2014181682A1
WO2014181682A1 PCT/JP2014/061378 JP2014061378W WO2014181682A1 WO 2014181682 A1 WO2014181682 A1 WO 2014181682A1 JP 2014061378 W JP2014061378 W JP 2014061378W WO 2014181682 A1 WO2014181682 A1 WO 2014181682A1
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gene
gene product
product
pluripotent stem
induced pluripotent
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Japanese (ja)
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大敬 伊関
康司 岡崎
晶彦 奥田
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学校法人埼玉医科大学
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Priority claimed from JP2013100311A external-priority patent/JP2014217344A/ja
Priority claimed from JP2013100312A external-priority patent/JP2014217345A/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/604—Klf-4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/606—Transcription factors c-Myc
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a composition for producing induced pluripotent stem cells and a method for producing induced pluripotent stem cells.
  • iPS induced pluripotent stem cells
  • induced pluripotent stem cells have properties such as pluripotency, self-replication, and high proliferation ability. It is known that differentiation can be differentiated into cardiomyocytes, blood cells, retinal pigment epithelial cells, nerve cells and the like by inducing differentiation. Therefore, for example, it is expected to be used as a regenerative medicine such as cell transplantation treatment, drug screening, and a tool for elucidating the cause of a disease.
  • a method for producing the induced pluripotent stem cell for example, a method for producing by introducing Oct3 / 4 gene, Sox2 gene, Klf4 gene, and c-Myc gene into somatic cells has been proposed (for example, patents).
  • Reference 1 a method for producing by introducing Oct3 / 4 gene, Sox2 gene, Klf4 gene, and c-Myc gene into somatic cells.
  • induced pluripotent stem cells can be produced, (1) production efficiency is low, (2) production period is long, and (3) human induced pluripotent stem cells are Leukemia inhibitory.
  • LIF naive-type induced pluripotent stem cells having a factor
  • the present invention provides a method for producing an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality, and a composition for producing induced pluripotent stem cells. Objective.
  • Means for solving the problems are as follows. That is, ⁇ 1> (A) Oct3 / 4 gene or its gene product; (B) the Sox2 gene or its gene product; (C) the Klf4 gene or its gene product; (D) the c-Myc gene or its gene product; (E) Jarid2 (Jumonji, AT rich interactive domain 2) mutant gene or its gene product, Prdm14 (PR domain containing 14) gene or its gene product, Esrrb (Estrogen-related receptor gene, or its gene) (Sal-like 4, transscript variant a) introducing into a somatic cell at least one selected from the group consisting of a gene or a gene product thereof, A method for producing an induced pluripotent stem cell, wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • ⁇ 2> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product, A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product.
  • an artificial pluripotent stem cell that can solve the above-mentioned problems and can achieve the above-mentioned object, is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality.
  • a production method and a composition for producing induced pluripotent stem cells can be provided.
  • FIG. 1-1 is a graph showing the results of Test Example 1-1.
  • FIG. 1-2 is a graph showing the results of Test Example 1-2.
  • FIG. 1-3 is a graph showing the results of Test Example 1-3.
  • FIG. 1-4 is a graph showing the results of Test Example 1-4.
  • FIG. 1-5 is a graph showing the results of Test Example 1-5.
  • FIG. 1-6 is a graph showing the results of Test Example 1-6.
  • FIG. 2-1 is a graph showing the results of Test Example 2-1.
  • FIG. 2-2 is a graph showing the results of Test Example 2-2.
  • FIG. 2-3 is a graph showing the results of Test Example 2-3.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3A is a photograph showing an example of the results obtained when the virus solution of (1) in Test
  • FIG. 3B is a photograph showing another example of the result obtained when the virus solution of (1) in Test Example 3-1 was used.
  • FIG. 3C is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-1 was used.
  • FIG. 3D is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-1 was used.
  • FIG. 3E is a photograph showing an example of the results obtained when the virus solution of (1) in Test Example 3-2 was used.
  • FIG. 3F is a photograph showing another example of the results obtained when the virus solution of (1) in Test Example 3-2 was used.
  • FIG. 3G is a photograph showing an example of the results obtained when the virus solution of (2) in Test Example 3-2 was used.
  • FIG. 3H is a photograph showing another example of the results obtained when the virus solution of (2) in Test Example 3-2 was used.
  • the method for producing induced pluripotent stem cells of the present invention comprises (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, (D Selected from the group consisting of :) c-Myc gene or its gene product, (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, or Sall4a gene or its gene product.
  • a somatic cell hereinafter sometimes referred to as a “gene or gene product introduction process”
  • the gene or its gene product introduction step includes at least (A) Oct3 / 4 gene or its gene product, (B) Sox2 gene or its gene product, (C) Klf4 gene or its gene product, and (D) c At least selected from the group consisting of the Myc gene or its gene product, and (E) the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • the gene product refers to mRNA (messenger RNA) transcribed from a gene and protein translated from the mRNA.
  • (E) Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product there is a particular limitation.
  • (1) an embodiment including a Jarid2 mutant gene or its gene product, (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or An embodiment including the gene product, (3) an embodiment including the Jarid2 mutant gene through the gene product, the Prdm14 gene through the gene product, the Esrrb gene through the gene product, and the Sall4a gene through the gene product are preferred, and (3) Jarid Variant gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product, and is more preferred embodiment including Sall4a gene or its gene product.
  • the more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time,
  • the origin of the Oct3 / 4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Oct3 / 4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_002701 (human) and NM_013633 (mouse).
  • Sox2 gene- The origin of the Sox2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Sox2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_003106 (human) and NM_011443 (mouse).
  • Klf4 gene- The origin of the Klf4 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • sequence information of the Klf4 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004235 (human) and NM_0103737 (mouse).
  • c-Myc gene- The origin of the c-Myc gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the c-Myc gene can be obtained from a known database. For example, NCBI can obtain the accession numbers NM_002467 (human) and NM_010849 (mouse).
  • the Jarid2 mutant gene is a gene (see SEQ ID NO: 12) encoding the 1st to 551st amino acids at the N-terminus of the Jarid2 protein.
  • the origin of the Jarid2 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Jarid2 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004973 (human) and NM_021878 (mouse).
  • Prdm14 gene- The origin of the Prdm14 gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Prdm14 gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_024504 (human) and NM_001081209 (mouse, see SEQ ID NO: 19).
  • -Esrrb gene- There is no restriction
  • the sequence information of the Esrrb gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_004452 (human) and NM_011934 (mouse, see SEQ ID NO: 20).
  • the origin of the Sall4a gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans and mice.
  • the sequence information of the Sall4a gene can be obtained from a known database. For example, NCBI can obtain accession numbers NM_020436 (human) and NM_175303 (mouse, see SEQ ID NO: 21).
  • Oct3 / 4 gene to its gene product Sox2 gene to its gene product, Klf4 gene to its gene product, c-Myc gene to its gene product, Jarid2 mutant gene to its gene product, Prdm14 gene to its gene product, Esrrb
  • the sequence of the gene or its gene product, and the sequence of the Sall4a gene or its gene product are not particularly limited as long as the effects of the present invention are not impaired, and may be only the portion of the sequence of each gene that is translated into a protein. However, other parts may be included. Further, the sequence of each gene or gene product thereof may contain a mutation.
  • the mutation examples include a mutation that does not affect the amino acid sequence of the protein of each gene, and one or several (2 to 5) amino acids are deleted from the amino acid sequence of the protein of each gene. Substitution, insertion, or mutation to be added.
  • the homology with the wild-type when each gene or its gene product has a mutation is not particularly limited and can be appropriately selected according to the purpose. 70% or more is preferable, 80% or more is more preferable, and 90% or more is particularly preferable.
  • tissue stem cells such as adipose tissue-derived stromal (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, sperm stem cells; tissue precursor cells; lymphocytes And already differentiated cells such as epithelial cells, muscle cells, fibroblasts, and the like.
  • the somatic cells may be recombinant so as to facilitate selection of induced pluripotent stem cells.
  • the recombinant cell include a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in a pluripotent cell.
  • the gene that is specifically highly expressed in the pluripotent cells include Fbx15 gene, Nanog gene, Oct3 / 4 gene, and the like.
  • the reporter gene include a green fluorescent protein (GFP) gene and a ⁇ -galactosidase gene.
  • the drug resistance gene include a puromycin resistance gene and a neomycin resistance gene.
  • the individual that collects the somatic cells is not particularly limited and can be appropriately selected according to the purpose.However, when the obtained induced pluripotent stem cells are used for regenerative medicine, from the viewpoint of rejection, the individual itself or another individual with the same or substantially the same type of MHC is preferred.
  • the type of the MHC is substantially the same when the cells obtained by inducing differentiation from the somatic cell-derived induced pluripotent stem cells are transplanted into an individual by using an immunosuppressant or the like. It means that the MHC types match to such an extent that cells can be engrafted.
  • the somatic cell culture conditions are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a culture temperature of about 37 ° C. and a CO 2 concentration of about 2% to 5%.
  • the medium used for culturing the somatic cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a medium (DMEM), RPMI 1640 medium, 199 medium, and F12 medium.
  • the method for introducing each gene or its gene product into a somatic cell is not particularly limited and can be appropriately selected depending on the purpose.
  • a method using a vector, a method using synthesized mRNA, a recombinant protein The method used, etc. are mentioned.
  • a viral vector for example, a viral vector, an artificial chromosome vector, a plasmid vector, an episomal vector etc. are mentioned.
  • the viral vectors include retrovirus (including lentivirus) vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, silvis Examples thereof include viral vectors, rhabdovirus vectors, paramyxovirus vectors, orthomyxovirus vectors, and the like.
  • the artificial chromosome vector examples include a YAC (Yeast Artificial Chromome) vector, a BAC (Bacterial Artificial Chromosome) vector, a PAC (P1-derived Artificial Chromome) vector, and the like.
  • the method for introducing the vector into the somatic cell is not particularly limited and may be appropriately selected depending on the purpose. For example, lipofection method, microinjection method, DEAE dextran method, gene gun method, electroporation method , Calcium phosphate method, and the like.
  • virus particles obtained using packaging cells may be used.
  • the packaging cell is a cell into which a gene encoding a structural protein of a virus has been introduced.
  • a recombinant virus vector incorporating a target gene is introduced into the cell, a recombinant virus particle incorporating the target gene is produced.
  • the packaging cell is not particularly limited and may be appropriately selected according to the purpose.
  • packaging cells based on human kidney-derived HEK293 cells or mouse fibroblast-derived NIH3T3 cells Ecotropic virus PLAT-E cells designed to express derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-E cells”), PLAT-A cells designed to express envelope glycoproteins derived from Amphotropic virus And PLAT-GP cells designed to express vesicular stomatitis virus-derived envelope glycoproteins (hereinafter sometimes referred to as “PLAT-GP cells”).
  • PLAT-A cells and PLAT-GP cells are preferable from the viewpoint of host orientation when a recombinant viral vector is introduced into human somatic cells.
  • the method for introducing the viral vector into the packaging cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a lipofection method, an electroporation method, and a calcium phosphate method.
  • the method for infecting the somatic cells with the obtained virus particles is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a polybrene method.
  • the vector may contain a marker gene for confirming the introduction of each gene.
  • the marker gene refers to a gene that enables selection and selection of cells by introducing the marker gene into cells.
  • Specific examples of the marker gene include a drug resistance gene, a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, and the like. These may be used individually by 1 type and may use 2 or more types together.
  • Specific examples of the drug resistance gene include a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, and a hygromycin resistance gene.
  • fluorescent protein gene examples include a GFP gene, a yellow fluorescent protein (YFP) gene, and a red fluorescent protein (RFP) gene.
  • luminescent enzyme gene examples include luciferase gene.
  • chromogenic enzyme gene examples include ⁇ -galactosidase gene, ⁇ -glucuronidase gene, alkaline phosphatase gene, and the like.
  • one gene may be incorporated into one vector, or two or more genes may be incorporated.
  • the two or more genes can be expressed simultaneously (hereinafter sometimes referred to as “co-expression”).
  • the c-Myc gene and the Jarid2 mutant gene are preferably introduced so as to be coexpressed.
  • the method for incorporating two or more genes into the one vector is not particularly limited and may be appropriately selected depending on the intended purpose. However, it is preferable to incorporate the two or more genes through a linking sequence.
  • the linking sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a gene sequence encoding 2A peptide derived from foot-and-mouth disease virus (Picornaviridae Aphthovirus), IRES (internal ribosome entry sites), and the like. It is done.
  • the number of introduction of each gene or its gene product into a somatic cell may be one time or two or more times. There is no particular limitation on the introduction timing of each gene or its gene product into a somatic cell, and it can be appropriately selected according to the purpose. Even if all the genes or their gene products are introduced simultaneously. It may be introduced at different times.
  • or its gene product may be used individually by 1 type, and may use 2 or more types together.
  • an embodiment using only a gene or an embodiment using only a gene product may be used, and among each of the above genes, a gene product may be used and another gene may be a gene. There may be.
  • each gene or its gene product As the introduction amount of each gene or its gene product into a somatic cell, all the genes or their gene products may be introduced in equal amounts or in different amounts.
  • the Oct3 / 4 gene is introduced in a large amount, for example, about 3 times the amount of the Sox2 gene, the Klf4 gene, or the c-Myc gene ( PNAS 106 (31): 12759-1276.2009, J. Biol. Chem. 287 (43): 36273-36282.201) are preferred.
  • genes or gene products other than the genes or gene products may be introduced as long as the effects of the present invention are not impaired.
  • the genes or gene products other than the genes or gene products thereof include, for example, the Oct family (Oct1A and Oct6) genes or their gene products, the Klf family (Klf1, Klf2, Klf4, and Klf5) genes or their gene products.
  • Myc family (N-Myc, and L-Myc) genes to their gene products
  • Sox family (Sox1, Sox3, Sox7, Sox15, Sox17, and Sox18) genes to their gene products
  • TERT genes to their gene products
  • SV40 Large T antigen gene or its gene product HPV16 E6 gene or its gene product, HPV16 E7 gene or its gene product, Bmil gene or its gene product, Fbx15 gene or its gene product , Nanog gene or its gene product, ERas gene or its gene product, ECAT15-2 gene or its gene product, Tcl1 gene or its gene product, ⁇ -catenin gene or its gene product, ECAT1 gene or its gene product, Esg1 gene or its Its gene product, Dnmt3L gene or its gene product, ECAT8 gene or its gene product, Gdf3 gene or its gene product, ECAT15-1 gene or its gene product, Fthl17 gene or its gene product, Rex1 gene or its gene product, UTF1 gene Or
  • Nr5a2 gene or its Gene product Rar family gene to its gene product (Proc. Natl. Acad. Sci. USA 108 (45): 18283-18288.2011); Utx gene to its gene product, Mdm2 gene to its gene product, Ring1b gene to Examples thereof include the gene product, the Wdr5 gene and the gene product thereof.
  • a low molecular compound may be administered as long as the effects of the present invention are not impaired.
  • the low molecular compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a GSK-3 inhibitor and an adenylate cyclase activator. Specific examples of the GSK-3 inhibitor include CHIR99021. Specific examples of the adenylate cyclase activator include Forskolin. There is no restriction
  • the other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose.
  • genes for culturing somatic cells into which each of the genes or gene products thereof has been introduced are introduced.
  • the gene product-introduced cell culture step is performed.
  • the gene or gene product-introduced cell culture step is a step of culturing somatic cells into which each gene or gene product has been introduced.
  • the culture conditions of the gene or its gene product-introduced cell are not particularly limited and can be appropriately selected according to the purpose.
  • the culture temperature is about 37 ° C.
  • the CO 2 concentration is about 2% to 5%
  • Etc is about 2% to 5%
  • the medium used for culturing the gene or its gene product-introduced cell is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the induced pluripotent stem cells produced by the method for producing induced pluripotent stem cells have differentiation pluripotency and self-renewal ability.
  • the term “pluripotency” means that all three germ layers can be differentiated.
  • the self-replicating ability means the ability to proliferate while maintaining an undifferentiated state.
  • the method for confirming whether or not the cell produced by the method for producing an induced pluripotent stem cell is an induced pluripotent stem cell is not particularly limited and can be appropriately selected depending on the purpose.
  • the somatic cell is a recombinant cell in which at least one of a reporter gene and a drug resistance gene is incorporated into a gene locus that is specifically highly expressed in differentiated pluripotent cells
  • This can be confirmed using the reporter gene and drug resistance gene.
  • a GFP gene is used as the reporter gene
  • a method of confirming GFP-positive cells with a flow cytometer can be mentioned, and a puromycin resistance gene can be used as the drug resistance gene. When used, it can be confirmed by administering puromycin to the cells.
  • the type of the induced pluripotent stem cell may be a prime type or a naive type, but a naive type is preferable in terms of excellent quality.
  • the method for confirming whether or not the artificial pluripotent stem cells are na ⁇ ve is not particularly limited and may be appropriately selected depending on the purpose. For example, the form of colonies, bFGF (basic fibroblast growth factor) Can be propagated in a single cell, whether it can be grown in a LIF-containing medium not containing LIF, or can be grown in a 2i-added medium (medium containing a MEK inhibitor and a GSK inhibitor) Whether or not it is used as an index can be confirmed.
  • bFGF basic fibroblast growth factor
  • the colony has a dome shape, can be grown in a LIF-containing medium not containing bFGF, can be grown in a 2i-added medium, and can be dispersed and passaged into single cells. If it is a cell that can be used, it can be determined to be naive.
  • the seed of the induced pluripotent stem cell is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably human.
  • composition for producing induced pluripotent stem cells is selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product. And at least one other component as necessary.
  • Jarid2 mutant gene or its gene product there is no particular limitation as at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product.
  • an embodiment including a Jarid2 mutant gene or its gene product (2) a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product
  • Jarid2 mutant gene or its gene product, Prdm14 gene or its gene product, Esrrb gene or its gene product, and Sall4a gene or its gene product are preferred
  • Jarid2 mutation Gene or its gene product, PRDM14 gene or its gene product, Esrrb gene or its gene product and is more preferred embodiment including Sall4a gene or its gene product.
  • the more preferable embodiment is advantageous in that it is superior in the production efficiency of induced pluripotent stem cells, can be produced in a shorter period of time, and is superior in quality.
  • the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product, as described in the method for producing induced pluripotent stem cells. It is.
  • the Jarid2 mutant gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the mode of the Jarid2 mutant gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose.
  • the mode in which the gene is incorporated into a vector Synthetic mRNA embodiments, recombinant protein embodiments, and the like. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • Prdm14 gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells.
  • the Prdm14 gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Prdm14 gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose. Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the Esrrb gene and its gene product are the same as those described in the method for producing induced pluripotent stem cells. Further, the Esrrb gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Esrrb gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected depending on the purpose.
  • Embodiment in which the gene is incorporated into a vector Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the Sall4a gene or its gene product is the same as that described in the method for producing induced pluripotent stem cells.
  • the Sall4a gene or its gene product may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the embodiment of the Sall4a gene or gene product thereof in the composition for producing induced pluripotent stem cells is not particularly limited and can be appropriately selected according to the purpose.
  • the embodiment in which the gene is incorporated into a vector synthesis Examples include mRNA and recombinant protein. Examples of the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the other configuration is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. Or a gene product thereof, and a c-Myc gene or a gene product thereof.
  • the Oct3 / 4 gene or its gene product, Sox2 gene or its gene product, Klf4 gene or its gene product, and c-Myc gene or its gene product are the same as those described in the method for producing induced pluripotent stem cells. belongs to. Further, each gene or gene product thereof may contain the same mutation as described in the method for producing induced pluripotent stem cells.
  • the Oct3 / 4 gene or its gene product, the Sox2 gene or its gene product, the Klf4 gene or its gene product, and the c-Myc gene or its gene product in the composition for producing induced pluripotent stem cells are particularly There is no restriction
  • the aspect by which the gene is integrated in the vector, the aspect of synthetic mRNA, the aspect of recombinant protein, etc. are mentioned.
  • the vector include those described in the method for producing induced pluripotent stem cells.
  • the synthetic mRNA and the recombinant protein can be produced by a known method.
  • the c-Myc gene and the Jarid2 mutant gene in the composition for producing induced pluripotent stem cells can be co-expressed.
  • the genes or gene products thereof may be divided into individual containers or may be combined into one container. Alternatively, any number may be collected in a container.
  • the amount of each gene or its gene product in the composition for producing induced pluripotent stem cells is not particularly limited, and all the genes or their gene products may be equal amounts or different amounts.
  • the composition for producing an induced pluripotent stem cell may contain a gene other than the gene or a gene product thereof or a gene product thereof.
  • a packaging cell is used. May be included. Examples of the genes or gene products other than the genes or gene products thereof and the packaging cells include those described in the method for producing induced pluripotent stem cells.
  • RNA was extracted from B6 mouse-derived embryonic stem cells (hereinafter sometimes referred to as “ESC”, obtained from the National University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and represented by SEQ ID NOs: 3 and 4 below.
  • ESC B6 mouse-derived embryonic stem cells
  • SEQ ID NOs: 3 and 4 SEQ ID NOs: 3 and 4 below.
  • Amplification and recovery of the Jarid2 mutant gene (the gene encoding the 1st to 551st amino acids of the N-terminal of the Jarid2 protein (see SEQ ID NO: 12)) by PCR using the primers obtained, and the pMXs vector (National University) Incorporated into a multicloning site of the University of Tokyo Medical Science Institute), a Jard2 mutant gene-containing virus vector was obtained.
  • Jarid2 mutant gene-containing virus vector as a template, and using primers represented by SEQ ID NOs: 9 and 10 below as primers, a part of Picoraviridae Aphthovirus-derived 2A peptide and a Jarid2 mutant gene (Jarid2 protein)
  • a gene encoding the N-terminal 1st to 551st amino acids (hereinafter sometimes referred to as “2A_Jarid2 ⁇ C gene”) was amplified and recovered.
  • the c-Myc_2A gene and the 2A_Jarid2 ⁇ C gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science).
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized. Then, using the primers represented by SEQ ID NOs: 1 and 2 below, Jarid2 A gene (a gene encoding the full length of the Jarid2 protein (see SEQ ID NO: 11)) is amplified, recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Jarid2 gene-containing viral vector Got.
  • pMXs vector obtained from the National University Corporation, Tokyo Institute of Medical Science
  • Jarid2 gene-containing viral vector as a template and using primers represented by the following SEQ ID NOs: 7 and 8, a part of Picoraviridae Aphthovirus-derived 2A peptide and the Jarid2 gene (encoding the full length of the Jarid2 protein)
  • the gene to be amplified (hereinafter sometimes referred to as “2A_Jarid2WT gene”) was amplified and recovered.
  • the c-Myc_2A gene and the 2A_Jarid2WT gene are incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation Tokyo Medical Research Institute).
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the National University of Tsukuba Life Science Animal Resource Center), and cDNA was synthesized, followed by PCR using the primers represented by SEQ ID NOs: 13 and 14 below by Prdm14.
  • a gene (gene encoding the full length of Prdm14 protein (see SEQ ID NO: 19)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and a Prdm14 gene-containing viral vector Got.
  • RNA was extracted from B6 mouse-derived embryonic stem cells (obtained from the University of Tsukuba Life Science Animal Resource Center), cDNA was synthesized, and Esrrb was synthesized by PCR using primers represented by SEQ ID NOs: 15 and 16 below.
  • a gene (a gene encoding the full length of the Esrrb protein (see SEQ ID NO: 20)) is amplified, recovered, incorporated into a multi-cloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Institute of Medical Science), and an Esrrb gene-containing viral vector Got.
  • a gene (a gene encoding the full length of the Sall4a protein (see SEQ ID NO: 21)) is amplified and recovered, incorporated into a multicloning site of a pMXs vector (obtained from the National University Corporation, Tokyo Medical Science Institute), and a Sall4a gene-containing viral vector Got.
  • the PLAT-E cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-E cell was collected the day after the medium was changed, and then filtered using a 0.45 ⁇ m filter, and the Oct3 / 4 gene-containing virus solution-1.
  • Production Example 2-6 Virus solution-1 containing c-Myc gene and Jarid2 mutant gene
  • the point that the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used in Production Example 2-1 was that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used.
  • the c-Myc gene and Jarid2 mutant gene-containing virus solution-1 were obtained in the same manner as in Production Example 2-1, except that the gene-containing virus vector was used.
  • the PLAT-GP cell culture medium was changed the day after the transfection, and the culture supernatant of the PLAT-GP cell was collected the day after the medium was replaced, and then filtered using a 0.45 ⁇ m filter, and Oct3 / 4 gene-containing virus solution-2.
  • Production Example 3-4 c-Myc gene-containing virus solution-2)
  • the point that the Oct3 / 4 gene-containing virus vector obtained in the production example 1-1 was used was replaced with the c-Myc gene-containing virus vector obtained in the production example 1-4.
  • a c-Myc gene-containing virus solution-2 was obtained in the same manner as in Production Example 3-1.
  • Production Example 3-5 Viral fluid-2 containing c-Myc gene and Jarid2 mutant gene
  • the Oct3 / 4 gene-containing virus vector obtained in Production Example 1-1 was used, except that the c-Myc gene obtained in Production Example 1-6 and the Jarid2 mutant were used.
  • a c-Myc gene and a Jarid2 mutant gene-containing virus solution-2 were obtained in the same manner as in Production Example 3-1, except that the gene-containing virus vector was used.
  • NG-MEF mouse fetal fibroblasts
  • STOCK Tg Nanog gene promoter-dependent manner
  • STOCK Tg Nanog-GFP, Puro 1Yam (No. RBRC02290) was obtained, mated, and isolated and prepared from trypsin on day 13.5).
  • the NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • the virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) to (3) below were each infected with the NG-MEF.
  • the day after the infection the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and Jarid2 gene-containing virus solution- 1 600 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and Jarid2 mutant gene-containing virus Liquid-1 600 ⁇ L
  • the ratio of the induced pluripotent stem cells to the total cells in any of the 6th day and 8th day after the virus infection is the above (1) And it was higher than in the case of (2), and it was shown that the production efficiency of induced pluripotent stem cells can be increased by using the Jarid2 mutant gene or its gene product.
  • Test Example 1-1 (Test Example 1-2: Production efficiency test-2) -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 300 ⁇ L of Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene-containing virus solution-1 300 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene and Jarid2 gene-containing virus solution-1 300 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-Myc gene and Jarid2 gene-containing virus solution-1 300 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 300 ⁇ L, Sox2 gene-containing virus solution-1 300 ⁇ L, Klf4 gene-containing virus solution-1 300 ⁇ L, and c-
  • Test Example 1-1 (Test Example 1-3: Production efficiency test-3) -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, and Foreign gene-free virus solution-1 600 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, K
  • Fig. 1-3 On the 8th day after the virus infection, the NG-MEF was detached with trypsin, and the number of GFP positive cells per 100,000 cells was measured with a flow cytometer (BD FACSCalibur (registered trademark), manufactured by Nippon Becton Dickinson). .
  • the results are shown in Fig. 1-3.
  • (1) to (3) show the results when the virus solutions (1) to (3) were used, respectively. From the results shown in FIGS. 1-3, in the cases (2) and (3), the ratio of the induced pluripotent stem cells to the whole cells is higher than that in the case (1).
  • NG-MEF expressing GFP and puromycin resistance gene in a Nanog gene promoter-dependent manner (transgenic mouse STOCK Tg (Nanog-GFP, Puro) 1Yam (No. RBRC02290) deposited at RIKEN BioResource Center) was obtained, mated, and isolated from 13.5 day embryos using trypsin).
  • the NG-MEF was cultured using DMEM containing 10% serum at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • the virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) to (8) below were each infected with the NG-MEF.
  • the day after the infection the supernatant was transferred to LIF (LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L and foreign gene-free virus solution-1 400 ⁇ L (3) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-1 200 ⁇ L and foreign gene-free virus solution-1
  • the number of iPS colonies is higher than in the cases of (1) to (7), and the Prdm14 gene to its gene product, the Sall4a gene to its gene product, It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
  • Test Example 1-5 Production efficiency test -5
  • NG-MEF was used in the same manner as in Test Example 1-4.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L, Sall4a gene-containing virus solution-1 200 ⁇ L, and Esrrb gene-containing virus solution-1 200 ⁇ L
  • FIGS. 1-5 The results are shown in FIGS. 1-5.
  • (1) and (2) show the results when the virus solutions (1) and (2) were used, respectively. From the result of FIG.
  • the ratio of induced pluripotent stem cells to the whole cells is higher than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its It was shown that the production efficiency of induced pluripotent stem cells can be increased by using the gene product and the Esrrb gene or the gene product thereof.
  • HDF human dermal fibroblasts
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd.
  • Test Example 1-4 the procedure was the same as in Test Example 1-4 except that the cells were changed from NG-MEF to HDF and the virus solution was replaced with one of the virus solutions (1) and (2) below. Then, the HDF was infected with a virus.
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene-containing virus solution-2 200 ⁇ L, Prdm14 gene-containing virus solution-2 200 ⁇ L, Sall4a gene-containing virus solution-2 200 ⁇ L, and Esrrb gene-containing virus solution-2 200 ⁇ L
  • FIGS. 1-6 show the results when the virus solutions (1) and (2) were used, respectively. From the results of FIG. 1-6, in the case of (2), the number of TRA-1-60 positive colonies is larger than in the case of (1), and the Prdm14 gene or its gene product, the Sall4a gene or its gene product. It has been shown that the production efficiency of induced pluripotent stem cells can be increased by using the Esrrb gene or its gene product.
  • Test Example 2-1 Production period test -1 -cell- As the cells, NG-MEF was used as in Test Example 1-1.
  • virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, and Foreign gene-free virus solution-1 600 ⁇ L
  • Test Example 2-2 Production period test-2
  • NG-MEF was used as in Test Example 1-1.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus solution -1 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, Sox2 gene-containing virus solution-1 200 ⁇ L, Klf4 gene-containing virus solution-1 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-1 200 ⁇ L, Prdm14 Gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-1 200 ⁇ L, and Sall4a gene-containing virus solution-1 200 ⁇
  • induced pluripotent stem cells were stably confirmed in three experiments ( In the case of (1) above, the number of induced pluripotent stem cell colonies (average 3) is stable in 3 experiments for the first time when puromycin is exposed on the 6th day after virus infection. Pluripotent stem cells were confirmed (number of induced pluripotent stem cell colonies: 8 on average). Therefore, by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product, an artificial pluripotent stem cell can be produced in a shorter period of time. It was shown that you can.
  • Test Example 2-3 Production period test-3 -cell- As the cell, NG-MEF was used in the same manner as in Test Example 1-4.
  • Virus solution (1) 200 ⁇ L of Oct3 / 4 gene-containing virus solution-1 200 ⁇ L, 200 ⁇ L of Sox2 gene-containing virus solution-1 200 ⁇ L, and c-Myc gene-containing virus solution-1 200 ⁇ L, and foreign gene-free virus Liquid-1 600 ⁇ L (2) Oct3 / 4 gene containing virus solution-1 200 ⁇ L, Sox2 gene containing virus solution-1 200 ⁇ L, Klf4 gene containing virus solution-1 200 ⁇ L, c-Myc gene containing virus solution-1 200 ⁇ L, Prdm14 gene containing virus solution-1 200 ⁇ L, Sall4a gene-containing virus solution-1 200 ⁇ L, and Esrrb gene-containing virus solution-1 200 ⁇ L
  • Fig. 2-3 D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “ ⁇ ” indicates the case where the virus solution of (1) was used. The result shows the result, and “ ⁇ ” shows the result when the virus solution of (2) was used. As shown in FIG. 2-3, D1 to D6 respectively indicate the start date of puromycin exposure (from day 1 to day 6 after virus infection), and “ ⁇ ” indicates the case where the virus solution of (1) was used. The result shows the result, and “ ⁇ ” shows the result when the virus solution of (2) was used. As shown in FIG.
  • Test Example 3-1 Quality test-1) -cell- HDF (Toyobo Co., Ltd.) was used as the cell.
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd. was used and cultured at a culture temperature of 37 ° C. and a CO 2 concentration of 5%.
  • -Virus infection- Virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • 20% KSR manufactured by GIBCO
  • 1 ⁇ NEAA manufactured by GIBCO
  • 2-mercaptoethanol manufactured by GIBCO
  • GlutaMax manufactured by GIBCO
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene, and Jarid2 mutant gene-containing virus solution-2 200 ⁇ L, Prdm14 Gene-containing virus solution-2 200 ⁇ L, Esrrb gene-containing virus solution-2 200 ⁇ L, and Sall4a gene-containing virus solution-2 200 ⁇ L
  • FIGS. 3A to 3D show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
  • 3A and 3B are examples of photographs when the virus solution of (1) is used
  • FIGS. 3C and 3D are examples of photographs when the virus solution of (2) is used.
  • the scale bar represents 200 ⁇ m.
  • the induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells.
  • the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell. Therefore, high-quality induced pluripotent stem cells are produced by using the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product. It was shown that it can.
  • Test Example 3-2 Quality test-2
  • -cell- HDF Toyobo Co., Ltd.
  • a human skin fibroblast basic medium manufactured by Toyobo Co., Ltd.
  • -Virus infection- Virus solutions prepared by adding polybrene having a final concentration of 4 ⁇ g / mL to the virus solutions of (1) and (2) below were each infected with the HDF.
  • LIF LIF protein-expressing vector pCAGGS-LIF (obtained from RIKEN Development and Regeneration Science) in Cos-7 cells by the calcium phosphate method, and the culture supernatant was used.
  • 20% KSR manufactured by GIBCO
  • 1 ⁇ NEAA manufactured by GIBCO
  • 2-mercaptoethanol manufactured by GIBCO
  • GlutaMax manufactured by GIBCO
  • Virus solution (1) Oct3 / 4 gene containing virus solution-2 200 ⁇ L, Sox2 gene containing virus solution-2 200 ⁇ L, Klf4 gene containing virus solution-2 200 ⁇ L, c-Myc gene containing virus solution-2 200 ⁇ L, and foreign gene-free virus solution -2 600 ⁇ L (2) Oct3 / 4 gene-containing virus solution-2 200 ⁇ L, Sox2 gene-containing virus solution-2 200 ⁇ L, Klf4 gene-containing virus solution-2 200 ⁇ L, c-Myc gene-containing virus solution-2 200 ⁇ L, Prdm14 gene-containing virus solution-1 200 ⁇ L, Esrrb gene-containing virus solution-2 200 ⁇ L, and Sall4a gene-containing virus solution-2 200 ⁇ L
  • FIGS. 3E to 3H show the results of photographing the cells 18 days after the virus infection and the cloned cells with an inverted microscope (Zeiss Axiovert 200M).
  • 3E and 3F are examples of photographs when the virus solution of (1) is used
  • FIGS. 3G and 3H are examples of photographs when the virus solution of (2) is used.
  • the scale bar represents 200 ⁇ m.
  • the induced pluripotent stem cells obtained using the virus solution of (1) above have a flat colony, can be grown on a bFGF-added medium, cannot grow on a 2i-added medium, and are single cells.
  • the induced pluripotent stem cells obtained using the virus solution of (2) above have a dome-like colony shape and can be grown in a LIF-added medium not containing bFGF, and can be grown in a 2i-added medium. It was found to be a naive-type induced pluripotent stem cell that can be dispersed and passaged into a single cell. Therefore, it was shown that high-quality induced pluripotent stem cells can be produced by using the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • an induced pluripotent stem cell can be produced with high efficiency, so that an induced pluripotent stem cell can be established from a small number of somatic cells, It is thought that the amount of rare cells and tissues collected can be reduced. Furthermore, since it is possible to suppress proliferation of non-induced pluripotent stem cells and selectively increase induced pluripotent stem cells, it is considered that establishment of induced pluripotent stem cell clones is facilitated. In addition, according to the method for producing induced pluripotent stem cells of the present invention, since induced pluripotent stem cells can be produced in a short period of time, self-derived to diseases such as spinal cord injury requiring early cell transplantation.
  • induced pluripotent stem cells Enables the use of induced pluripotent stem cells, reduces the risk of genome damage due to the long-term induced pluripotent stem cell induction process, and facilitates the method of creating induced pluripotent stem cells by transient gene transfer It is considered possible to produce safe induced pluripotent stem cells. Furthermore, according to the method for producing induced pluripotent stem cells of the present invention, induced pluripotent stem cells can be produced efficiently and in a short period of time, so that the cost can be reduced as compared with conventional methods. Further, according to the method for producing induced pluripotent stem cells of the present invention, naive induced pluripotent stem cells can be produced even in the case of human cells, so that handling of induced pluripotent stem cells is simplified. It is considered that differentiation induction efficiency can be improved.
  • Examples of the aspect of the present invention include the following. ⁇ 1> (A) Oct3 / 4 gene or its gene product; (B) the Sox2 gene or its gene product; (C) the Klf4 gene or its gene product; (D) the c-Myc gene or its gene product; (E) introducing at least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product into a somatic cell.
  • a method for producing an induced pluripotent stem cell wherein the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • the Jarid2 mutant gene or a gene product thereof is a gene encoding the N-terminal amino acids 1 to 551 of the Jarid2 protein or a gene product thereof.
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutation
  • ⁇ 3> At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product,
  • the method for producing an induced pluripotent stem cell according to the above ⁇ 2> comprising the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product.
  • ⁇ 4> The method for producing induced pluripotent stem cells according to any one of ⁇ 2> to ⁇ 3>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
  • ⁇ 5> At least one selected from the group consisting of the Jarid2 mutant gene or its gene product, the Prdm14 gene or its gene product, the Esrrb gene or its gene product, and the Sall4a gene or its gene product is the Prdm14 gene Or the gene product, the Esrrb gene or the gene product thereof, and the Sall4a gene or the gene product thereof, or the method for producing an induced pluripotent stem cell according to ⁇ 1>.
  • ⁇ 6> The method for producing an induced pluripotent stem cell according to any one of ⁇ 1> to ⁇ 5>, wherein the induced pluripotent stem cell is a naive type.
  • ⁇ 7> The method for producing an induced pluripotent stem cell according to any one of ⁇ 1> to ⁇ 6>, wherein the induced pluripotent stem cell is a human induced pluripotent stem cell.
  • ⁇ 8> comprising at least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product, A composition for producing induced pluripotent stem cells, wherein the Jarid2 mutant gene or its gene product is a gene encoding the N-terminal 1st to 551st amino acids of the Jarid2 protein or its gene product. .
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is a Jarid2 mutant gene or The composition for producing induced pluripotent stem cells according to ⁇ 8>, including the gene product.
  • At least one selected from the group consisting of a Jarid2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, and a Sall4a gene or its gene product is further added to the Prdm14 gene or its gene product.
  • the composition for producing induced pluripotent stem cells according to ⁇ 9> including the gene product, Esrrb gene or gene product, and Sall4a gene or gene product.
  • At least one selected from the group consisting of a Jard2 mutant gene or its gene product, a Prdm14 gene or its gene product, an Esrrb gene or its gene product, or a Sall4a gene or its gene product is a Prdm14 gene or its gene
  • the composition for producing induced pluripotent stem cells according to ⁇ 8> comprising a product, an Esrrb gene or a gene product thereof, and a Sall4a gene or a gene product thereof.
  • ⁇ 12> Any one of ⁇ 8> to ⁇ 11>, further including Oct3 / 4 gene or gene product, Sox2 gene or gene product, Klf4 gene or gene product, and c-Myc gene or gene product
  • ⁇ 13> The composition for producing induced pluripotent stem cells according to ⁇ 12>, wherein the c-Myc gene and the Jarid2 mutant gene are coexpressed.
  • an induced pluripotent stem cell of the present invention it is possible to produce an induced pluripotent stem cell that is excellent in production efficiency, can be produced in a short period of time, and is excellent in quality. It can be suitably used in a method for producing induced pluripotent stem cells used as a regenerative medicine such as treatment, drug screening, and a tool for elucidating the cause of a disease.

Abstract

La présente invention concerne une méthode de production de cellules souches pluripotentes induites comprenant un processus d'introduction (A) du gène Oct3/4 ou d'un produit génique associé, (B) du gène Sox2 ou d'un produit génique associé, (C) du gène Klf4 ou d'un produit génique associé, (D) du gène c-Myc ou d'un produit génique associé, et (E) d'au moins un gène choisi dans le groupe constitué par le gène mutant Jarid2 et des produits géniques associés, le gène Prdm14 et des produits géniques associés, le gène Esrrb et des produits géniques associés, et le gène Sall4a et des produits géniques associés à l'intérieur d'une cellule somatique. Le gène mutant Jarid2 ou un produit génique associé est un gène ou un produit génique codant pour les acides aminés allant du premier au 551ème acide aminé N-terminal de la protéine Jarid2.
PCT/JP2014/061378 2013-05-10 2014-04-23 Composition pour la production de cellules souches pluripotentes induites et méthode de production de cellules souches pluripotentes induites WO2014181682A1 (fr)

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JP2013-100312 2013-05-10
JP2013-100311 2013-05-10
JP2013100312A JP2014217345A (ja) 2013-05-10 2013-05-10 人工多能性幹細胞製造用組成物、及び人工多能性幹細胞の製造方法

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US10738280B2 (en) 2015-03-18 2020-08-11 Ono Pharmaceutical Co., Ltd. Method for producing naïve pluripotent stem cells

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