WO2013031826A1 - Substance de reprogrammation nucléaire - Google Patents

Substance de reprogrammation nucléaire Download PDF

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WO2013031826A1
WO2013031826A1 PCT/JP2012/071829 JP2012071829W WO2013031826A1 WO 2013031826 A1 WO2013031826 A1 WO 2013031826A1 JP 2012071829 W JP2012071829 W JP 2012071829W WO 2013031826 A1 WO2013031826 A1 WO 2013031826A1
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cells
cell
ips
oct3
klf4
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伸弥 山中
直樹 五島
崇裕 佐藤
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国立大学法人京都大学
独立行政法人産業技術総合研究所
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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
    • 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
    • C12N2501/602—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
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/604—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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a novel nuclear reprogramming substance and its use. More specifically, the present invention relates to a novel nuclear reprogramming substance that can replace c-Myc, and a method for establishing an induced pluripotent stem (hereinafter referred to as iPS) cell using the same.
  • iPS induced pluripotent stem
  • Irx family members Example: IRX6
  • Glis family members eg GLIS1
  • Ptx family eg PITX2
  • DMRT-like ⁇ familyDMB with proline-rich C-terminal, 1 gene (DMRTB1)
  • DMRTB1 DMRT-like ⁇ familyDMB with proline-rich C-terminal, 1 gene
  • iPS cells could be established efficiently when introduced into mouse and human skin-derived fibroblasts together with 3,4 Sox2 and c-Myc genes, these transcription factors could replace Klf4
  • Patent Document 7 a novel nuclear reprogramming factor
  • Glis family members eg GLIS1
  • Glis family members have a function to increase iPS cell establishment efficiency as a substitute factor for c-Myc as much as or higher
  • An object of the present invention is to identify a novel nuclear reprogramming substance, particularly a novel nuclear reprogramming substance that can replace c-Myc, and to provide a novel method for establishing iPS cells using the same.
  • the present inventors have widely used human gene libraries such as receptors or enzymes as a substitute for c-Myc in the same manner as described in WO ⁇ 2010/098419.
  • any of the 80 genes shown in Fig. 3 is introduced into mouse fetal fibroblasts (MEF) together with three genes Oct3 / 4, Sox2 and Klf4, iPS cells can be established efficiently I was able to.
  • Zinc finger and SCAN domain containing 4 Zscan4 compared c-Myc and Glis1 with iPS cell-inducing activity.
  • Zscan4 can induce iPS cells as well as Glis1 or better. It became. Based on these findings, the present inventors have identified Zscan4 as a novel nuclear reprogramming factor that can replace c-Myc, and have completed the present invention.
  • the present invention is as follows.
  • (b) A method for producing iPS cells, comprising a step of introducing a substance capable of inducing iPS cells from somatic cells by combining with c-Myc into the somatic cells.
  • the substance of (b) is selected from the group consisting of Oct family members, Sox family members, Klf family members, Glis family members, Nanog and Lin family members, and nucleic acids encoding them.
  • [3] The method according to [1] above, wherein the substance (b) is Oct3 / 4 or a nucleic acid encoding the same.
  • a method for producing a somatic cell comprising subjecting the iPS cell according to [11] above to differentiation induction treatment to differentiate it into a somatic cell.
  • a somatic cell-to-iPS cell inducer comprising Zscan4 or a nucleic acid encoding the same, together with a substance capable of inducing iPS cells from somatic cells in combination with c-Myc. An agent, characterized in that it is introduced.
  • Zscan4 or a nucleic acid encoding the same for the production of iPS cells, the substance being introduced into somatic cells together with a substance capable of inducing iPS cells from somatic cells in combination with c-Myc Characterized by being used.
  • Zscan4 or a nucleic acid that encodes Zscan4 as an inducer of iPS cells from somatic cells and is introduced into somatic cells together with a substance capable of inducing iPS cells from somatic cells in combination with c-Myc A substance characterized by that.
  • Zscan family members can replace c-Myc in nuclear reprogramming, enabling efficient establishment of iPS cells while avoiding the risk of tumor formation by c-Myc. It is useful for application.
  • FIG. 1 is a conceptual diagram showing the steps from the human Gateway O entry clone (N. Goshima et al., Nature methods, 2008) to the selection of function-specific entry clones.
  • FIG. 2 is a diagram showing a procedure for preparing a transcription factor library for screening somatic cell reprogramming factors from entry clones of transcription factors.
  • FIG. 3 shows the names of 80 genes hit in the 2nd screening.
  • FIG. 4 is a graph showing the result of comparing the iPS cell inducing activity of ZSCAN4, c-MYC and GLIS1 on the 14th day (Day 14) after virus infection.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKZ means OCT3 / 4, SOX2, KLF4 and It means the introduction of ZSCAN4, OSKG means the introduction of OCT3 / 4, SOX2, KLF4 and GLIS1.
  • shaft in a figure shows fluorescence intensity (Mean
  • FIG. 5 is a graph showing the results of comparing the iPS cell inducing activity of ZSCAN4, c-MYC and GLIS1 on the 15th day (Day 15) after virus infection.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKZ means OCT3 / 4, SOX2, KLF4 and It means the introduction of ZSCAN4,
  • OSKG means the introduction of OCT3 / 4, SOX2, KLF4 and GLIS1.
  • shaft in a figure shows fluorescence intensity (Mean
  • FIG. 6 is a graph showing the results of comparing the iPS cell inducing activities of ZSCAN4, c-MYC and GLIS1 on the 16th day after virus infection (Day 16).
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKZ means OCT3 / 4, SOX2, KLF4 and It means the introduction of ZSCAN4,
  • OSKG means the introduction of OCT3 / 4, SOX2, KLF4 and GLIS1.
  • FIG. 7 is a graph showing the results of comparing the iPS cell inducing activity of ZSCAN4, c-MYC and GLIS1 on the 17th day after virus infection (Day 17).
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKZ means OCT3 / 4, SOX2, KLF4 and It means the introduction of ZSCAN4, OSKG means the introduction of OCT3 / 4, SOX2, KLF4 and GLIS1.
  • shaft in a figure shows fluorescence intensity (Mean
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • FIG. 9 is a graph showing the results of comparing the iPS cell inducing activity of ZSCAN4, c-MYC, and GLIS1 on the 15th day after virus infection (Day 15).
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • shaft in a figure shows fluorescence intensity (Mean
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • FIG. 11 is a graph showing the results of comparing iPS cell inducing activities of ZSCAN4, c-MYC, and GLIS1 on the 17th day (Day 17) after virus infection.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • shaft in a figure shows fluorescence intensity (Mean
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • FIG. 13 is a graph showing the results of comparing iPS cell inducing activities of ZSCAN4, c-MYC, and GLIS1 on the 8th day (Day 8) after virus infection.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • shaft in a figure shows fluorescence intensity (Mean
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • FIG. 15 is a graph showing the results of comparing iPS cell inducing activities of ZSCAN4, c-MYC, and GLIS1 on the 10th day (Day 10) after virus infection.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC
  • OSKG means OCT3 / 4
  • SOX2, KLF4 and OSKZ means the introduction of OCT3 / 4, SOX2, KLF4 and ZSCAN4, and OSKGZ means the introduction of OCT3 / 4, SOX2, KLF4, GLIS1 and ZSCAN4.
  • shaft in a figure shows fluorescence intensity (Mean
  • FIG. 16 is a graph showing the results of comparing iPS cell inducing activities of ZSCAN4, c-MYC, and GLIS1 on the 29th day after virus infection.
  • the upper part shows an immunostained fluorescent image of SSEA-4
  • the lower part shows an ALP-stained image.
  • OSK means the introduction of OCT3 / 4, SOX2 and KLF4
  • OSKM means the introduction of OCT3 / 4, SOX2, KLF4 and c-MYC.
  • the number in parentheses means the number of colonies measured.
  • the present invention is a Zscan family member that is a nuclear reprogramming substance that can replace c-Myc, and a nuclear reprogramming capable of inducing iPS cells from somatic cells by combining the substance with c-Myc.
  • a method for producing iPS cells by introducing a substance into somatic cells is provided.
  • the “nuclear reprogramming substance” is a substance (group) capable of inducing iPS cells from somatic cells, and is a proteinous factor or a nucleic acid encoding the same (including forms incorporated in a vector) Alternatively, it may be composed of any substance such as a low molecular compound.
  • a nuclear reprogramming substance that can replace c-Myc identified by the present invention is any protein shown in FIG. 3 or a nucleic acid encoding it.
  • the Zscan family member has a structure including a SCAN box and about 4 C2H2 type Zinc finger regions, such as Zscan1, Zscan2, Zscan3, Zscan4, Zscan5 and Zscan6.
  • Zscan4 zinc finger and SCAN domain containing 4
  • Zscan4 is a gene that encodes a protein contained in telomeres and has been shown to be involved in maintaining the stability of telomeres and genomes in ES cells (Nature, 464, 858-863 (2010 ), WO2011 / 028880).
  • the amino acid sequence and cDNA sequence information of Zscan4 derived from human and mouse can be obtained by referring to NCBI accession numbers described in Table 1, and those skilled in the art can easily obtain each protein based on the cDNA sequence information. Can be isolated and, if necessary, recombinant proteins can be produced.
  • Zscan4 has 90% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 99% or more identity with each of the above amino acid sequences, and wild-type Zscan4 as an alternative factor for c-Myc.
  • Natural or artificial mutant proteins having equivalent nuclear reprogramming ability and nucleic acids encoding the same can also be used as Zscan4 of the present invention.
  • Only one member of the Zscan family (including the nucleic acid encoding it) may be used, or two or more members may be used in combination.
  • a nuclear reprogramming substance that can induce iPS cells by combining with c-Myc a combination of substances excluding c-Myc from the combination of (1)-(12) above, that is, (i) Oct3 / 4, Klf4 (ii) Oct3 / 4, Klf4, Sox2 (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17 or Sox18.
  • c-Myc can be replaced with T58A (active mutant), N-Myc, L-Myc.)
  • Oct3 / 4, Klf4, Sox2, TERT, SV40 Large T (v) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6 (vi) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7 (vii) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7 (viii) Oct3 / 4, Klf4, Sox2, TERT, Bmil (ix) Oct3 / 4, Klf4, Sox2, Nanog, Lin28 (x) Oct3 / 4, Klf4, Sox2, SV40 Large T (xi) Oct3 / 4, Klf4, Sox2,
  • Oct family members such as Oct1A and Oct6 can be used instead of Oct3 / 4.
  • Sox family members such as Sox7 can be used instead of Sox2 (or Sox1, Sox3, Sox15, Sox17, Sox18).
  • Klf family members eg, Klf1, Klf2, Klf5 or known alternative factors thereof (eg, Esrr family members such as Esrrb, Esrrg, IRX1, IRX2, IRX3, IRX4, IRX5, Irx family members such as IRX6, Glis family members such as GLIS1, GLIS2, and GLIS3, Ptx family members such as PITX1, PITX2, and PITX3, and DMRTB1) can also be used.
  • Esrr family members such as Esrrb, Esrrg, IRX1, IRX2, IRX3, IRX4, IRX5, Irx family members such as IRX6, Glis family members such as GLIS1, GLIS2, and GLIS3, Ptx family members such as PITX1, PITX2, and PITX3, and DMRTB1
  • Esrr family members such as Esrrb, Esrrg, IRX1, IRX2, IRX3, IRX4, IR
  • nuclear reprogramming substances that can induce iPS cells by combining with c-Myc are preferably Oct family members (eg, Oct3 / 4, Oct1A, Oct6), Sox family members (eg: Sox2, Sox1, Sox3, Sox7, Sox15, Sox17, Sox18), Klf family members (eg, Klf4, Klf1, Klf2, Klf5), Nanog and Lin family members (eg, Lin28, Lin28b).
  • a combination comprising at least Oct3 / 4 and optionally comprising Sox2 and / or Klf4 (ie (a) Oct3 / 4, (b) Oct3 / 4 + Sox2, (c) Oct3 / 4 + Klf4, (d) Oct3 / 4 + Sox2 + Klf4), and Nanog and / or Lin28 may be used in combination.
  • nuclear reprogramming substance in the present invention.
  • other substances such as members of the Myc family other than c-Myc (eg, L-Myc, N-Myc) or known substitutes thereof (eg, members of the Glis family such as GLIS1, GLIS2, GLIS3), etc. You may combine.
  • an alternative factor for c-Myc is, for example, compared to the iPS cell induction activity (including the case where there is no induction activity) obtained based on the combination of nuclear reprogramming substances (i) to (xii) above.
  • Any iPS cell obtained by adding the factor to each of the iPS cells can be used as long as the factor increases iPS cell-inducing activity, and is not necessarily limited to the combination of the above-mentioned nuclear reprogramming substances (1)-(12) containing c-Myc. It does not mean that it becomes stronger than the induction activity.
  • somatic cells subject to nuclear reprogramming may partially express any of the components in any of (i)-(x) above at a level sufficient for nuclear reprogramming.
  • the combination of only the remaining components excluding the component can also be included in the category of “nuclear reprogramming substance capable of inducing iPS cells by combining with c-Myc” in the present invention.
  • iPS cells when the obtained iPS cells are used for therapeutic purposes, a combination of two factors Oct3 / 4 and Sox2, or a combination of three factors Oct3 / 4, Sox2 and Klf4 is preferable.
  • the four factors Oct3 / 4, Klf4, Sox2 and Lin28, or Nanog are preferred.
  • Mouse and human cDNA sequence information of each of the above proteinaceous factors can be obtained by referring to NCBI accession numbers described in WO 2007/069666 (Nanog is described as “ECAT4” in the publication)
  • mouse and human cDNA sequence information of Lin28, Lin28B, Esrrb, Esrrg, L-Myc, and Nr5a2 can be obtained by referring to the following NCBI accession numbers, respectively).
  • cDNA can be isolated.
  • the obtained cDNA is inserted into an appropriate expression vector, introduced into a host cell, and cultured from the resulting culture. Can be prepared by recovering.
  • the obtained cDNA is inserted into a viral vector, episomal vector or plasmid vector to construct an expression vector, which is then used for the nuclear reprogramming step.
  • Somatic cell source Somatic cells that can be used as a starting material for producing iPS cells in the present invention are any cells other than germ cells derived from mammals (eg, humans, mice, monkeys, pigs, rats, etc.).
  • keratinized epithelial cells eg, keratinized epidermal cells
  • mucosal epithelial cells eg, epithelial cells of the tongue surface layer
  • exocrine glandular epithelial cells eg, mammary cells
  • hormone secreting cells Eg, adrenal medullary cells
  • cells for metabolism / storage eg, hepatocytes
  • luminal epithelial cells that make up the interface eg, type I alveolar cells
  • luminal epithelial cells in the inner chain eg, Vascular endothelial cells
  • cilia cells with transport ability eg, airway epithelial cells
  • cells for extracellular matrix secretion eg, fibroblasts
  • contractile cells e
  • undifferentiated progenitor cells including somatic stem cells
  • final differentiated mature cells It can be used as the source of somatic cells in the invention.
  • undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • dental pulp stem cells can be isolated and prepared from teeth extracted due to wisdom teeth, periodontal disease, etc., so they are readily available and are expected to be used as somatic cell sources for iPS cell banks in the future. .
  • blood cells peripheral mononuclear cells, peripheral blood lymphocytes, umbilical cord blood cells, etc.
  • peripheral blood lymphocytes peripheral blood lymphocytes
  • umbilical cord blood cells etc.
  • somatic cells there are no particular restrictions on the mammalian individual from which somatic cells are collected, but when the resulting iPS cells are used for human regenerative medicine, the patient or the type of HLA is used from the viewpoint that rejection does not occur. It is particularly preferred to collect somatic cells from others who are identical or substantially identical.
  • the type of HLA is “substantially the same” means that when the cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like, the transplanted cells are This means that the HLA types match to the extent that they can be engrafted.
  • the main HLA for example, 3 loci of HLA-A, HLA-B and HLA-DR and 4 loci including HLA-Cw
  • the main HLA for example, 3 loci of HLA-A, HLA-B and HLA-DR and 4 loci including HLA-Cw
  • iPS cells when not being administered (transplanted) to humans, for example, when iPS cells are used as a source of screening cells for evaluating the patient's drug sensitivity and the presence or absence of side effects, It is desirable to collect somatic cells from others who have the same genetic polymorphism that correlates with side effects.
  • Somatic cells isolated from mammals can be pre-cultured in a medium known per se suitable for culturing according to the type of cells prior to being subjected to the nuclear reprogramming step.
  • a medium known per se suitable for culturing according to the type of cells prior to being subjected to the nuclear reprogramming step.
  • Examples of such a medium include a minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, and F12 medium containing about 5 to 20% fetal calf serum. It is not limited to.
  • MEM minimum essential medium
  • DMEM Dulbecco's modified Eagle medium
  • RPMI1640 RPMI1640 medium
  • 199 medium 199 medium
  • F12 medium containing about 5 to 20% fetal calf serum containing about 5 to 20% fetal calf serum.
  • a medium for mesenchymal stem cells such as Mesenchymal stem cells basal medium (Lonza).
  • Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject TM Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), and lipid-based Profect-1 (Targeting Systems) ), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on membrane-permeable peptides, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus), and the like are commercially available.
  • the introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows.
  • Dilute the nuclear reprogramming substance in an appropriate solvent for example, buffer solution such as PBS, HEPES, etc.
  • an appropriate solvent for example, buffer solution such as PBS, HEPES, etc.
  • CPP derived from PTD include polyarginine such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)).
  • a fusion protein expression vector incorporating a nuclear reprogramming substance cDNA and a PTD sequence or CPP sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. Introduction can be performed in the same manner as described above except that no protein introduction reagent is added.
  • Microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 ⁇ m and puncture is introduced into a cell, and the protein can be reliably introduced into the cell.
  • electroporation method in addition, electroporation method, semi-intact cell method (Kano, F. et al. Methods in Molecular Biology, Vol. 322, 357-365 (2006)), introduction method using Wr-t peptide (Kondo, E. et al. Protein introduction methods such as Mol. Cancer Ther. 3 (12), 1623-1630 (2004)) can also be used.
  • the protein introduction operation can be performed any number of one or more times (for example, 1 to 10 times or 1 to 5 times), and preferably the introduction operation is performed 2 or more times (for example, 3 or 4 times). ) Can be done repeatedly. Examples of the interval when the introduction operation is repeated include 6 to 48 hours, preferably 12 to 24 hours.
  • the nuclear reprogramming substance in the form of a nucleic acid that encodes it rather than as a protein factor itself.
  • the nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera, and the nucleic acid may be double-stranded or single-stranded.
  • the nucleic acid is double stranded DNA, in particular cDNA.
  • the cDNA of the nuclear reprogramming substance is inserted into an appropriate expression vector containing a promoter that can function in somatic cells as a host.
  • expression vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) or the like.
  • Examples of the promoter used in the expression vector include EF1 ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Molone murine leukemia virus) LTR. HSV-TK (herpes simplex virus thymidine kinase) promoter and the like are used. Of these, EF1 ⁇ promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR ⁇ promoter and the like are preferable.
  • the expression vector may contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like as desired.
  • the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.
  • Nucleic acid that is a nuclear reprogramming substance may be incorporated on separate expression vectors, or two or more, preferably 2-3 types of genes may be incorporated into one expression vector. It is preferable to select the former when using a retrovirus or lentiviral vector with high gene transfer efficiency, and the latter when using a plasmid, adenovirus, episomal vector, or the like. Furthermore, an expression vector incorporating two or more types of genes and an expression vector incorporating only one gene can be used in combination.
  • sequences enabling polycistronic expression include 2A sequences of foot-and-mouth disease virus (PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), IRES sequences (US Patent No. 4,937,190), preferably A 2A sequence can be used.
  • An expression vector containing a nucleic acid that is a nuclear reprogramming substance can be introduced into a cell by a method known per se according to the type of the vector.
  • a virus produced in the culture supernatant by introducing a plasmid containing the nucleic acid into an appropriate packaging cell (eg, Plat-E cell) or a complementary cell line (eg, 293 cell) The vector is collected and cells are infected with the vector by an appropriate method according to each viral vector.
  • an appropriate packaging cell eg, Plat-E cell
  • a complementary cell line eg, 293 cell
  • the vector is collected and cells are infected with the vector by an appropriate method according to each viral vector.
  • specific means of using a retroviral vector as a vector are disclosed in WO2007 / 69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007).
  • reprogramming genes may increase the risk of carcinogenesis in tissues regenerated from differentiated cells derived from iPS cells. It is preferable that the gene is transiently expressed without being integrated into the cell chromosome. From this point of view, it is preferable to use an adenovirus vector that rarely integrates into the chromosome. Specific means using an adenoviral vector is described in Science, 322, 945-949 (2008).
  • adeno-associated virus also has a low frequency of integration into chromosomes, and has lower cytotoxicity and inflammation-inducing action than adenovirus vectors, and thus can be mentioned as another preferred vector.
  • the Sendai virus vector can exist stably outside the chromosome, and can be preferably used in the same manner because it can be decomposed and removed by siRNA as necessary. Sendai virus vectors are described in J. Biol. Chem., 282, 27383-27391 (2007), Proc. Jpn. Acad., Ser. B 85, 348-362 (2009), or Japanese Patent No. 3602058 Can be used.
  • a method of excising a nucleic acid encoding a nuclear reprogramming substance at a time point can be preferably used. That is, loxP sequences are arranged at both ends of the nucleic acid, and after iPS cells are induced, Cre recombinase is allowed to act on the cells using a plasmid vector or an adenovirus vector to cut out the region sandwiched between the loxP sequences. be able to.
  • the enhancer-promoter sequence in the LTR ⁇ U3 region may up-regulate nearby host genes by insertion mutation, so the 3′-self is deleted or replaced with a polyadenylation sequence such as SV40.
  • an inactivated (SIN) LTR is used to avoid expression control of the endogenous gene by an LTR outside the loxP sequence that is not excised and remains in the genome. Specific means using the Cre-loxP system and SIN LTR are disclosed in Chang et al., Stem Cells, 27: 1042-1049 (2009).
  • plasmid vector which is a non-viral vector
  • the vector is transferred to cells using lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, etc.
  • lipofection method liposome method
  • electroporation method calcium phosphate coprecipitation method
  • DEAE dextran method microinjection method
  • gene gun method etc.
  • Specific means using a plasmid as a vector are described in, for example, Science, 322, 949-953 (2008).
  • gene introduction can be carried out any number of one or more times (for example, 1 to 10 times or 1 to 5 times).
  • time for example, 1 to 10 times, or 1 to 5 times, etc.
  • the number of times can be performed, and preferably the introduction operation can be repeated 2 times or more (for example, 3 times or 4 times).
  • transgene may be integrated into the chromosome, it is necessary to finally confirm that there is no gene insertion into the chromosome by Southern blotting or PCR. Therefore, it may be advantageous to use a means for removing the gene after the transgene has been once integrated into the chromosome, as in the Cre-loxP system.
  • there is a method for completely removing a transgene from a chromosome by incorporating a transgene into a chromosome using a transposon and then allowing a transferase to act on the cell using a plasmid vector or an adenovirus vector. Can be used.
  • Preferred transposons include, for example, piggyBac, which is a transposon derived from a lepidopteran insect. Specific means using the piggyBac transposon are disclosed in Kaji, K. et al., Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766-770 (2009).
  • Another preferred non-integrated vector is an episomal vector capable of autonomous replication outside the chromosome.
  • episomal vectors Specific means using episomal vectors are disclosed in Yu et al., Science, 324, 797-801 (2009), WO 2011/016588 A1 or Nature Methods, 8 (5), 409-412 (2011), etc. ing.
  • the episomal vector examples include a vector containing a sequence necessary for autonomous replication derived from EBV, SV40 or the like as a vector element.
  • vector elements necessary for autonomous replication include a replication origin and a gene encoding a protein that binds to the replication origin and controls replication.
  • the replication origin oriP And EBNA-1 gene SV40 includes the origin of replication ori and SV40 large T antigen gene.
  • S / MAR sequences derived from pEPI-based vectors and ⁇ -satellite DNA for human artificial chromosomes can also be used as vector elements necessary for autonomous replication (Mol Ther 16 (9), 1525-1538 (2008) ).
  • the episomal expression vector also contains a promoter that controls transcription of the reprogramming gene.
  • the promoter the same promoter as described above can be used.
  • the episomal expression vector may further contain an enhancer, a poly A addition signal, a selection marker gene in mammalian cells, and the like, if desired.
  • the selection marker gene include a dihydrofolate reductase gene and a neomycin resistance gene.
  • the selection marker gene include a dihydrofolate reductase gene and a neomycin resistance gene.
  • selectable marker genes eg, ampicillin resistance
  • Gene auxotrophic complementary gene
  • the loxP sequence used in the present invention includes a wild type loxP sequence derived from bacteriophage P1 and recombination when placed in the same direction at a position sandwiching a vector element required for replication of the reprogramming gene. And any mutated loxP sequence that can delete sequences between loxP sequences. Examples of the mutant loxP sequence include lox71 having a mutation in the 5 'repeat, lox66 having a mutation in the 3' repeat, lox2272 and lox511 having a mutation in the spacer portion, and the like.
  • the two loxP sequences arranged on the 5 ′ side and 3 ′ side of the vector element may be the same or different, but in the case of a mutant loxP sequence having a mutation in the spacer portion (for example, , Lox2272 and lox511) are used.
  • a mutant loxP sequence eg, lox71
  • a mutant loxP sequence eg, lox66
  • the loxP sequence remaining on the chromosome as a result of recombination has double mutations in the 5 'and 3' repeats, making it difficult to recognize by Cre recombinase, and chromosomal deletion mutations due to unnecessary recombination. The risk of causing this is reduced.
  • any of the mutant loxP sequences may be arranged on the 5 ′ side and 3 ′ side of the vector element, but the mutation site is mutated so that the mutation site is located at the outer end of the loxP sequence. It is necessary to insert the loxP sequence.
  • the two loxP sequences are the 5 'and 3' sides of the vector elements necessary for replication of the reprogramming gene (ie, the gene sequence encoding the replication origin or a protein that binds to the replication origin and controls replication). Are arranged in the same direction.
  • the vector element sandwiched by the loxP sequence may be either one of the replication origin, the gene sequence encoding the protein that binds to the replication origin and controls replication, or both.
  • Episomal vectors can be introduced into cells using, for example, lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method and the like. Specifically, for example, the methods described in Science, 324: 797-801 (2009), WO 2011/016588 A1 or Nature Methods, 8 (5), 409-412 (2011) can be used.
  • Whether or not the vector element necessary for replication of the reprogramming gene has been removed from the iPS cell is determined by using a nucleic acid containing a base sequence in the vector element and / or in the vicinity of the loxP sequence as a probe or primer. It can be carried out by conducting Southern blot analysis or PCR analysis using the isolated episomal fraction as a template and examining the presence or absence of the band or the length of the detection band (WO 2011/016588 A1, Nature Methods, 8 (5) , See 409-412 (2011)). Episomal fractions may be prepared using methods well known in the art.For example, Science, 324: 797-801 (2009), WO 2011/016588 A1, Nature Methods, 8 (5), 409-412 ( 2011) etc. can be used.
  • the nuclear reprogramming substance that can induce iPS cells by combining with c-Myc is a low molecular weight compound
  • introduction of the substance into somatic cells can be achieved by dissolving the substance in an aqueous or non-aqueous solvent at an appropriate concentration.
  • MEM minimal essential medium
  • DMEM Dulbecco's modified Eagle medium
  • RPMI1640 medium suitable for culturing somatic cells isolated from humans or mice
  • the concentration of the nuclear reprogramming substance varies depending on the type of the nuclear reprogramming substance used, but is appropriately selected within the range of about 0.1 nM to about 100 nM.
  • the contact period is not particularly limited as long as it is a time sufficient for the nuclear reprogramming of the cells to be achieved, but it is usually sufficient that the contact period coexists in the medium until a positive colony appears.
  • HDAC histone deacetylase
  • VPA valproic acid
  • trichostatin Nucleic acid expression inhibitors such as A, sodium butyrate
  • small molecule inhibitors such as MC 1293, M344, siRNA and shRNA against HDAC (eg, HDAC1 siRNA Smartpool O (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.) Etc.]
  • G9a histone methyltransferase inhibitors for example, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) ( Nucleic acid expression inhibitors such as Santa Cruz Biotechnology)], L-calcium channel agonist (eg Bayk8644) (Cell Stem Cell, 3,
  • the nucleic acid expression inhibitor may be in the form of an expression vector containing DNA encoding siRNA or shRNA.
  • SV40 large T is not an essential factor for somatic cell nuclear reprogramming, but is an auxiliary factor. It can also be included in a category.
  • auxiliary factors other than those essential for nuclear reprogramming are positioned as nuclear reprogramming substances or substances that improve the establishment efficiency of iPS cells. It may be convenient.
  • the nuclear reprogramming process of somatic cells is regarded as an overall event caused by the contact of somatic cells with the nuclear reprogramming substance and the substance that improves the establishment efficiency of iPS cells. There will be no gender.
  • Contact of the iPS cell establishment efficiency improving substance with the somatic cell may be carried out when the substance is (a) a protein factor, (b) a nucleic acid encoding the protein factor, or (c) a low molecular weight compound.
  • the nuclear initialization material can be carried out by the same method as described above.
  • the iPS cell establishment efficiency improving substance may be brought into contact with the somatic cells simultaneously with the nuclear reprogramming substance. Alternatively, either one may be contacted first.
  • the nuclear reprogramming substance is a nucleic acid encoding a proteinous factor
  • the substance that improves the establishment efficiency of iPS cells is a chemical inhibitor
  • the former removes the proteinous factor from the gene transfer treatment.
  • a substance that improves the establishment efficiency of iPS cells is added to the medium can do.
  • a nuclear reprogramming substance and an iPS cell establishment efficiency improving substance are used in the form of a viral vector, a plasmid vector, or an episomal vector, both are introduced into the cell simultaneously. Also good.
  • the iPS cell establishment efficiency can be further improved by culturing cells under hypoxic conditions in the somatic cell nuclear reprogramming step.
  • the “hypoxic condition” means that the oxygen concentration in the atmosphere when cells are cultured is significantly lower than that in the air. Specifically, the oxygen concentration condition is lower than the oxygen concentration in the atmosphere of 5-10% CO 2 / 95-90% air generally used in normal cell culture. For example, oxygen in the atmosphere Conditions with a concentration of 18% or less apply.
  • the oxygen concentration in the atmosphere is 15% or less (eg, 14% or less, 13% or less, 12% or less, 11% or less, etc.), 10% or less (eg, 9% or less, 8% or less, 7% or less) 6% or less), or 5% or less (eg, 4% or less, 3% or less, 2% or less, etc.).
  • the oxygen concentration in the atmosphere is preferably 0.1% or more (eg, 0.2% or more, 0.3% or more, 0.4% or more), 0.5% or more (eg, 0.6% or more, 0.7% or more, 0.8% or more, 0.95 Or 1% or more (eg, 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, etc.).
  • a method for creating a hypoxic state in the cell environment is not particularly limited, but a method of culturing the cells in a CO 2 incubator in which the oxygen concentration can be adjusted is the easiest and is a preferable example.
  • CO 2 incubators with adjustable oxygen concentration are sold by various equipment manufacturers (for example, CO for low oxygen culture by manufacturers such as Thermo scientific, Ikemoto Rika Kogyo, Toji Field, and Waken Pharmaceutical Co., Ltd.) 2 incubators can be used).
  • the time when cell culture is started under hypoxic conditions is not particularly limited as long as it does not prevent the establishment efficiency of iPS cells from being improved compared to the case of normal oxygen concentration (20%).
  • the contact may be before the contact with the reprogramming substance, at the same time as the contact, or after the contact. For example, immediately after the somatic cell is contacted with the nuclear reprogramming substance, or the contact It is preferable to culture under hypoxic conditions after a certain period of time (for example, 1 to 10 (eg, 2,3,4,5,6,7,8 or 9) days).
  • the period for culturing cells under hypoxic conditions is not particularly limited as long as it does not prevent the establishment efficiency of iPS cells from being improved compared to the case of normal oxygen concentration (20%). Examples include, but are not limited to, a period of 7 days or more, 10 days or more, 50 days or less, 40 days or less, 35 days or less, or 30 days or less.
  • a preferable culture period under low oxygen conditions varies depending on the oxygen concentration in the atmosphere, and those skilled in the art can appropriately adjust the culture period according to the oxygen concentration used. In one embodiment, when selection of iPS cell candidate colonies is performed using drug resistance as an index, it is preferable to return from a low oxygen condition to a normal oxygen concentration before drug selection is started.
  • the preferred time and preferred culture period for starting cell culture under hypoxic conditions vary depending on the type of nuclear reprogramming substance used, iPS cell establishment efficiency under normoxic conditions, and the like.
  • the cells can be cultured under conditions suitable for culturing ES cells, for example.
  • LIF Leukemia Inhibitory Factor
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • STO cell lines (ATCC CRL-1503) are usually used as MEFs.
  • SNL cells SNL76 / 7 STO cells
  • ECACC 07032801 McMahon, AP & Bradley, A. Cell 62, 1073-1085 (1990)
  • the co-culture with feeder cells may be started before the contact with the nuclear reprogramming substance, or may be started at the time of the contact or after the contact (for example, after 1-10 days).
  • the cells are cultured without any non-human animal-derived components (ie, under completely xeno-free conditions) ), It is possible to produce human iPS cells.
  • cells are derived from FBS or other non-human animals after contact with a nuclear reprogramming substance (and, if necessary, a substance that improves iPS cell establishment efficiency). Cultivated in medium without ingredients.
  • a human-derived purified protein preferably a recombinant protein
  • feeder cells any human-derived somatic cells can be used.
  • human dermal fibroblasts (HDF) and human dental pulp stem cells can be preferably used.
  • HDF human dermal fibroblasts
  • a commercially available xeno-free coating agent can be used instead of matrigel or gelatin as the coating agent for the cell container.
  • the selection of iPS cell candidate colonies includes a method using drug resistance and reporter activity as indicators and a method using visual morphological observation.
  • Examples of the former include a drug resistance gene and / or a gene locus that is specifically highly expressed in differentiated pluripotent cells (for example, Fbx15, Nanog, Oct3 / 4, etc., preferably Nanog or Oct3 / 4).
  • a recombinant cell targeted with a reporter gene is used to select colonies positive for drug resistance and / or reporter activity.
  • Such recombinant cells include, for example, MEF (Takahashi & naka Yamanaka, Cell, 126, 663) derived from a mouse in which a ⁇ geo (encoding a fusion protein of ⁇ -galactosidase and neomycin phosphotransferase) gene is knocked in at the Fbx15 locus. -676 (2006)), or MEFs derived from transgenic mice incorporating the green fluorescent protein (GFP) gene and puromycin resistance gene at the Nanog locus (Okita et al., Nature, 448, 313-317 (2007)) Etc.
  • MEF green fluorescent protein
  • examples of a method for selecting candidate colonies by visual morphological observation include the methods described in Takahashi et al., Cell, 131, 861-872-8 (2007).
  • a method using a reporter cell is simple and efficient, when iPS cells are produced for the purpose of human therapeutic use, visual colony selection is desirable from the viewpoint of safety.
  • a Zscan family member is introduced into a somatic cell in the form of a nucleic acid, and as a nuclear reprogramming substance capable of inducing iPS cells by combining with c-Myc, at least a Klf family member (eg, Klf4) is in a somatic cell in the form of a nucleic acid.
  • a Klf family member eg, Klf4
  • the iPS cells obtained are new cells that differ from conventionally known iPS cells in that they contain these exogenous nucleic acids.
  • the exogenous nucleic acid when introduced into a somatic cell using a retrovirus, a lentivirus or the like, the exogenous nucleic acid is usually incorporated into the genome of the resulting iPS cell, and therefore contains the exogenous nucleic acid. This trait is stably maintained.
  • the iPS cells thus established can be used for various purposes.
  • differentiation induction methods reported for pluripotent stem cells such as ES cells eg, differentiation induction methods for neural stem cells are disclosed in JP-A-2002-291469, and differentiation induction methods for pancreatic stem-like cells are Examples of methods for inducing differentiation into hematopoietic cells include the methods described in JP 2003-505006 and other methods for inducing differentiation by formation of embryoid bodies. The method described in 2003-523766 is exemplified.
  • To differentiate iPS cells into various cells eg, cardiomyocytes, blood cells, nerve cells, vascular endothelial cells, insulin secreting cells, etc.). Can be guided.
  • iPS cells are induced using somatic cells collected from the patient or another person who has the same or substantially the same type of HLA
  • the desired cells ie, the organ in which the patient is affected
  • Stem cell therapy based on autologous transplantation or allogeneic transplantation, such as differentiation into cells and cells that exhibit therapeutic effects on diseases, etc., and transplantation into the patient becomes possible.
  • functional cells differentiated from iPS cells eg, hepatocytes
  • drug candidates It can also be suitably used for in vitro screening of the efficacy and toxicity of compounds.
  • Example 1 Screening of novel reprogramming factor Human Gateway O entry clone prepared by Goshima et al. (Using the library described in N. Goshima et al., Nature methods, 2008. Y. Maruyama et al., Nucleic Acid Res. , 2009 database), about 20,000 clones of human comprehensive genes were aligned by the method shown in FIG. In other words, about 50,000 clones including full-length ORF in human Gateway O entry clones were subjected to blastp search based on NCBI RefSeq 37,900 sequences (24,200 genes) with a coverage of 80% or more and amino acid identity of 95% or more.
  • a sub-library consisting of approximately 20,000 entry clones with no sequence duplication was constructed in each type of N-type having a stop codon at the 3 ′ end and F-type not having a stop codon.
  • This aligned 20,000-entry clone is classified into protein kinases, protein phosphatases, transcription factors, GPCRs, and other clone groups using bioinformatics techniques, and a sub-library consisting of entry clones of transcription factors (all human transcription factors) (Over 50% of the cover) was constructed (Fig. 1).
  • the number of retroviruses used for initialization was 2.5 x 10 6 per 100 mm culture dish (Falcon) the day before and 2.5 x 10 4 per well for the 96-well plate (Falcon).
  • Each pMXs expression vector described above was individually introduced into seeded Plat-E cells (Morita, S. et al., Gene Ther. 7, 1063-1066 (2000)).
  • the culture solution was DMEM / 10% FBS (DMEM (Nacalai tesque) added with 10% fetal calf serum) and cultured at 37 ° C. and 5% CO 2 .
  • IPS cell induction experiment was performed using MEFs derived from Nanog-GFP mice (Okita et al., Nature, 448, 313-317 (2007)) and 96-well plate as the 1st screening.
  • MEFs derived from Nanog-GFP mice were seeded on a 96-well plate at 800 per well.
  • DMEM / 10% FBS was used and cultured at 37 ° C. and 5% CO 2 .
  • infection was made with retroviruses prepared from various expression vectors (Day 0).
  • 3 genes of human OCT3 / 4, SOX2 and KLF4 and 1 gene from the aforementioned human transcription factor expression library or kinase expression library were infected at a ratio of 1: 1: 1: 1.
  • As a negative control three genes OCT3 / 4, SOX2 and KLF4 were infected at a ratio of 1: 1: 1.
  • OCT3 / 4, SOX2, KLF4 and c-MYC 4 genes were infected at a ratio of 1: 1: 1: 1.
  • the day after virus infection (Day 1), the retrovirus solution was removed, replaced with DMEM / 10% FBS, and cultured on DMEM / 10% FBS until the third day of infection.
  • feeder cells were seeded on 96-well plate in which MEF was cultured. SNL cells (McMahon, cA. P. & Bradley, A. Cell 62, 1073-1085 (1990)) treated with mitomycin C to stop cell division were used as feeder cells.
  • ES cell medium (DMEM Na (Nacarai tesque) 15% fetal calf serum, 2 mM L-glutamine (Invitrogen), 100 ⁇ M non-essential amino acid (Invitrogen), 100 ⁇ M 2-mercaptoethanol ( Invitrogen), 50 / mL penicillin (Invitrogen) and 50 ⁇ g / mL streptomycin (Invitrogen), Cell, 126, 663-676 (2006)) were used. Thereafter, the ES cell medium was changed every two days. Drug selection using puromycin was performed from Day 21, and the number of GFP positive colonies was counted on Day 28. As a result, about 430 genes out of about 1,700 genes screened, more GFP positive colonies were observed than the negative control.
  • 2nd screening was performed for about 430 genes.
  • the 2nd screening was performed in the same manner as the 1st screening by seeding MEFs derived from Nanog-GFP mice at 3,300 per 1 well using a 24-well plate. Criteria were determined that (1) the number of GFP-positive colonies was larger than that of the negative control, and (2) the colony morphology was dome-shaped and the outer edge was clear. As a result, the 80 genes shown in FIG. 3 were found to satisfy this criterion, and were found to be novel reprogramming factors that could replace c-Myc.
  • Example 2 Activity of promoting the efficiency of establishment of iPS cells by ZSCAN4 factor Regarding human ZSCAN4 (Zinc finger and SCAN domain containing 4), which is one of the factors obtained by the 2nd screening, human c-MYC and GLIS1 (Nature, 474, 225) -229 (2011) doi: 10.1038 / nature10106) and iPS cell establishment efficiency promoting activity were compared.
  • the combinations of human genes used for the introduction are shown below.
  • OCT3 / 4, SOX2, KLF4 (negative control) (OSK) OCT3 / 4, SOX2, KLF4, c-MYC (positive control) (OSKM)
  • OCT3 / 4, SOX2, KLF4, ZSCAN4 (OSKZ) OCT3 / 4, SOX2, KLF4, GLIS1 (OSKG)
  • the iPS cell induction experiment was performed in the same manner as in Example 1 by seeding 1 ⁇ 10 3 or 1.5 ⁇ 10 3 Nanog-GFP mouse-derived MEF per well of a 96-well plate (Thermo Fisher Scientific). However, this time, selection with puromycin was not performed, and the number of GFP positive cells was measured with POWERSCAN4 (DS Pharma Biomedical) from day 14 to day 17 after infection (Day 0). The results are shown in FIGS. In combination with OCT3 / 4, SOX2 and KLF4, it was revealed that ZSCAN4 has iPS cell induction activity (iPS cell establishment efficiency promoting activity) equivalent to or higher than GLIS1.
  • Example 3 Activity of promoting iPS cell establishment efficiency by ZSCAN4 factor
  • the iPS cell establishment efficiency promoting activity of ZSCAN4 was compared under the same conditions as in Example 2.
  • the combinations of human genes used for the introduction are shown below.
  • Example 2 were seeded 96-well 1 well per 1.5 ⁇ 10 3 cells or 2.0x10 3 pieces of Nanog-GFP mice-derived MEF of plate, the iPS cell induction was carried out in the same manner as in Example 1. However, on the next day of infection, feeder cells are seeded on a 96-well plate in which MEF is cultured, and from the second day of infection (Day 2), the culture medium is switched to the mouse ES cell culture medium. The medium for ES cells was changed. The iPS cell induction efficiency was determined by measuring the number of GFP positive cells with POWERSCAN4 on the 14th to 17th days after infection (Day 0), as in Example 2. The results are shown in FIGS.
  • Example 4 Activity of promoting iPS cell establishment efficiency by inhibition of ZSCAN4 factor and p53
  • the activity of inducing iPS cells by the combination of ZSCAN4 and / or GLIS1 and inhibition of p53 was examined.
  • iPS cell induction was performed using a p53-deficient Nanog-GFP mouse-derived MEF (p53-null MEF).
  • p53-null MEF is a p53 gene having a Nanog reporter, obtained by producing a knockout mouse in which exon5 of the p53 gene is replaced with a neomycin resistance gene and mating with a Nanog reporter mouse according to the method described in International Publication WO2009 / 157593.
  • ZSCAN4 has a higher iPS cell-inducing activity (iPS cell) than OCT3 / 4, SOX2 and KLF4 alone when combined with OCT3 / 4, SOX2 and KLF4. Activity for promoting establishment efficiency). Furthermore, it has been clarified that the combination of ZSCAN4 and GLIS1 enhances iPS cell induction activity (iPS cell establishment efficiency promoting activity).
  • Example 5 iPS cell establishment efficiency promoting activity by ZSCAN4 factor (iPS cell establishment from keratinocytes) IPS cells were established using keratinocytes (epidermal keratinocytes) derived from human epidermis, and the iPS cell establishment efficiency promoting activity by ZSCAN4 was examined.
  • the combinations of human genes used for the introduction are shown below.
  • human epidermis-derived keratinocytes purchased from Cell Applications were seeded on a 6-well plate at a rate of 0.4 ⁇ 10 5 cells per well.
  • a keratinocyte medium (Cell Applications) was used as a culture solution, and the cells were cultured at 37 ° C. and 5% CO 2 .
  • retroviruses prepared from various expression vectors were infected by spinfection (rotated at 1000 g for 30 minutes at 32 ° C.) (Day 0).
  • the day after virus infection (Day 1) SNL cells treated with mitomycin C to stop cell division were seeded as feeder cells, and cultured on days 3, 5 and 7 with the keratinocyte medium replaced. From day 9 of infection (Day 9), the culture medium was switched to a medium for human ES cells (ReproCell), and thereafter the medium for human ES cells was changed every day.

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Abstract

La présente invention concerne un membre de la famille Zscan (par exemple, ZSCAN4) qui est un facteur de remplacement de c-Myc en tant qu'inducteur de cellules iPS, et un acide nucléique codant pour celui-ci. La présente invention concerne en outre un procédé de production de cellules iPS, comprenant une étape d'introduction dans des cellules somatiques d'une substance capable d'induire des cellules iPS à partir des cellules somatiques par combinaison du membre de la famille Zscan et de c-Myc. De plus, la présente invention concerne des cellules iPS comprenant un acide nucléique étranger qui code pour des membres de la famille Klf et des membres de la famille Zscan, qui peuvent être obtenues par le procédé, et un procédé de production de cellules somatiques par induction de la différenciation des cellules iPS.
PCT/JP2012/071829 2011-08-29 2012-08-29 Substance de reprogrammation nucléaire WO2013031826A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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