WO2015152146A1 - Method for culturing haploid embryonic stem cells - Google Patents

Method for culturing haploid embryonic stem cells Download PDF

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WO2015152146A1
WO2015152146A1 PCT/JP2015/059901 JP2015059901W WO2015152146A1 WO 2015152146 A1 WO2015152146 A1 WO 2015152146A1 JP 2015059901 W JP2015059901 W JP 2015059901W WO 2015152146 A1 WO2015152146 A1 WO 2015152146A1
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cells
haploid
embryonic stem
cell
stem cells
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Japanese (ja)
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史敏 石野
知英 李
沙央里 ▲高▼橋
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国立大学法人東京医科歯科大学
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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the present invention relates to a method for culturing haploid embryonic stem cells, and more particularly, to a method for culturing haploid embryonic stem cells while maintaining the monoploidy.
  • the present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method.
  • the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells.
  • Mammals usually have a diploid genome derived from their father and mother. This has the significance that when a disease such as an autosomal recessive genetic disease occurs, the genome derived from one parent does not become a fatal symptom by supplementing the function. However, when performing forward genetics, if it is diploid, it is difficult to directly analyze the relationship between the genotype and the phenotype.
  • Haploid (haploid) cell lines have only one pair of chromosomes, and since genotypes and phenotypes can be seen directly, they are expected to be applied to forward genetics research.
  • Establishment of a haploid embryonic stem cell line has been reported.
  • a haploid embryo is produced by chemically stimulating a mouse unfertilized egg, and an ES cell line is established therefrom, whereby a haploid embryonic cell derived from a female parthenogenetic embryo ( It has been reported that female haploid ES cells, parthenogenetic haploid ES cells, phES cells, phESC) were obtained (Non-Patent Documents 1 to 4).
  • haploid ES cells chromosome doubling (diploidization) occurs naturally, and it is difficult to culture while maintaining the haploid state (monoploidy). Therefore, in order to maintain the haploidity of haploid ES cells, it is necessary to regularly collect cells in the G1 phase of haploid cells (1N cells) using a flow cytometer (FACS). There is.
  • haploid ES cells when applying haploid ES cells to forward genetics research, as described above, there is a need for a method of stably culturing the cells in a haploid for a long period of time, as well as normal ES cells ( Similarly, diploid ES cells established from embryos obtained by natural mating) are also required to have stable properties as pluripotent stem cells. That is, it is necessary to show a gene expression profile similar to that of normal ES cells and to have differentiation pluripotency and high proliferation ability (self-replication ability). Furthermore, naturally, when applied to forward genetics research that searches for a causative gene from a phenotype, it is possible to prevent mutations such as deletion and amplification in genomic DNA. Desired.
  • the present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a method capable of stably culturing haploid ES cells over a long period of time.
  • the present inventors have found that diploid ES cells naturally diploid because the transition from the G2 phase to the M phase of the cell cycle is insufficient.
  • the cell cycle is regularly rotated from G1 phase, S phase, G2 phase, and M phase (see the upper part of FIG. 1), but when haploid cells diploid, G1 It is expected that the cell cycle has failed, such as the period, S period, G2 period, G1 period, S period, G2 period, and M period.
  • DNA synthesis occurs without passing through cell division by passing the S phase twice before entering the M phase in this way, and genomic DNA is doubling in haploid cells. (See the middle part of FIG. 1).
  • Cdc2 is a factor responsible for the transition from the G2 phase to the M phase in the progression of the cell cycle. That is, by culturing haploid ES cells in the presence of inhibitors against Wee1 and Myt1, which are factors that inactivate Cdc2, Cdc2 was constantly activated in the cells. As a result, diploidization of haploid ES cells was suppressed, and culturing was possible for a long time while maintaining monoploidy.
  • haploid ES cells cultured by this method maintain undifferentiated properties, have multipotency in vivo, and can contribute to the formation of chimeric mice. Furthermore, it was also found that the haploid ES cells can be transferred to the germline in chimeric mice.
  • haploid ES cells cultured by this method can be differentiated into Epiblast Stem cells (EpiSC) while maintaining their haploidity, and even in the differentiated cells, the haploidy can be differentiated. It has also been found that it can be maintained, and the present invention has been completed. That is, the present invention relates to a method for culturing haploid embryonic stem cells. The present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method. Furthermore, the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells. More specifically, the present invention provides the following inventions.
  • EpiSC Epiblast Stem cells
  • a method for culturing haploid embryonic stem cells comprising culturing the cells under conditions that constantly activate Cdc2.
  • (2) A method for producing a non-human chimeric animal (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo; (Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
  • the non-human chimeric animal is a non-human germ line chimeric animal whose germ cells are derived from the haploid embryonic stem cells.
  • a method for producing haploid differentiated cells (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) a step of inducing differentiation of the haploid embryonic stem cells. (5) A drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells, comprising a Cdc2 activator as an active ingredient.
  • haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability.
  • differentiated cells can be obtained from such haploid ES cells while maintaining their haploidity, and the diploidy can also be maintained in the differentiated cells. It becomes possible.
  • FIG. 1 It is a schematic diagram showing cell cycle progression assumed in haploid embryonic stem (ES) cells. That is, if normal cell cycle progression is carried out in haploid ES cells, as shown in the upper part of FIG. 1, the haploidity is maintained and the cells are divided and proliferated. Become. However, since haploid ES cells naturally diploidize, for example, as shown in the middle part of FIG. 1, G1 phase, S phase, G2 phase, G1 phase, S phase, G2 phase, As in the M phase, it is assumed that the cell cycle is broken and diploidization occurs.
  • FIG. 2 is a schematic diagram showing that it was possible to suppress somaticization (see the lower part of FIG. 1) and succeeded in culturing for a long time while maintaining monoploidy. It is the schematic which shows the process of collect
  • results of FACS analysis of haploid ES cells (phES-B6GFP-3) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS. Furthermore, it is a histogram showing the results of FACS analysis of haploid ES cells after three subcultures in the presence of PD166285 (see the right in the figure). Results of FACS analysis of haploid ES cells (phES-B6GFP) subcultured 8 times in the absence of PD166285 (see the left in the figure), and 1N cells were sorted from the cells by FACS.
  • Results of FACS analysis of haploid ES cells (phES-B6GFP-4) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS.
  • the upper left panel is a photograph showing the results of observing the appearance after 30 days of immunodeficient mice transplanted subcutaneously with haploid ES cells.
  • the upper right panel is a photomicrograph showing endothelium epithelium tissue observed by HE staining of teratoma extracted from the mouse.
  • the lower left panel is a photomicrograph showing the epidermis tissue observed by HE staining of teratoma extracted from the mouse.
  • the lower right panel is a photomicrograph showing a cartilage tissue observed by HE staining of teratoma extracted from the mouse.
  • FIG. 6 is a Scatcher plot diagram showing the result of comprehensive analysis of the difference in gene expression level with ES cells (mES-B6-ave) established from the obtained embryos using a microarray.
  • “1-3”, “1-4”, “2-1”, “2-2”, “1-5”, and “2-3” on the horizontal axis represent chimera fetus individuals.
  • “Body” indicates the chimera contribution ratio of all remaining parts excluding the head and internal organs from the chimeric individual.
  • Total indicates the chimera contribution ratio (chimera contribution ratio in all tissues) obtained by analyzing the whole individual by grinding it without removing the organ from the chimera individual.
  • the bar at the right end indicates the average value of the ratio of GFP positive cells of 1-3, 1-4, 2-1, 2-2 individuals. It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. . It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. .
  • mESCs indicates the analysis results of mouse ES cells established from embryos obtained by natural mating
  • mEpiSCs indicates the analysis results of epiblast stem cells obtained by differentiating the mouse ES cells.
  • PhESCs indicates the analysis result of haploid ES cells obtained by culturing according to the method of the present invention
  • phEpiSCsP3 indicates epiblast stem cells obtained by differentiating the haploid ES cells.
  • the analysis result of (passage number 3) is shown, “phEpiSCsP5” shows the analysis result of epiblast stem cells (passage number 5) obtained by differentiating the haploid ES cells, and “MEF” is mouse embryo 13
  • the analysis result of the fibroblast obtained from the day embryo is shown, and “H 2 O” shows the result of analyzing water (no template DNA) (negative control).
  • the notation on the left side of the figure indicates the analyzed marker genes (Oct4, Nanog, Rex1, Fgf5), “GAPDH” indicates a positive control in this analysis, and “RT ( ⁇ )” indicates a negative control (reverse transcription). Results without reaction).
  • passage number here means the passage number after differentiating phESC into phEpiSC.
  • the notation on the right side of the figure indicates that each marker gene shown on the left side of the figure is an ES cell-specific marker gene (indicated as “ES”) and an epiblast stem cell-specific marker gene (“EpiSC”). And a marker gene whose expression is recognized in ES cells and epiblast stem cells (denoted as “ES / EpiSC”).
  • the present invention provides a method for culturing haploid embryonic stem cells, the method comprising culturing the cells under conditions that constitutively activate Cdc2.
  • the transition from the G2 phase to the M phase is performed in the progression of the cell cycle. It is possible to promote and maintain the monoploidy. Furthermore, in the haploid embryonic stem cells obtained by culturing in this way, mutations such as deletion and amplification do not occur in the genomic DNA, and the pluripotency and high proliferation ability remain maintained.
  • haploid embryonic stem cell means an embryonic stem cell (ES cell) established from an early embryo that has developed as a haploid, and is described in, for example, Non-Patent Documents 1 to 4.
  • Female parthenogenetic embryo-derived haploid ES cells female haploid ES cells, parthenogenetic haploid ES cells, which are established from the early embryos obtained by generating activated unfertilized eggs as described above PhES cells, phESC).
  • Non-Patent Documents 5 and 6 male parthenogenesis established from early embryos obtained by transplanting, activating and generating sperm nuclei into enucleated unfertilized eggs Examples also include embryos derived from haploid ES cells (male haploid ES cells, androgenetic haploid ES cells, ahES cells, ahESC).
  • the origin of unfertilized eggs and sperm used for establishment of these haploid ES cells is not particularly limited, and examples thereof include humans and non-human animals.
  • non-human animals include rodents such as mice and rats, cows, horses, pigs, sheep, monkeys, dogs, mammals such as cats, and birds such as chickens.
  • any method for activating the unfertilized egg any method may be used as long as the second polar body is released from the egg. For example, a method of chemically stimulating with strontium, ethanol, or the like, The method of giving is mentioned.
  • examples of the early embryo prepared by generating activated unfertilized eggs and used for establishing haploid ES cells include, for example, the 8-cell stage, 16-cell stage, morula stage, or blastocyst
  • the embryo in the stage is mentioned.
  • embryos in the blastocyst stage are preferable from the viewpoint of high ES cell establishment efficiency.
  • they are produced from a pure mouse such as C57BL / 6, they are in the 16 cell stage or morula stage. Embryos are preferred, and embryos in the morula stage are more preferred (see Table 1 below).
  • Examples of the “haploid ES cells” used in the culture method of the present invention include not only naturally derived cells but also cells that have been genetically modified, such as phESC-B6GFP used in the examples described later ( Genetically modified cells).
  • the genetically modified cell may be a cell into which a gene encoding a protein to be expressed is introduced exogenously, such as GFP in the phESC-B6GFP, and may be obtained by a knockout method, an RNA method, an antisense method, etc.
  • a cell in which the function of a specific gene is suppressed may be used, or a cell in which the function of a gene is suppressed or activated randomly.
  • Examples of cells whose gene functions are randomly suppressed or activated include cells treated with chemical mutagens such as EMU, EMS, NMU, and NTG, and DNA-cleaving enzymes such as zinc finger nuclease and TALEN. Cells, cells irradiated with fast neutron rays, gamma rays or ion beams, and cells in which transposons are randomly inserted into genomic DNA. Further, such gene modification may be performed after the establishment of haploid cells, for example, at the individual stage of collecting the unfertilized egg or the sperm before the establishment. It may be a thing.
  • Constant activation of Cdc2 means that Cdc2 is always in a state capable of promoting cell division. That is, in normal cell cycle progression, it means that Cdc2 is in a state where Cdc2 can promote cell division, not limited to G2 to M phase where Cdc2 is activated.
  • Cdc2 In the control of the cell cycle, Cdc2 is inactivated and the transition to M phase is suppressed by phosphorylation of 14th threonine or 15th tyrosine of Cdc2 by Wee1 or Myt1 (in G2 phase) Will stop).
  • Cdc2 is activated by Cdc25, whereby Cdc2 is activated by dephosphorylating the 14th threonine and 15th tyrosine of Cdc2.
  • a condition for activating Cdc2 constantly includes a condition under which phosphorylation of Cdc2 at 14th threonine or 15th tyrosine is constantly suppressed.
  • Such suppression of phosphorylation can be achieved by suppressing the activity of Wee1 or Myt1, enhancing the activity of Cdc25, etc.
  • Wee1 it is preferable to suppress the activity of Myt1, and it is more preferable to suppress both the activities of Wee1 and Myt1.
  • “activity suppression” means not only complete suppression (inhibition) of the activity but also partial suppression. Moreover, suppression or enhancement of the activity of these proteins can also be achieved by suppression or enhancement of the expression of the protein. Therefore, “inhibition or enhancement of activity” in the present invention includes cases achieved through inhibition or enhancement of expression. Furthermore, the suppression or enhancement of expression may be at the transcription level or at the translation level.
  • the constant suppression of phosphorylation of Cdc2 at 14th threonine or 15th tyrosine can be achieved by adding a Cdc2 activator described later to a medium in which haploid embryonic stem cells are cultured, etc. This can be achieved by introducing the activator into cells.
  • the haploid embryonic stem cells in the medium supplemented with PD166285 are used in the culture method of the present invention. Is preferably cultured.
  • the concentration of PD166285 added to the medium is not particularly limited as long as Cdc2 can be constantly activated. From the viewpoint of culturing without causing cell death in haploid embryonic stem cells and the like, 300 nM The following is preferable.
  • the haploid embryonic stem cells may be cultured under the conditions where Cdc2 is constantly activated as described above, and other culture conditions can be determined by those skilled in the art. If it exists, the culture
  • a medium used for culturing haploid embryonic stem cells can be prepared based on a basal medium for culturing known embryonic stem cells.
  • basal media include DMEM medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, ⁇ MEM medium, Ham medium, RPMI 1640 medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improved medium, Examples include IMDM medium, medium 199 medium, and any mixed medium.
  • the medium used for culturing haploid embryonic stem cells may be a serum-containing medium or a serum-free medium.
  • a serum-free medium means a medium that does not contain unprepared or unpurified serum, and may include a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors). From the viewpoint of preventing contamination with components derived from different animals, the serum is preferably derived from the same species as the stem cells.
  • the medium used for culturing haploid embryonic stem cells may or may not contain a serum substitute. Examples of serum substitutes include commercially available products such as knockout serum substitutes (KSR, manufactured by Invitrogen), chemical lipid concentrates (Chemically-defined Lipid concentrated, manufactured by Gibco), and glutamax (produced by Gibco). It is done.
  • a medium used for culturing haploid embryonic stem cells is added with a compound that suppresses differentiation of embryonic stem cells and the like.
  • Such compounds include leukemia inhibitory factor (LIF), MEK inhibitor (PD0325901, AZD6244, CI-1040 (PD184352), RDEA119 (BAY869766), SL327, U0126, PD98059, U0124, U0125), GSK3 inhibitor (CHIR99021, SB216763).
  • TGF ⁇ receptor inhibitors SB431542, etc.
  • FGF receptor inhibitors SU5402, PD1733074, etc.
  • ROCK inhibitors GSK26962A, Y-27632, H-1152, etc.
  • the medium used for the culture of haploid embryonic stem cells includes amino acids (L-glutamine, non-essential amino acids, etc.), reducing agents (2-mercaptoethanol), antibiotics (penicillin, streptomycin, etc.), fatty acids or lipids. , Sugars, vitamins, growth factors, cytokines, antioxidants, organic acids (pyruvic acid, lactic acid, etc.), buffers, inorganic salts, and the like.
  • incubator used for culturing haploid embryonic stem cells known ones used for culturing embryonic stem cells and the like can be used as appropriate, and cell-adhesive incubators (for example, ECM, Matrigel) , A culture vessel coated with a cell support substrate such as gelatin or collagen) or a non-cell-adhesive culture vessel.
  • cell-adhesive incubators for example, ECM, Matrigel
  • non-cell-adhesive culture vessel for example, ECM, Matrigel
  • culture conditions for haploid embryonic stem cells can be appropriately selected and adjusted by those skilled in the art in accordance with known culture conditions for embryonic stem cells.
  • the culture temperature is particularly limited. However, it is usually about 30 to 40 ° C, preferably about 37 ° C.
  • concentration of CO 2 is usually about 1-10%, preferably about 2-5%.
  • the oxygen concentration is usually 1 to 10%.
  • the culture of haploid embryonic stem cells may be an adhesion culture or a non-adhesion culture.
  • haploid embryonic stem cells can also be cultured in the presence of feeder cells.
  • the feeder cells are not particularly limited.
  • mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation (gamma rays or the like) or treatment with antibiotics (mitomycin C or the like) are used.
  • non-adherent culture include dispersion culture, aggregated suspension culture, and suspension culture on a carrier.
  • haploid embryonic stem cells cultured using the above-described culture method are not only maintained monoploidy but also pluripotent without causing deletion or the like in genomic DNA.
  • a chimeric animal can be obtained from the haploid embryonic stem cells.
  • the present invention is a method for producing a non-human chimeric animal comprising: (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; (Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo; (Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
  • step (i) is as described in ⁇ Method for culturing haploid embryonic stem cells> above.
  • the “early embryo” into which the haploid embryonic stem cells are introduced may be any non-human animal, such as rodents such as mice and rats, cows, horses, pigs, Examples include sheep, monkeys, dogs, mammals such as cats, and birds such as chickens.
  • the origin of the early embryo is preferably the same (same type) as that of the introduced haploid embryonic stem cell, but may be of a heterogeneous relationship.
  • the early embryonic development stage examples include the 8-cell stage, the 16-cell stage, the morula stage, or the blastocyst stage.
  • an early embryo in the blastocyst stage is used for producing a chimeric embryo.
  • a tetraploid embryo may be used as an early embryo into which a haploid embryonic stem cell is introduced. This is because an animal derived from the haploid embryonic stem cell can be directly obtained by generating a tetraploid chimeric embryo into which the haploid embryonic stem cell has been introduced (A. Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8424-8428).
  • the tetraploid embryo should be prepared by a method known to those skilled in the art, such as electrofusion of blastocysts or electrofusion of 2-cell blastocysts by applying an electric pulse in a mannitol solution. Can do.
  • the “introduction” from the haploid embryonic stem cells to the aforementioned early embryo can be performed using a known chimeric embryo production method such as a microinjection method or an aggregation method.
  • step (iii) the chimeric embryo obtained as described above is transplanted into the maternal womb (uterus or fallopian tube) of the non-human animal and generated, whereby a non-human chimeric animal can be obtained.
  • the non-human animal to which the chimeric embryo is transplanted is preferably a pseudopregnant animal, and is preferably the same animal as the origin of the early embryo.
  • the present invention can also provide a method for producing a non-human germline chimeric animal, wherein the germ cells are derived from haploid embryonic stem cells, comprising the steps (i) to (iii).
  • a non-human germline chimeric animal obtained by this method is mated with a normal animal (wild-type animal) or the non-human germline chimeric animal, and a gene derived from the haploid embryonic stem cell is generated from its offspring. If an individual to be possessed is selected, an animal having a gene derived from the haploid embryonic stem cell (an animal derived from the haploid embryonic stem cell) can also be obtained. For selection of an individual having a gene derived from the haploid embryonic stem cell, various traits can be used as an index. For example, body color or the like can be selected as an index. It is also possible to select by extracting DNA from a part of the body and performing Southern blot analysis or PCR analysis.
  • a genetically modified cell into which the above-mentioned specific foreign gene is introduced is used as the haploid embryonic stem cell introduced into the early embryo
  • an animal having the introduced foreign gene can be obtained.
  • a gene-deficient heterozygous animal can be obtained by using a genetically modified cell in which the function of the specific gene is suppressed.
  • gene-deficient homozygous animals can also be obtained by mating the obtained gene-deficient heterozygous animals.
  • the haploid embryonic stem cell introduced into the early embryo if a genetically modified cell whose gene function is randomly suppressed or activated is used, the phenotype of the obtained non-human chimeric animal or its offspring Based on the above, it is possible to search for a gene that causes the disease (a gene whose function is suppressed or activated). That is, the method of the present invention can be suitably used for forward genetics.
  • the present invention is a method for producing haploid differentiated cells comprising: (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2; And (ii) a step of inducing differentiation of the haploid embryonic stem cell.
  • the step (i) is as described in ⁇ Method for culturing haploid embryonic stem cells> above.
  • the differentiation induction in the step (ii) is preferably not performed under conditions that constitutively activate Cdc2 from the viewpoint of not suppressing the differentiation of haploid embryonic stem cells.
  • differentiation induction may be performed by in vitro culture or by in vivo culture. A person skilled in the art can perform a known induction method from a universal stem cell such as an embryonic stem cell to a target differentiated cell. It can be performed by selecting appropriately.
  • the above-described haploid embryonic stem cells are cultured in a non-adhered state by removing factors that suppress the differentiation of cells such as LIF from the above-described haploid embryonic stem cell medium.
  • an embryoid body (EB) containing a tissue differentiated into three germ layers can be formed.
  • this EB is cultured on a substrate such as laminin or fibronectin using a medium supplemented with a differentiation inducer such as retinoic acid or activin to obtain cells differentiated into nervous system cells or blood cells. be able to.
  • target differentiated cells can be directly obtained from the haploid embryonic stem cells by a combination of cytokines, growth factors and the like as described in Examples below. It can also be obtained.
  • the differentiated cells obtained in this way may be stem cells (such as epiblast stem cells) as shown in Examples described later, or may be cells obtained by further differentiation induction from the stem cells.
  • a teratoma formation method as shown in Examples described later can also be used. That is, by transplanting the haploid embryonic stem cells into an immunodeficient animal, a teratoma can be formed in the animal, and a wide variety of differentiated cells can be isolated from the teratoma.
  • the differentiated cells thus obtained can be cultured under conditions that constantly activate Cdc2, whereby the monoploidy of the differentiated cells can be maintained.
  • the conditions for activation are as described above.
  • Such culture can be performed by those skilled in the art by appropriately selecting a medium, temperature, CO 2 concentration and the like suitable for the differentiated cells to be cultured.
  • Cdc2 ⁇ Drug to maintain monoploidy>
  • the present invention provides a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells containing a Cdc2 activator as an active ingredient.
  • the “Cdc2 activator” of the present invention is a compound having an action of suppressing the activity of Wee1 or Myt1, which is a factor inhibiting Cdc2 activation, and more specifically, Wee1 as shown in Examples described later.
  • a low molecular weight compound that binds to Myt1 and RNA that binds to a transcription product of a gene encoding Wee1 or Myt1.
  • Cdc2 activator of the present invention In order to act on cells, it does not require complicated and damaging work such as gene introduction using viral vectors, introduction by electroporation, introduction using transfection reagents, etc., and it is added to the medium From the standpoint that Cdc2 of the cell can be constitutively activated alone, a low molecular weight compound that binds to Wee1 or Myt1 is preferred as the Cdc2 activator of the present invention.
  • Examples of the “low molecular compound having an action of suppressing the activity of Wee1 or Myt1” include PD166285 (2-[[4- [2- (diethylamino) ethoxy] phenyl] amino] -6- (2,6-dichlorophenyl).
  • a low molecular weight compound having an action of suppressing the activity of Wee1 International Publication
  • the low molecular weight compounds having the action of suppressing the activity of Myt1 described in 2000/33842 and physiologically acceptable salts or solvates of these compounds can be mentioned, but both the activities of Wee1 and Myt1 can be suppressed.
  • PD166285 is preferable, and PD166285 dihydrochloride is more preferable.
  • RNA that binds to the transcript of the gene encoding Wee1 or Myt1 may be any RNA that targets Wee1 or Myt1 and can suppress the activity of these proteins through suppression of its expression.
  • examples include dsRNA (double stranded RNA) such as siRNA and shRNA (short haipin RNA) complementary to a transcription product of a gene encoding Myt1.
  • the dsRNA chain length is not particularly limited as long as it can suppress the expression of the target gene and does not exhibit toxicity, and is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs. More preferably, it is 21 to 30 base pairs.
  • the dsRNA need not be completely identical to the base sequence of the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more of sequence identity. Sequence identity can be determined by the BLAST program.
  • an antisense RNA complementary to the transcript of the gene encoding Wee1 or Myt1 or the transcript is specifically cleaved.
  • An RNA having ribozyme activity is also included.
  • RNA may be substituted in part or all by artificial nucleic acids such as PNA, LNA, ENA and the like.
  • these RNAs may be in the form of an expression vector that holds DNA encoding the RNA for expression in cells.
  • those skilled in the art can prepare such RNA by chemical synthesis using a commercially available synthesizer or the like.
  • the “Cdc2 activator” in the present invention may be a compound having an action of suppressing phosphorylation of Cdc2 at these phosphorylated sites. Examples of such a compound include Cdc2 decoy and Cdc2 An antibody is mentioned.
  • Examples of the Cdc2 decoy include a partial polypeptide of Cdc2 containing a site (14th threonine or 15th tyrosine) that is phosphorylated by Wee1 or Myt1.
  • the chain length of such a partial polypeptide is not particularly limited and includes, for example, 5 to 30 amino acids, but does not include the 161st tyrosine, which is a phosphorylation site necessary for activating Cdc2. It is preferable that Such a polypeptide can also be chemically synthesized using a commercially available synthesizer or the like, and can also be prepared using a known recombinant protein production method using Escherichia coli or the like as a host.
  • a cell membrane-permeable peptide may be added to the Cdc2 decoy to increase the efficiency of introduction into the cell.
  • the cell membrane permeable peptide include VP22, Kaposi FGF, TAT, Drosophila Antitinapedia, Penetratin, M918, Transportan-10, Poly-Arginine, and derivatives of these peptides.
  • the cell membrane permeable peptide may be appropriately prepared based on the description of International Publication No. 2007/13255.
  • Examples of antibodies against Cdc2 include antibodies that can specifically recognize and bind to the non-phosphorylated state of the 14th threonine or 15th tyrosine of Cdc2.
  • the form of the antibody may be a polyclonal antibody, a monoclonal antibody, or a functional fragment (nanoantibody, single chain antibody) of these antibodies.
  • Those skilled in the art can appropriately prepare such an antibody by a known antibody production method (hybridoma method, phage display method, etc.) using a partial polypeptide of Cdc2 containing the 14th threonine or the 15th tyrosine. can do.
  • Cdc2 is also activated by dephosphorylation of the 14th threonine or 15th tyrosine of Cdc2 phosphorylated by Wee1 or Myt1 by Cdc25. Therefore, a compound having an action of enhancing the activity of Cdc25 can also be used as the “Cdc2 activator” in the present invention.
  • An example of such a compound is Cdc25. That is, by introducing the protein itself into the cell, the overall activity of Cdc25 in the cell can be enhanced through an increase in the expression level thereof.
  • Cdc25 not only a protein aspect but also an expression vector aspect that holds a DNA encoding Cdc25 in order to express the protein in a cell.
  • the “drug for maintaining monoploidy” of the present invention may contain a physiologically acceptable carrier in addition to the aforementioned Cdc2 activator.
  • physiologically acceptable carriers include isotonic solutions containing physiological isotonic solutions (physiological saline, medium, glucose and other adjuvants (D-sorbitol, D-mannitol, sodium chloride, etc.), etc. ), Excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol, etc.), binders, solubilizers, nonionic surfactants, buffers (phosphate buffers, sodium acetate buffers, etc.) , Preservatives and antioxidants.
  • physiological isotonic solutions containing physiological isotonic solutions (physiological saline, medium, glucose and other adjuvants (D-sorbitol, D-mannitol, sodium chloride, etc.), etc. ), Excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol
  • a transfection reagent for example, a polyamine-based transfection reagent or a cationic lipid-based transfection reagent
  • a transfection reagent may be appropriately added in order to introduce the Cdc2 activator into cells.
  • Example 1 ⁇ Establishment of female parthenogenetic embryo-derived haploid ES cells (phES cells, phESC) and culture thereof>
  • PMSG serum gonadotropin
  • hCG human chorionic gonadotropin
  • Ovulation was induced and unfertilized eggs were collected.
  • the collected unfertilized eggs were treated with strontium chloride (SrCl 2 ) for 30 to 60 minutes, and then cultured in vitro in KSOM medium until they developed in morula.
  • the developed morulae are treated with 1/2 dilution acidic Tyrode to dissolve the zona pellucida, and cultured on ICR mouse-derived feeder cells treated with mitomycin to obtain pure mouse C57BL / 6 Haploid ES cells (phESC-B6) derived from female parthenogenetic embryos were established.
  • the mixed group of phES cells and diploid ES cells prepared in this manner is 20% KSOM, 2 mM L-glutamine, 1 ⁇ non-essential amino acid, 100 uM mercaptoethanol in knockout DMEM. Further, the cells were cultured in a medium in which 3 ⁇ M CHIR99021 and 1 ⁇ M PD0325901 called 2i were further added to 0.1% penicillin / streptomycin, 1000 unit LIF (ESGRO, registered trademark). After 1N cells were collected by FACS, 300 ⁇ M PD166285 was added to the medium and cultured. The mixed group was also cultured in the absence of feeder cells.
  • Cultivation in the absence of feeder cells was performed using a 0.1% gelatin solution in an incubator and treating at 37 ° C. for 30 minutes, then discarding the gelatin solution and using the incubator.
  • Other culture conditions (addition concentration of PD166285, etc.) were the same as those in the presence of the feeder cells described above.
  • 1N cells were collected by FACS by staining with Hoechst 33342 for 30 minutes, washing the cells with PBS ( ⁇ ) with 4% FBS, and then using FACS Aria II (manufactured by Nippon Becton Dickinson).
  • the cells were fixed by treating with 70% ethanol at 4 ° C. for 8 hours or more and analyzed by FACS Calibur (manufactured by Becton Dickinson, Japan) after PI staining.
  • Example 2 ⁇ Establishment of male parthenogenetic embryo-derived haploid ES cells (ahES cells, ahESC) and culture thereof>
  • ahESC male parthenogenetic embryo-derived haploid ES cells
  • ahESC male parthenogenetic embryo-derived haploid ES cells
  • Example 3 ⁇ Establishment of female parthenogenetic embryo-derived epiblast stem cells (phEpiSC, parthenogenetic haploid epiblast stem cell) and culture method thereof> Differentiation induction from phESC established as described above to phEpiSC was performed as follows. That is, differentiation induction into phEpiSC was performed by culturing phESC in DMEM / F12 medium supplemented with N2, B27, NEAA, GlutaMAXI, and ⁇ -mercaptoethanol for 7 days or more.
  • the cells after differentiation induction into phEpiSC are selected from haploid cells using FACS, and then cultured in a medium further supplemented with 20 ng / ml activin A, 20 ng / ml bFGF, 10 nM XAV939 and 300 nM PD166285.
  • 20 ng / ml activin A 20 ng / ml activin A
  • 20 ng / ml bFGF 10 nM XAV939
  • 300 nM PD166285. See Sumi, T. et al., PLoS One, 2013, 8, e63378 for components other than PD166285 regarding the medium in the culture).
  • the cells after differentiation induction into phEpiSC were cultured on feeder cells treated with mitomycin C. Furthermore, in this culture in the presence of PD166285, haploid cells were purified using FACS every 7 days.
  • the haploid phEpiSC was purified by treating with Hoechst at 37 ° C. for 30 minutes, and then selecting only the G1 compartment containing the haploid cells with FACS Aria II. Further, in the differentiation induction from phESC to phEpiSC and the culture after the differentiation induction, the cells are subcultured by treating with complete acactase (registered trademark, complete accurate) at 37 ° C. for 2 minutes. Passage was once at a pace.
  • complete acactase registered trademark, complete accurate
  • the genomic DNA thus prepared was treated with an In-situ oligo DNA microarray kit (manufactured by Agilent) and analyzed by array CGH using 4 ⁇ 180 L slides.
  • the reference genome was labeled with Cy3, and the phESC-derived genome was labeled with Cy5.
  • PCR analysis was also performed using the genomic DNA as a template.
  • anti-Oct3 / 4 antibody goat-derived anti-mouse Oct3 / 4 antibody
  • anti-Nanog antibody rabbit-derived anti-mouse Nanog antibody
  • anti-SSEA1 antibody donkey-derived anti-mouse SSEA1 antibody
  • Alexafluoro 568-conjugated goat-derived anti-rabbit IgG antibody, Alexafluoro 488-conjugated goat-derived anti-mouse IgM antibody and Alexafluoro 488-conjugated donkey-derived anti-goat IgG antibody were each used as secondary antibodies, and the concentration was 1/500. And reacted with phESC at room temperature for 1 hour. These cells were DAPI stained for 5 minutes at room temperature after the secondary antibody reaction. Alkaline phosphatase staining was performed using the SK-5300 kit (manufactured by Vector).
  • teratoma formation 1N cells of haploid ES cells were collected by FACS, and within 7 days, these cells were administered subcutaneously to KSN male mice at 2 ⁇ 10 6 cells / mouse. Then, teratoma was analyzed 30 days after the administration of the cells. The HE staining at the time of analysis was performed according to a conventional method.
  • RNA expression analysis using microarray The gene expression level in phESC obtained by the method of the present invention was analyzed. Specifically, first, 2 ⁇ 10 5 phESC 1N cells and 2N cells of phESC-derived ploidy ES cells were collected by FACS, and total RNA was extracted with Trizol. ES cells established from embryos obtained by natural mating were subjected to RNA extraction without collecting 2N cells by FACS. The RNA thus extracted was treated with a whole mouse genomic DNA oligo microarray kit (manufactured by Agilent) and analyzed with a microarray using 4 ⁇ 44K slides.
  • mice dissected at d14.5 After 14.5 days, or a fetus was obtained by spontaneous delivery and GFP fluorescence was confirmed.
  • the proportion of GFP-positive cells was confirmed for each organ by FACS Aria II. Confirmation of germline migration in chimeric mice was confirmed by detecting GFP expression in fetuses obtained by natural mating of chimeric mice.
  • haploid embryos having one set of maternal genome can be produced by generating parthenogenesis by giving strontium (Sr) stimulation to mouse unfertilized eggs (see Non-Patent Documents 1 to 4). ).
  • the present inventors have partially generated eggs of C57BL / 6 female mice, cultured until in vitro culture until they became morulae, and placed on feeder cells to obtain ES cells (female parthenogenetic embryo-derived 1). It has succeeded in establishing polyploid ES cells (phES cells, phESC).
  • PD166285 an inhibitor against factors (Wee1 and Myt1) that inhibit the transition from G2 phase to M phase in the cell cycle, is used in the culture of haploid ES cell lines. In order to prevent the diploid formation, the culturing was continued for a long time while maintaining the haploid state.
  • phES cells were subcultured 2 to 4 times in a knockout DMEM supplemented with 2i and the like, and then a haploid ES cell line (1N cell) was recovered by FACS and then cultured. Cultivated in the presence (300 nM, 500 nM or 1000 nM) or absence of PD166285, an inhibitor against Wee1 and Myt1. See FIG. 2 for the culturing process, and FIGS. 3 to 6 show the results of FACS analysis in the culturing process. Furthermore, the shape of the cells obtained by culturing in this way and their growth rate were also analyzed. The obtained results are shown in FIGS.
  • FIGS. 3 to 6 it was revealed that a high 1N cell rate can be maintained by recovering the haploid ES cell line by FACS and culturing in the presence of PD166285 from the subsequent culture. Further, as is apparent from the results shown in FIGS. 7 and 8, the cells when 300 nM PD166285 was added to the medium formed more three-dimensional colonies than those not added. Although not shown in the figure, the cell growth rate did not change with or without the inhibitor.
  • the haploid ES cell culture method of the present invention is extremely effective in maintaining the cells for a long time and stably.
  • male parthenogenetic embryo-derived haploid ES cells prepared by the method described in Example 2 in the same manner as the above-described phESC female parthenogenetic embryo-derived haploid ES cells.
  • AhESC male parthenogenetic embryo-derived haploid ES cells
  • histological analysis of the obtained teratomas revealed that three germ layers derived from the injected phESC-B6GFP were formed. Therefore, it was revealed that phESC-B6 established by culturing in the presence of PD166285 maintains undifferentiation and further has differentiation ability in vivo.
  • the method of the present invention it was possible to establish a haploid ES cell line that can be transferred to the germline even with the pure mouse phESC-B6GFP, which is considered to be difficult to establish ES cells and the like. It was revealed that the high quality cell line can be stably maintained.
  • haploid cells differentiated from phESC could be obtained with high efficiency by the method described in Example 3 and FIG. Furthermore, as a result of detecting the expression of the stem cell marker gene in these haploid differentiated cells, as shown in FIG. 22, the expression of Fgf5, a stem cell marker gene unique to EpiSC, was observed in the haploid differentiated cells. On the other hand, the expression of Rex1, a stem cell marker gene unique to ES cells, was not detected in these cells.
  • a differentiated cell can be obtained from a haploid ES cell while maintaining its haploid state, and the diploidy is also maintained in the differentiated cell. It became clear that it was possible.
  • haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability.
  • the present invention it is possible to culture C57BL / 6-derived haploid ES cells while maintaining the haploidity thereof. Therefore, the present invention is extremely useful in forward genetics research and the like.
  • a differentiated cell can be obtained from a haploid ES cell while maintaining its haploidity, and further, the uniploidy can be maintained in the differentiated cell. Can do. Therefore, the present invention is also useful in regenerative medicine, drug discovery development, and the like where there is a strong demand for providing a wide variety of cells, tissues, organs, and the like.

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Abstract

It was discovered that, when Cdc2 was constantly activated in haploid embryonic stem cells, the cell cycle transition from G2 phase to M phase was promoted and thus the haploidy could be maintained. Also, it was discovered that the cultured haploid embryonic stem cells thus obtained showed no variations such as deletion or amplification in the genomic DNA thereof and, furthermore, kept the differentiation/pluripotency and high proliferation potency. According to the present invention, therefore, haploid embryonic stem cells can be cultured while maintaining the haploidy and keeping the differentiation/pluripotency and high proliferation potency without causing any variations in the genomic DNA.

Description

１倍体胚性幹細胞の培養方法Method for culturing haploid embryonic stem cells
　本発明は、１倍体胚性幹細胞の培養方法に関し、より詳しくは、１倍体胚性幹細胞の１倍性を維持したまま培養する方法に関する。また、本発明は、該方法により培養した１倍体胚性幹細胞から非ヒトキメラ動物又は１倍体分化細胞を作製する方法にも関する。さらに、本発明は、１倍体胚性幹細胞又は１倍体分化細胞の１倍性を維持するための薬剤に関する。 The present invention relates to a method for culturing haploid embryonic stem cells, and more particularly, to a method for culturing haploid embryonic stem cells while maintaining the monoploidy. The present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method. Furthermore, the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells.
　哺乳類は通常、父親と母親に由来する２倍体ゲノムを持つ。これには、常染色体劣性遺伝病のような疾患を起こした場合、片親由来のゲノムが機能を補うことで致命的な症状とならないという意義がある。ところが、フォワードジェネティクス（Ｆｏｒｗａｒｄ　ｇｅｎｅｔｉｃｓ）を行う場合、２倍体であると、遺伝型と表現型との関係を直接的に解析することが困難になる。 Mammals usually have a diploid genome derived from their father and mother. This has the significance that when a disease such as an autosomal recessive genetic disease occurs, the genome derived from one parent does not become a fatal symptom by supplementing the function. However, when performing forward genetics, if it is diploid, it is difficult to directly analyze the relationship between the genotype and the phenotype.
　１倍体（半数体）細胞株は染色体を１組しか持たず、遺伝型と表現型とが直接的に見られることから、フォワードジェネティクス研究への応用が期待されており、最近、哺乳類において１倍体胚性幹細胞株（１倍体ＥＳ細胞株）の樹立が報告されている。例えば、マウス未受精卵に化学的な刺激を与えることで１倍体胚（ｈａｐｌｏｉｄ　ｅｍｂｒｙｏ）を作製し、そこからＥＳ細胞株を樹立することで、雌性単為発生胚由来１倍体ＥＳ細胞（雌性１倍体ＥＳ細胞、ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃ　ｈａｐｌｏｉｄ　ＥＳ細胞、ｐｈＥＳ細胞、ｐｈＥＳＣ）を得られたことが報告されている（非特許文献１～４）。また、除核した分裂中期にあるマウス未受精卵に円形精子細胞の核を移植し、化学的な刺激を与えることで１倍体胚を作製し、そこからＥＳ細胞株を樹立することで、雄性単為発生胚由来１倍体ＥＳ細胞（雄性１倍体ＥＳ細胞、ａｎｄｒｏｇｅｎｅｔｉｃ　ｈａｐｌｏｉｄ　ＥＳ細胞、ａｈＥＳ細胞、ａｈＥＳＣ）が得られることも報告されている（非特許文献５及び６）。 Haploid (haploid) cell lines have only one pair of chromosomes, and since genotypes and phenotypes can be seen directly, they are expected to be applied to forward genetics research. Establishment of a haploid embryonic stem cell line (haploid ES cell line) has been reported. For example, a haploid embryo is produced by chemically stimulating a mouse unfertilized egg, and an ES cell line is established therefrom, whereby a haploid embryonic cell derived from a female parthenogenetic embryo ( It has been reported that female haploid ES cells, parthenogenetic haploid ES cells, phES cells, phESC) were obtained (Non-Patent Documents 1 to 4). In addition, by transplanting the nucleus of a round sperm cell to a mouse non-fertilized egg that has been enucleated and producing a haploid embryo by applying chemical stimulation, an ES cell line is established from it, It has also been reported that male parthenogenetic embryo-derived haploid ES cells (male haploid ES cells, androgenetic haploid ES cells, ahES cells, ahESC) are obtained (Non-patent Documents 5 and 6).
　しかしながら、１倍体ＥＳ細胞においては、染色体の倍化（２倍体化）が自然に生じてしまうため、１倍体の状態（１倍性）を維持したまま培養することは困難である。したがって、１倍体ＥＳ細胞のその１倍性を維持するためには、フローサイトメーター（ＦＡＣＳ）を用い、定期的に１倍体細胞のＧ１期にある細胞（１Ｎ細胞）を回収し続ける必要がある。 However, in haploid ES cells, chromosome doubling (diploidization) occurs naturally, and it is difficult to culture while maintaining the haploid state (monoploidy). Therefore, in order to maintain the haploidity of haploid ES cells, it is necessary to regularly collect cells in the G1 phase of haploid cells (1N cells) using a flow cytometer (FACS). There is.
　ところが、このようにＦＡＣＳを用いて回収しても、染色体の倍化が自然に生じる培養条件下では、１Ｎ細胞の収率は１０％未満となり、回収できる細胞の数が少な過ぎるため、結果として、その後の１倍体ＥＳ細胞の維持は困難となる。 However, even when recovered using FACS in this way, under the culture conditions in which chromosome doubling occurs naturally, the yield of 1N cells is less than 10%, and the number of cells that can be recovered is too small. Thereafter, maintenance of haploid ES cells becomes difficult.
　また、１倍体ＥＳ細胞をフォワードジェネティクス研究に応用する際には、前述の通り、該細胞を１倍体のまま長期間安定に培養する方法が必要とされる他、通常のＥＳ細胞（自然交配にて得られた胚から樹立される２倍体のＥＳ細胞）同様に、多能性幹細胞としての性質を安定的に有していることも求められる。すなわち、通常のＥＳ細胞同様の遺伝子発現プロファイルを示し、分化多能性及び高い増殖能（自己複製能）を有していることが必要となる。さらに、当然のことながら、表現型から原因遺伝子を探索するフォワードジェネティクス研究への応用に際し、ゲノムＤＮＡにおいて欠失、増幅等の変異が生じさせないことも、１倍体ＥＳ細胞の培養方法には求められる。 In addition, when applying haploid ES cells to forward genetics research, as described above, there is a need for a method of stably culturing the cells in a haploid for a long period of time, as well as normal ES cells ( Similarly, diploid ES cells established from embryos obtained by natural mating) are also required to have stable properties as pluripotent stem cells. That is, it is necessary to show a gene expression profile similar to that of normal ES cells and to have differentiation pluripotency and high proliferation ability (self-replication ability). Furthermore, naturally, when applied to forward genetics research that searches for a causative gene from a phenotype, it is possible to prevent mutations such as deletion and amplification in genomic DNA. Desired.
　このように、フォワードジェネティクス研究等への応用に際し、１倍体ＥＳ細胞を長期に渡って安定に培養する方法が求められているが、そのような方法は未だ開発されていないのが現状である。 Thus, a method for stably culturing haploid ES cells over a long period of time is required for application to forward genetics research, etc., but such a method has not been developed yet. is there.
　本発明は、前記従来技術の有する課題に鑑みてなされたものであり、１倍体ＥＳ細胞を長期に渡って安定的に培養することが可能な方法を提供することを目的とする。 The present invention has been made in view of the above-described problems of the prior art, and an object thereof is to provide a method capable of stably culturing haploid ES cells over a long period of time.
　本発明者らは、前記目的を達成すべく鋭意研究を重ねた結果、１倍体ＥＳ細胞が自然に２倍体化するのは、細胞周期のＧ２期からＭ期への移行が不十分であることに基づくという可能性を想定した。すなわち、通常の２倍体細胞ではＧ１期、Ｓ期、Ｇ２期、Ｍ期と規則正しく細胞周期が回っているが（図１上段参照）、１倍体細胞が２倍体化する際にはＧ１期、Ｓ期、Ｇ２期、Ｇ１期、Ｓ期、Ｇ２期、Ｍ期というように、細胞周期が破綻していることが予想される。そして、このようにＭ期に入る前に２度のＳ期を経過することによって、細胞分裂を介さずにＤＮＡ合成が生じ、１倍体細胞においてゲノムＤＮＡが２倍体化していることが想定される（図１中段参照）。 As a result of intensive studies to achieve the above object, the present inventors have found that diploid ES cells naturally diploid because the transition from the G2 phase to the M phase of the cell cycle is insufficient. We assumed the possibility of being based on something. That is, in normal diploid cells, the cell cycle is regularly rotated from G1 phase, S phase, G2 phase, and M phase (see the upper part of FIG. 1), but when haploid cells diploid, G1 It is expected that the cell cycle has failed, such as the period, S period, G2 period, G1 period, S period, G2 period, and M period. It is assumed that DNA synthesis occurs without passing through cell division by passing the S phase twice before entering the M phase in this way, and genomic DNA is doubling in haploid cells. (See the middle part of FIG. 1).
　そこで、かかる想定に基づき、細胞周期の進行においてＧ２期からＭ期への移行を担う因子であるＣｄｃ２を活性化させることを試みた。すなわち、Ｃｄｃ２を不活性化する因子であるＷｅｅ１及びＭｙｔ１に対する阻害剤の存在下にて、１倍体ＥＳ細胞を培養することにより、該細胞において、Ｃｄｃ２を恒常的に活性化させた。その結果、１倍体ＥＳ細胞の２倍体化を抑制し、１倍性を維持したまま長期に培養できた。 Therefore, based on this assumption, an attempt was made to activate Cdc2, which is a factor responsible for the transition from the G2 phase to the M phase in the progression of the cell cycle. That is, by culturing haploid ES cells in the presence of inhibitors against Wee1 and Myt1, which are factors that inactivate Cdc2, Cdc2 was constantly activated in the cells. As a result, diploidization of haploid ES cells was suppressed, and culturing was possible for a long time while maintaining monoploidy.
　また、この方法で培養した１倍体ＥＳ細胞は、未分化性を維持しており、ｉｎ　ｖｉｖｏにおける多分化能も有し、キメラマウス形成にも寄与することができることも明らかにした。さらに、当該１倍体ＥＳ細胞は、キメラマウスにおいて生殖系列に移行（ｇｅｒｍｌｉｎｅ　ｔｒａｎｓｍｉｓｓｉｏｎ）できることも判明した。 It was also revealed that haploid ES cells cultured by this method maintain undifferentiated properties, have multipotency in vivo, and can contribute to the formation of chimeric mice. Furthermore, it was also found that the haploid ES cells can be transferred to the germline in chimeric mice.
　また、この方法で培養した１倍体ＥＳ細胞から、その１倍性を維持させたままエピブラスト幹細胞（Ｅｐｉｂｌａｓｔ　Ｓｔｅｍ　ｃｅｌｌ；ＥｐｉＳＣ）に分化させることができ、該分化細胞においてもその１倍性を維持させることができることも見出し、本発明を完成するに至った。すなわち、本発明は、１倍体胚性幹細胞の培養方法に関する。また、本発明は、該方法により培養した１倍体胚性幹細胞から非ヒトキメラ動物又は１倍体分化細胞を作製する方法にも関する。さらに、本発明は、１倍体胚性幹細胞又は１倍体分化細胞の１倍性を維持するための薬剤に関し、より詳しくは以下の発明を提供するものである。
（１）　１倍体胚性幹細胞の培養方法であって、Ｃｄｃ２を恒常的に活性化する条件下にて該細胞を培養する工程を含む方法。
（２）　非ヒトキメラ動物を作製する方法であって、
（i)Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
（ii)該１倍体胚性幹細胞を初期胚に導入し、キメラ胚を作製する工程と、
（iii)該キメラ胚を非ヒト動物の母胎内に移植し、発生させ、非ヒトキメラ動物を得る工程とを含む方法。
（３）　前記非ヒトキメラ動物が、生殖細胞が前記１倍体胚性幹細胞に由来する非ヒト生殖系列キメラ動物である、（２）に記載の方法。
（４）　１倍体分化細胞を作製する方法であって、
（i)Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
（ii)該１倍体胚性幹細胞を分化誘導する工程とを含む方法。
（５）　Ｃｄｃ２活性化剤を有効成分とする、１倍体胚性幹細胞又は１倍体分化細胞の１倍性を維持するための薬剤。 In addition, haploid ES cells cultured by this method can be differentiated into Epiblast Stem cells (EpiSC) while maintaining their haploidity, and even in the differentiated cells, the haploidy can be differentiated. It has also been found that it can be maintained, and the present invention has been completed. That is, the present invention relates to a method for culturing haploid embryonic stem cells. The present invention also relates to a method for producing non-human chimeric animals or haploid differentiated cells from haploid embryonic stem cells cultured by the method. Furthermore, the present invention relates to a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells. More specifically, the present invention provides the following inventions.
(1) A method for culturing haploid embryonic stem cells, the method comprising culturing the cells under conditions that constantly activate Cdc2.
(2) A method for producing a non-human chimeric animal,
(I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
(Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo;
(Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
(3) The method according to (2), wherein the non-human chimeric animal is a non-human germ line chimeric animal whose germ cells are derived from the haploid embryonic stem cells.
(4) A method for producing haploid differentiated cells,
(I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
(Ii) a step of inducing differentiation of the haploid embryonic stem cells.
(5) A drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells, comprising a Cdc2 activator as an active ingredient.
　本発明によれば、１倍体ＥＳ細胞を長期に渡って安定的に培養することが可能となる。すなわち、１倍性を維持しつつ、ゲノムＤＮＡにおいて欠失等を生じさせることなく、分化多能性及び高い増殖能を保持したまま、１倍体ＥＳ細胞を培養することが可能となる。また、本発明によれば、かかる１倍体ＥＳ細胞から、その１倍性を維持させたまま分化した細胞を得ることができ、さらには該分化細胞においてもその１倍性を維持させることも可能となる。 According to the present invention, haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability. In addition, according to the present invention, differentiated cells can be obtained from such haploid ES cells while maintaining their haploidity, and the diploidy can also be maintained in the differentiated cells. It becomes possible.
１倍体胚性幹（ＥＳ）細胞において想定される、細胞周期の進行を示す模式図である。すなわち、１倍体ＥＳ細胞において通常の細胞周期の進行が行われていれば、図１の上段に示されているように、その１倍性は維持されたまま分裂、増殖していくこととなる。しかしながら、１倍体ＥＳ細胞が自然に２倍体化していくため、例えば、図１の中段に示されているように、Ｇ１期、Ｓ期、Ｇ２期、Ｇ１期、Ｓ期、Ｇ２期、Ｍ期というように、細胞周期が破綻し、２倍体化が生じていることが想定される。そこで、本発明においては、かかる想定に基づき、細胞周期の進行においてＧ２期からＭ期への移行を担う因子であるＣｄｃ２を活性化させることを試み、その結果、１倍体ＥＳ細胞の２倍体化を抑制することができ（図１の下段参照）、１倍性を維持したまま長期に培養できることに成功したことを示す模式図である。It is a schematic diagram showing cell cycle progression assumed in haploid embryonic stem (ES) cells. That is, if normal cell cycle progression is carried out in haploid ES cells, as shown in the upper part of FIG. 1, the haploidity is maintained and the cells are divided and proliferated. Become. However, since haploid ES cells naturally diploidize, for example, as shown in the middle part of FIG. 1, G1 phase, S phase, G2 phase, G1 phase, S phase, G2 phase, As in the M phase, it is assumed that the cell cycle is broken and diploidization occurs. Therefore, in the present invention, based on this assumption, an attempt was made to activate Cdc2, which is a factor responsible for the transition from the G2 phase to the M phase in the progression of the cell cycle, and as a result, twice that of haploid ES cells. FIG. 2 is a schematic diagram showing that it was possible to suppress somaticization (see the lower part of FIG. 1) and succeeded in culturing for a long time while maintaining monoploidy. １倍体ＥＳ細胞（１Ｎ細胞）をＦＡＣＳにて回収し、その後の培養からＷｅｅ１及びＭｙｔ1の阻害剤であるＰＤ１６６２８５の存在下又は非存在下にて培養した工程を示す、概略図である。It is the schematic which shows the process of collect | recovering a haploid ES cell (1N cell) by FACS, and culture | cultivating in the presence or absence of PD166285 which is an inhibitor of Wee1 and Myt1 from subsequent culture | cultivation. ＰＤ１６６２８５の非存在下にて継代培養した１倍体ＥＳ細胞（ｐｈＥＳ－Ｂ６ＧＦＰ－３）をＦＡＣＳにて解析した結果（図中、左参照）と、当該細胞からＦＡＣＳにて１Ｎ細胞をソーティングし、さらにＰＤ１６６２８５の存在下にて３回継代培養した後の１倍体ＥＳ細胞をＦＡＣＳにて解析した結果（図中、右参照）とを示す、ヒストグラムである。Results of FACS analysis of haploid ES cells (phES-B6GFP-3) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS. Furthermore, it is a histogram showing the results of FACS analysis of haploid ES cells after three subcultures in the presence of PD166285 (see the right in the figure). ＰＤ１６６２８５の非存在下にて８回継代培養した１倍体ＥＳ細胞（ｐｈＥＳ－Ｂ６ＧＦＰ）をＦＡＣＳにて解析した結果（図中、左参照）と、該細胞からＦＡＣＳにて１Ｎ細胞をソーティングし、さらにＰＤ１６６２８５の存在下にて６回継代培養（計１４回継代培養）した後の１倍体ＥＳ細胞をＦＡＣＳにて解析した結果（図中、真ん中参照）と、該細胞からＦＡＣＳにて１Ｎ細胞をソーティングし、さらにＰＤ１６６２８５の存在下にて２９回継代培養（計４３回継代培養）した後の１倍体ＥＳ細胞をＦＡＣＳにて解析した結果（図中、右参照）を示す、ヒストグラムである。Results of FACS analysis of haploid ES cells (phES-B6GFP) subcultured 8 times in the absence of PD166285 (see the left in the figure), and 1N cells were sorted from the cells by FACS. Further, the results of FACS analysis of haploid ES cells after 6 subcultures (total 14 subcultures) in the presence of PD166285 (see the middle in the figure), and from these cells to FACS 1N cells were sorted, and the results of FACS analysis of haploid ES cells after 29 passages (43 passages in total) in the presence of PD166285 (see the right in the figure) This is a histogram. ＰＤ１６６２８５の非存在下にて継代培養した１倍体ＥＳ細胞（ｐｈＥＳ－Ｂ６ＧＦＰ－４）をＦＡＣＳにて解析した結果（図中、左参照）と、当該細胞からＦＡＣＳにて１Ｎ細胞をソーティングし、さらにＰＤ１６６２８５の存在下にて３回継代培養した後の１倍体ＥＳ細胞をＦＡＣＳにて解析した結果（図中、右参照）とを示す、ヒストグラムである。Results of FACS analysis of haploid ES cells (phES-B6GFP-4) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS. Furthermore, it is a histogram showing the results of FACS analysis of haploid ES cells after three subcultures in the presence of PD166285 (see the right in the figure). ＰＤ１６６２８５の非存在下にて継代培養した１倍体ＥＳ細胞（ｐｈＥＳ－Ｂ６－９）をＦＡＣＳにて解析した結果（図中、左参照）と、当該細胞からＦＡＣＳにて１Ｎ細胞をソーティングし、さらにＰＤ１６６２８５の存在下にて１０回継代培養した後の１倍体ＥＳ細胞をＦＡＣＳにて解析した結果（図中、右参照）とを示す、ヒストグラムである。Results of FACS analysis of haploid ES cells (phES-B6-9) subcultured in the absence of PD166285 (see the left in the figure) and 1N cells were sorted from the cells by FACS. Furthermore, it is a histogram showing the results of FACS analysis of haploid ES cells after 10 subcultures in the presence of PD166285 (see the right in the figure). ＰＤ１６６２８５の存在下（ＰＤ（＋））又は非存在下（ＰＤ（－））にて培養した結果、得られた１倍体ＥＳ細胞のコロニーを顕微鏡にて観察した結果を示す写真である。図中、スケールバーは１００μｍを示し、上段はフィーダー細胞存在下で培養し、位相差顕微鏡にて観察した結果を示し、下段はフィーダー細胞非存在化で培養し、位相差顕微鏡にて観察した結果を示す。It is a photograph showing the result of observing with a microscope the colonies of haploid ES cells obtained as a result of culturing in the presence (PD (+)) or absence (PD (−)) of PD166285. In the figure, the scale bar indicates 100 μm, the upper row shows the results of culturing in the presence of feeder cells and observed with a phase-contrast microscope, and the lower row shows the results of culturing in the absence of feeder cells and observed with a phase-contrast microscope. Indicates. ＰＤ１６６２８５の存在下（ＰＤ（３００ｎＭ）、ＰＤ（５００ｎＭ）及びＰＤ（１０００ｎＭ））又は非存在下（ＰＤ（－））にて培養した結果、得られた１倍体ＥＳ細胞のコロニーを顕微鏡にて観察した結果を示す写真である。As a result of culturing in the presence of PD166285 (PD (300 nM), PD (500 nM) and PD (1000 nM)) or in the absence (PD (−)), colonies of the resulting haploid ES cells were observed with a microscope. It is a photograph which shows the result of observation. 図２に示した工程にて、ＰＤ１６６２８５の存在下にて３３日間培養した１倍体ＥＳ細胞（図中、右側参照）と、ＰＤ１６６２８５の非存在下にて３３日間培養した１倍体ＥＳ細胞（図中、左側参照）とを、ＦＡＣＳにて解析した結果を示すヒストグラムである。In the process shown in FIG. 2, haploid ES cells cultured for 33 days in the presence of PD166285 (see the right side in the figure) and haploid ES cells cultured for 33 days in the absence of PD166285 ( Is a histogram showing the result of FACS analysis. 本発明の方法により培養し、得られた１倍体ＥＳ細胞の核型を観察した結果を示す、顕微鏡写真である。It is a microscope picture which shows the result of having observed by the method of this invention, and having observed the karyotype of the obtained haploid ES cell. 本発明の方法により培養し、得られた１倍体ＥＳ細胞のゲノムＤＮＡをＣＧＨアレイにて解析した結果を示すドット図である。図中、下部パネルは該１倍体ＥＳ細胞のゲノムＤＮＡを解析した結果を示し、上部パネルは野生型マウス肝臓由来のゲノムＤＮＡについて解析した結果（実験コントロール）を示す。レファレンスゲノムＤＮＡには上部、下部パネル共に野生型Ｃ５７ＢＬ／６マウスの肝臓由来のゲノムＤＮＡを使用している。It is a dot figure which shows the result of having analyzed by the CGH array about the genomic DNA of the haploid ES cell obtained by culture | cultivating by the method of this invention. In the figure, the lower panel shows the result of analyzing the genomic DNA of the haploid ES cell, and the upper panel shows the result of analyzing the genomic DNA derived from the wild-type mouse liver (experimental control). For reference genomic DNA, genomic DNA derived from the liver of wild-type C57BL / 6 mice is used for both the upper and lower panels. 本発明の方法により培養し、得られた１倍体ＥＳ細胞における、幹細胞マーカー（Ｏｃｔ４、Ｎａｎｏｇ及びＳＳＥＡ１）の発現を、免疫染色にて解析した結果を示す、顕微鏡写真である。It is a microscope picture which shows the result of having analyzed the expression of the stem cell marker (Oct4, Nanog, and SSEA1) in the haploid ES cell obtained by culture | cultivating by the method of this invention by the immuno-staining. 本発明の方法により培養し、得られた１倍体ＥＳ細胞における、未分化マーカー（ＡＬＰ）の発現を、アルカリフォスファターゼ染色にて解析した結果を示す、顕微鏡写真である。図中、スケールバーは１００μｍを示す。It is a microscope picture which shows the result of having analyzed by the alkaline phosphatase dyeing | staining the expression of the undifferentiation marker (ALP) in the haploid ES cell obtained by culture | cultivating by the method of this invention. In the figure, the scale bar indicates 100 μm. 本発明の方法により培養し、得られた１倍体ＥＳ細胞を、免疫不全マウスの皮下に移植し、その３０日後に形成されたテラトーマを観察した結果を示す写真である。図中、左上のパネルは、１倍体ＥＳ細胞を皮下に移植した免疫不全マウスの３０日後の外観を観察した結果を示す写真である。右上のパネルは、該マウスより摘出されたテラトーマをＨＥ染色して観察された内皮性上皮（ｅｎｄｏｔｈｅｌｉａｌ　ｅｐｉｔｈｅｌｉｕｍ）組織を示す顕微鏡写真である。左下のパネルは、該マウスより摘出されたテラトーマをＨＥ染色して観察された表皮（ｅｐｉｄｅｒｍｉｓ）組織を示す顕微鏡写真である。右下のパネルは、該マウスより摘出されたテラトーマをＨＥ染色して観察された軟骨（ｃａｒｔｉｌａｇｅ）組織を示す顕微鏡写真である。It is a photograph which shows the result of having observed the teratoma formed 30 days after transplanting the haploid ES cell obtained by culture | cultivating by the method of this invention to the skin of an immunodeficient mouse | mouth. In the figure, the upper left panel is a photograph showing the results of observing the appearance after 30 days of immunodeficient mice transplanted subcutaneously with haploid ES cells. The upper right panel is a photomicrograph showing endothelium epithelium tissue observed by HE staining of teratoma extracted from the mouse. The lower left panel is a photomicrograph showing the epidermis tissue observed by HE staining of teratoma extracted from the mouse. The lower right panel is a photomicrograph showing a cartilage tissue observed by HE staining of teratoma extracted from the mouse. 本発明の方法により培養して得られた１倍体ＥＳ細胞（ｐｈＥＳ－Ｂ６－ａｖｅ）と、１倍体ＥＳ細胞が２倍体化した細胞（ｐｄＥＳ－Ｂ６－ａｖｅ）と、自然交配により得られた胚から樹立したＥＳ細胞（ｍＥＳ－Ｂ６－ａｖｅ）との間における、遺伝子発現量の差をマイクロアレイにて網羅的に解析した結果を示す、スキャッチャープロット図である。Obtained by natural mating between haploid ES cells (phES-B6-ave) obtained by culturing according to the method of the present invention and cells in which haploid ES cells have doubled (pdES-B6-ave). FIG. 6 is a Scatcher plot diagram showing the result of comprehensive analysis of the difference in gene expression level with ES cells (mES-B6-ave) established from the obtained embryos using a microarray. 本発明の方法により培養して得られた１倍体ＥＳ細胞（ｐｈＥＳＣ１、ｐｈＥＳＣ２）と、１倍体ＥＳ細胞が２倍体化した細胞（ｐｄＥＳＣ１、ｐｄＥＳＣ２）と、自然交配により得られた胚から樹立したＥＳ細胞（ｍＥＳＣ１、ｍＥＳＣ２）との間にて、遺伝子発現量を比較した結果を示すグラフである。図中、横軸の表記は、発現量を解析した遺伝子を示し、また各棒は左から順に、ｐｈＥＳＣ１、ｐｈＥＳＣ２、ｐｄＥＳＣ１、ｐｄＥＳＣ２、ｍＥＳＣ１、ｍＥＳＣ２における各遺伝子の発現量を示している。From embryos obtained by natural mating with haploid ES cells (phESC1, phESC2) obtained by culturing according to the method of the present invention and cells with diploid ES cells doubling (pdESC1, pdESC2) It is a graph which shows the result of having compared the gene expression level between the established ES cells (mESC1, mESC2). In the figure, the abscissa indicates the gene whose expression level was analyzed, and each bar indicates the expression level of each gene in phESC1, phESC2, pdESC1, pdESC2, mESC1, and mESC2 in order from the left. 本発明の方法により培養して得られた１倍体ＥＳ細胞を用いて作製されたキメラマウスにおける、各臓器のキメラ寄与率を示すグラフである。図中、横軸の表記の「１－３」、「１－４」、「２－１」、「２－２」、「１－５」及び「２－３」は、キメラ胎仔の個体を示す。「体」は、キメラ個体から頭部及び内臓を除いた残り総ての部分のキメラ寄与率を示す。「全」は、キメラ個体から臓器を取り出さずに、個体を丸ごとすり潰して解析し、得られたキメラ寄与率（全ての組織におけるキメラ寄与率）を示す。また、各臓器におけるキメラ寄与率を示す棒において一番右端にある棒は、１－３、１－４、２－１、２－２個体のＧＦＰ陽性細胞の割合の平均値を示す。It is a graph which shows the chimera contribution rate of each organ in the chimera mouse produced using the haploid ES cell obtained by culture | cultivating by the method of this invention. In the figure, “1-3”, “1-4”, “2-1”, “2-2”, “1-5”, and “2-3” on the horizontal axis represent chimera fetus individuals. Show. “Body” indicates the chimera contribution ratio of all remaining parts excluding the head and internal organs from the chimeric individual. “Total” indicates the chimera contribution ratio (chimera contribution ratio in all tissues) obtained by analyzing the whole individual by grinding it without removing the organ from the chimera individual. In the bar indicating the chimera contribution ratio in each organ, the bar at the right end indicates the average value of the ratio of GFP positive cells of 1-3, 1-4, 2-1, 2-2 individuals. 本発明の方法により培養して得られた１倍体ＥＳ細胞を用いて作製されたキメラマウス（雌及び雄）における、各臓器のキメラ寄与率をＦＡＣＳにて解析した結果を示す、ヒストグラムである。It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. . 本発明の方法により培養して得られた１倍体ＥＳ細胞を用いて作製されたキメラマウス（雌及び雄）における、各臓器のキメラ寄与率をＦＡＣＳにて解析した結果を示す、ヒストグラムである。It is a histogram which shows the result of having analyzed the chimera contribution rate of each organ in FACS in the chimera mouse (female and male) produced using the haploid ES cell obtained by culture by the method of the present invention. . 本発明の方法により培養して得られた１倍体ＥＳ細胞（ｐｈＥＳ）から、１倍体エピブラスト幹細胞（ｐｈＥｐｉＳＣ）へと分化誘導する工程を示す、概略図である。It is the schematic which shows the process of differentiation-inducing from the haploid ES cell (phES) obtained by culture | cultivating by the method of this invention to a haploid epiblast stem cell (phEpiSC). 本発明の方法により培養して得られた１倍体ＥＳ細胞から分化させて得られた、１倍体エピブラスト幹細胞のＤＮＡ量をＦＡＣＳにて解析した結果を示す、ヒストグラムである。It is a histogram which shows the result of having analyzed the DNA amount of the haploid epiblast stem cell obtained by making it differentiate from the haploid ES cell obtained by culture | cultivating by the method of this invention by FACS. 本発明の方法により培養して得られた１倍体ＥＳ細胞から分化させて得られた、１倍体エピブラスト幹細胞の遺伝子発現量の解析結果を示す、ゲル電気泳動の写真である。図中、「ｍＥＳＣｓ」は、自然交配により得られた胚から樹立したマウスＥＳ細胞の解析結果を示し、「ｍＥｐｉＳＣｓ」は、該マウスＥＳ細胞を分化させて得られたエピブラスト幹細胞の解析結果を示し、「ｐｈＥＳＣｓ」は、本発明の方法により培養して得られた１倍体ＥＳ細胞の解析結果を示し、「ｐｈＥｐｉＳＣｓＰ３」は、該１倍体ＥＳ細胞を分化させて得られたエピブラスト幹細胞（継代数３）の解析結果を示し、「ｐｈＥｐｉＳＣｓＰ５」は、該１倍体ＥＳ細胞を分化させて得られたエピブラスト幹細胞（継代数５）の解析結果を示し、「ＭＥＦ」はマウス胎生１３日目の胚から得られた繊維芽細胞の解析結果を示し、「Ｈ２Ｏ」は、水（鋳型ＤＮＡなし）を解析した結果（陰性対照）を示す。また、図中左側の表記は、解析したマーカー遺伝子（Ｏｃｔ４、Ｎａｎｏｇ、Ｒｅｘ１、Ｆｇｆ５）を示し、「ＧＡＰＤＨ」は、本解析における陽性対照を示し、「ＲＴ（－）」は陰性対照（逆転写反応なしの結果）を示す。なお、ここでの継代数は、ｐｈＥＳＣをｐｈＥｐｉＳＣに分化させてからの継代数を意味する。また、図中右側の表記は、図中左側に示す各マーカー遺伝子が、ＥＳ細胞特異的マーカー遺伝子であること（「ＥＳ」と表記）、エピブラスト幹細胞特異的マーカー遺伝子であること（「ＥｐｉＳＣ」と表記）、ＥＳ細胞及びエピブラスト幹細胞において発現が認められるマーカー遺伝子であること（「ＥＳ／ＥｐｉＳＣ」と表記）を示す。It is the photograph of gel electrophoresis which shows the analysis result of the gene expression level of the haploid epiblast stem cell obtained by making it differentiate from the haploid ES cell obtained by culture | cultivating by the method of this invention. In the figure, “mESCs” indicates the analysis results of mouse ES cells established from embryos obtained by natural mating, and “mEpiSCs” indicates the analysis results of epiblast stem cells obtained by differentiating the mouse ES cells. “PhESCs” indicates the analysis result of haploid ES cells obtained by culturing according to the method of the present invention, and “phEpiSCsP3” indicates epiblast stem cells obtained by differentiating the haploid ES cells. The analysis result of (passage number 3) is shown, “phEpiSCsP5” shows the analysis result of epiblast stem cells (passage number 5) obtained by differentiating the haploid ES cells, and “MEF” is mouse embryo 13 The analysis result of the fibroblast obtained from the day embryo is shown, and “H 2 O” shows the result of analyzing water (no template DNA) (negative control). The notation on the left side of the figure indicates the analyzed marker genes (Oct4, Nanog, Rex1, Fgf5), “GAPDH” indicates a positive control in this analysis, and “RT (−)” indicates a negative control (reverse transcription). Results without reaction). In addition, the passage number here means the passage number after differentiating phESC into phEpiSC. In addition, the notation on the right side of the figure indicates that each marker gene shown on the left side of the figure is an ES cell-specific marker gene (indicated as “ES”) and an epiblast stem cell-specific marker gene (“EpiSC”). And a marker gene whose expression is recognized in ES cells and epiblast stem cells (denoted as “ES / EpiSC”).
　＜１倍体胚性幹細胞の培養方法＞
　本発明は、１倍体胚性幹細胞の培養方法であって、Ｃｄｃ２を恒常的に活性化する条件下にて該細胞を培養する工程を含む方法を提供するものである。 <Culture method of haploid embryonic stem cells>
The present invention provides a method for culturing haploid embryonic stem cells, the method comprising culturing the cells under conditions that constitutively activate Cdc2.
　そして、該培養方法によれば、後述の実施例において示す通り、１倍体胚性幹細胞において、Ｃｄｃ２を恒常的に活性化させることにより、細胞周期の進行においてＧ２期からＭ期への移行を促進させ、その１倍性を維持することが可能となる。さらにこのように培養して得られる１倍体胚性幹細胞では、そのゲノムＤＮＡにおいて欠失、増幅等の変異が生じることはなく、さらに分化多能性及び高い増殖能は維持されたままである。 And according to this culture method, in the haploid embryonic stem cell, as shown in the below-mentioned Example, by making Cdc2 constitutively activated, the transition from the G2 phase to the M phase is performed in the progression of the cell cycle. It is possible to promote and maintain the monoploidy. Furthermore, in the haploid embryonic stem cells obtained by culturing in this way, mutations such as deletion and amplification do not occur in the genomic DNA, and the pluripotency and high proliferation ability remain maintained.
　本発明において「１倍体胚性幹細胞」とは、１倍体のまま発生した初期胚から樹立される胚性幹細胞（ＥＳ細胞）のことを意味し、例えば、非特許文献１～４に記載されているような、活性化させた未受精卵を発生させ、得られた初期胚から樹立される、雌性単為発生胚由来１倍体ＥＳ細胞（雌性１倍体ＥＳ細胞、ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃ　ｈａｐｌｏｉｄ　ＥＳ細胞、ｐｈＥＳ細胞、ｐｈＥＳＣ）が挙げられる。また、非特許文献５及び６に記載されているような、除核した未受精卵に精子の核を移植し、活性化させ発生させることにより得られた初期胚から樹立される、雄性単為発生胚由来１倍体ＥＳ細胞（雄性１倍体ＥＳ細胞、ａｎｄｒｏｇｅｎｅｔｉｃ　ｈａｐｌｏｉｄ　ＥＳ細胞、ａｈＥＳ細胞、ａｈＥＳＣ）も挙げられる。 In the present invention, “haploid embryonic stem cell” means an embryonic stem cell (ES cell) established from an early embryo that has developed as a haploid, and is described in, for example, Non-Patent Documents 1 to 4. Female parthenogenetic embryo-derived haploid ES cells (female haploid ES cells, parthenogenetic haploid ES cells, which are established from the early embryos obtained by generating activated unfertilized eggs as described above PhES cells, phESC). In addition, as described in Non-Patent Documents 5 and 6, male parthenogenesis established from early embryos obtained by transplanting, activating and generating sperm nuclei into enucleated unfertilized eggs Examples also include embryos derived from haploid ES cells (male haploid ES cells, androgenetic haploid ES cells, ahES cells, ahESC).
　これら１倍体ＥＳ細胞の樹立に用いられる未受精卵及び精子の由来としては特に制限はなく、ヒト及び非ヒト動物が挙げられる。非ヒト動物としては、例えば、マウス及びラット等のげっ歯類、ウシ、ウマ、ブタ、ヒツジ、サル、イヌ、並びにネコ等の哺乳類、ニワトリ等の鳥類が挙げられる。また、未受精卵を活性化させる方法としては、該卵から第２極体を放出させる方法であればよく、例えば、ストロンチウムやエタノール等により化学的に刺激を与える方法や、電気的な刺激を与える方法が挙げられる。さらに、活性化させた未受精卵を発生させることにより調製され、１倍体ＥＳ細胞の樹立に用いられる初期胚としては、例えば、８細胞期、１６細胞期、桑実胚期又は胚盤胞期にある胚が挙げられる。一般的にＥＳ細胞の樹立効率が高いという観点から、胚盤胞期にある胚が好ましいが、Ｃ５７ＢＬ／６のような純系マウスから作製する場合には、１６細胞期又は桑実胚期にある胚が好ましく、桑実胚期にある胚がより好ましい（後述の表１　参照）。 The origin of unfertilized eggs and sperm used for establishment of these haploid ES cells is not particularly limited, and examples thereof include humans and non-human animals. Examples of non-human animals include rodents such as mice and rats, cows, horses, pigs, sheep, monkeys, dogs, mammals such as cats, and birds such as chickens. Moreover, as a method for activating the unfertilized egg, any method may be used as long as the second polar body is released from the egg. For example, a method of chemically stimulating with strontium, ethanol, or the like, The method of giving is mentioned. Furthermore, examples of the early embryo prepared by generating activated unfertilized eggs and used for establishing haploid ES cells include, for example, the 8-cell stage, 16-cell stage, morula stage, or blastocyst The embryo in the stage is mentioned. In general, embryos in the blastocyst stage are preferable from the viewpoint of high ES cell establishment efficiency. However, when they are produced from a pure mouse such as C57BL / 6, they are in the 16 cell stage or morula stage. Embryos are preferred, and embryos in the morula stage are more preferred (see Table 1 below).
　本発明の培養方法に供する「１倍体ＥＳ細胞」としては、天然由来の細胞のみならず、後述の実施例において用いられているｐｈＥＳＣ－Ｂ６ＧＦＰのように、遺伝的に改変されている細胞（遺伝子改変細胞）であってもよい。遺伝子改変細胞としては、前記ｐｈＥＳＣ－Ｂ６ＧＦＰにおけるＧＦＰのように、発現させたいタンパク質をコードする遺伝子が外来的に導入されている細胞であってもよく、ノックアウト法、ＲＮＡ法、アンチセンス法等により、特定の遺伝子の機能が抑制されている細胞であってもよく、またランダムに遺伝子の機能が抑制又は活性化されている細胞であってもよい。遺伝子の機能がランダムに抑制又は活性化されている細胞としては、例えば、ＥＭＵ、ＥＭＳ、ＮＭＵ及びＮＴＧ等の化学変異剤によって処理された細胞、ジンクフィンガーヌクレアーゼやＴＡＬＥＮ等のＤＮＡ切断酵素が導入された細胞、速中性子線、ガンマー線又はイオンビームが照射された細胞、トランスポゾンがゲノムＤＮＡにランダムに挿入された細胞が挙げられる。また、かかる遺伝子の改変は、１倍体細胞が樹立された後に施されたものであってもよく、樹立される前、例えば、前記未受精卵や前記精子を採取する個体段階において施されたものであってもよい。 Examples of the “haploid ES cells” used in the culture method of the present invention include not only naturally derived cells but also cells that have been genetically modified, such as phESC-B6GFP used in the examples described later ( Genetically modified cells). The genetically modified cell may be a cell into which a gene encoding a protein to be expressed is introduced exogenously, such as GFP in the phESC-B6GFP, and may be obtained by a knockout method, an RNA method, an antisense method, etc. A cell in which the function of a specific gene is suppressed may be used, or a cell in which the function of a gene is suppressed or activated randomly. Examples of cells whose gene functions are randomly suppressed or activated include cells treated with chemical mutagens such as EMU, EMS, NMU, and NTG, and DNA-cleaving enzymes such as zinc finger nuclease and TALEN. Cells, cells irradiated with fast neutron rays, gamma rays or ion beams, and cells in which transposons are randomly inserted into genomic DNA. Further, such gene modification may be performed after the establishment of haploid cells, for example, at the individual stage of collecting the unfertilized egg or the sperm before the establishment. It may be a thing.
　本発明において「Ｃｄｃ２の恒常的な活性化」とは、Ｃｄｃ２が常に細胞の分裂を促進し得る状態にあることを意味する。すなわち、通常の細胞周期進行において、Ｃｄｃ２が活性化されているＧ２期からＭ期に限らず、Ｃｄｃ２が細胞の分裂を促進し得る状態にあることを意味する。 In the present invention, “constant activation of Cdc2” means that Cdc2 is always in a state capable of promoting cell division. That is, in normal cell cycle progression, it means that Cdc2 is in a state where Cdc2 can promote cell division, not limited to G2 to M phase where Cdc2 is activated.
　細胞周期の制御において、Ｃｄｃ２の１４番目のスレオニン又は１５番目のチロシンがＷｅｅ１又はＭｙｔ１によりリン酸化されることによって、Ｃｄｃ２は不活性化され、Ｍ期への移行は抑制される（Ｇ２期にて停止する）こととなる。一方、Ｃｄｃ２５によって、Ｃｄｃ２の１４番目のスレオニン及び１５番目のチロシンが脱リン酸化されることにより、Ｃｄｃ２は活性化され、Ｍ期への移行が促進されることが明らかになっている。 In the control of the cell cycle, Cdc2 is inactivated and the transition to M phase is suppressed by phosphorylation of 14th threonine or 15th tyrosine of Cdc2 by Wee1 or Myt1 (in G2 phase) Will stop). On the other hand, it has been clarified that Cdc2 is activated by Cdc25, whereby Cdc2 is activated by dephosphorylating the 14th threonine and 15th tyrosine of Cdc2.
　したがって、「Ｃｄｃ２を恒常的に活性化する条件」として、より具体的には、Ｃｄｃ２の１４番目のスレオニン又は１５番目のチロシンにおけるリン酸化が恒常的に抑制される条件が挙げられる。このようなリン酸化の抑制は、Ｗｅｅ１又はＭｙｔ１の活性を抑制すること、Ｃｄｃ２５の活性を亢進すること等によって達成することができるが、Ｍ期への移行をより促進し易いという観点から、Ｗｅｅ１又はＭｙｔ１の活性を抑制することが好ましく、Ｗｅｅ１及びＭｙｔ１の活性を共に抑制することがより好ましい。 Therefore, more specifically, “a condition for activating Cdc2 constantly” includes a condition under which phosphorylation of Cdc2 at 14th threonine or 15th tyrosine is constantly suppressed. Such suppression of phosphorylation can be achieved by suppressing the activity of Wee1 or Myt1, enhancing the activity of Cdc25, etc. From the viewpoint of facilitating the transition to the M phase, Wee1 Alternatively, it is preferable to suppress the activity of Myt1, and it is more preferable to suppress both the activities of Wee1 and Myt1.
　なお、本発明において「活性の抑制」とは、当該活性の完全な抑制（阻害）のみならず、部分的な抑制も含む意味である。また、これらタンパク質の活性の抑制又は亢進は、当該タンパク質の発現の抑制又は亢進によっても達成することができる。したがって、本発明における「活性の抑制又は亢進」には、発現の抑制又は亢進を通して達成される場合も含まれる。さらに、発現の抑制又は亢進は、転写レベルであってもよく、翻訳レベルであってもよい。 In the present invention, “activity suppression” means not only complete suppression (inhibition) of the activity but also partial suppression. Moreover, suppression or enhancement of the activity of these proteins can also be achieved by suppression or enhancement of the expression of the protein. Therefore, “inhibition or enhancement of activity” in the present invention includes cases achieved through inhibition or enhancement of expression. Furthermore, the suppression or enhancement of expression may be at the transcription level or at the translation level.
　また、Ｃｄｃ２の１４番目のスレオニン又は１５番目のチロシンにおけるリン酸化の恒常的な抑制は、後述のＣｄｃ２活性化剤を１倍体胚性幹細胞を培養している培地に添加する等して、該細胞に該活性化剤を導入することにより達成することができる。特に、後述のＰＤ１６６２８５は、Ｗｅｅ１及びＭｙｔ１の活性を共に抑制し、効率良くＣｄｃ２を活性化することができるため、本発明の培養方法においては、ＰＤ１６６２８５を添加した培地にて１倍体胚性幹細胞を培養することが好ましい。 In addition, the constant suppression of phosphorylation of Cdc2 at 14th threonine or 15th tyrosine can be achieved by adding a Cdc2 activator described later to a medium in which haploid embryonic stem cells are cultured, etc. This can be achieved by introducing the activator into cells. In particular, since PD166285, which will be described later, can suppress both Wee1 and Myt1 activities and can efficiently activate Cdc2, the haploid embryonic stem cells in the medium supplemented with PD166285 are used in the culture method of the present invention. Is preferably cultured.
　ＰＤ１６６２８５の培地への添加濃度としては、Ｃｄｃ２を恒常的に活性化することができる濃度であればよいが、１倍体胚性幹細胞等に細胞死を生じさせずに培養できるという観点から、３００ｎＭ以下であることが好ましい。 The concentration of PD166285 added to the medium is not particularly limited as long as Cdc2 can be constantly activated. From the viewpoint of culturing without causing cell death in haploid embryonic stem cells and the like, 300 nM The following is preferable.
　また、本発明において、１倍体胚性幹細胞の培養方法としては、前述の通り、Ｃｄｃ２が恒常的に活性化している条件下での培養であればよく、他の培養条件は、当業者であれば公知の胚性幹細胞（特に、１倍体胚性幹細胞）の培養方法を適宜選択して利用することができる。 In the present invention, the haploid embryonic stem cells may be cultured under the conditions where Cdc2 is constantly activated as described above, and other culture conditions can be determined by those skilled in the art. If it exists, the culture | cultivation method of a well-known embryonic stem cell (especially haploid embryonic stem cell) can be selected suitably, and can be utilized.
　例えば、１倍体胚性幹細胞の培養に用いられる培地としては、公知の胚性幹細胞を培養するための基礎培地を基に調製することができる。公知の基礎培地としては、ＤＭＥＭ培地、ＫＳＯＭ培地、イーグルＭＥＭ培地、グラスゴーＭＥＭ培地、αＭＥＭ培地、ハム培地、ＲＰＭＩ　１６４０培地、フィッシャーズ培地、ＢＭＥ培地、ＢＧＪｂ培地、ＣＭＲＬ　１０６６培地、ＭＥＭ　Ｚｉｎｃオプション改善培地、ＩＭＤＭ培地、メヂィウム１９９培地、並びにこれら任意の混合培地が挙げられる。 For example, a medium used for culturing haploid embryonic stem cells can be prepared based on a basal medium for culturing known embryonic stem cells. Known basal media include DMEM medium, KSOM medium, Eagle MEM medium, Glasgow MEM medium, αMEM medium, Ham medium, RPMI 1640 medium, Fisher's medium, BME medium, BGJb medium, CMRL 1066 medium, MEM Zinc option improved medium, Examples include IMDM medium, medium 199 medium, and any mixed medium.
　１倍体胚性幹細胞の培養に用いられる培地は、血清含有培地であってもよく、また無血清培地でもよい。無血清培地とは、無調製又は未精製の血清を含まない培地を意味し、精製された血液由来成分や動物組織由来成分（例えば、増殖因子）を含有する培地が挙げられ得る。異種動物由来成分の混入防止の観点から、血清は、幹細胞と同種動物由来のものが好ましい。また、１倍体胚性幹細胞の培養に用いられる培地は、血清代替物を含んでいてもよく、含んでいなくともよい。血清代替物としては、例えば、ノックアウト血清代替物（ＫＳＲ、インビトロジェン社製）、化学的脂質濃縮物（Ｃｈｅｍｉｃａｌｌｙ－ｄｅｆｉｎｅｄ　Ｌｉｐｉｄ　ｃｏｎｃｅｎｔｒａｔｅｄ、Ｇｉｂｃｏ社製）、グルタマックス（Ｇｉｂｃｏ社製）等の市販品が挙げられる。 The medium used for culturing haploid embryonic stem cells may be a serum-containing medium or a serum-free medium. A serum-free medium means a medium that does not contain unprepared or unpurified serum, and may include a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors). From the viewpoint of preventing contamination with components derived from different animals, the serum is preferably derived from the same species as the stem cells. In addition, the medium used for culturing haploid embryonic stem cells may or may not contain a serum substitute. Examples of serum substitutes include commercially available products such as knockout serum substitutes (KSR, manufactured by Invitrogen), chemical lipid concentrates (Chemically-defined Lipid concentrated, manufactured by Gibco), and glutamax (produced by Gibco). It is done.
　１倍体胚性幹細胞の培養に用いられる培地には、胚性幹細胞等の分化を抑制する化合物が添加されていることが好ましい。かかる化合物としては、白血病阻害因子（ＬＩＦ）、ＭＥＫ阻害剤（ＰＤ０３２５９０１、ＡＺＤ６２４４、ＣＩ－１０４０（ＰＤ１８４３５２）、ＲＤＥＡ１１９（ＢＡＹ８６９７６６）、ＳＬ３２７、Ｕ０１２６、ＰＤ９８０５９、Ｕ０１２４、Ｕ０１２５）、ＧＳＫ３阻害剤（ＣＨＩＲ９９０２１、ＳＢ２１６７６３、ＣＨＩＲ９８０１４、ＳＢ４１５２８６等）、ＴＧＦβ受容体阻害剤（ＳＢ４３１５４２等）、ＦＧＦ受容体阻害剤（ＳＵ５４０２、ＰＤ１７３０７４等）、ＲＯＣＫ阻害剤（ＧＳＫ２６９９６２Ａ、Ｙ－２７６３２、Ｈ－１１５２等）が挙げられる。 It is preferable that a medium used for culturing haploid embryonic stem cells is added with a compound that suppresses differentiation of embryonic stem cells and the like. Such compounds include leukemia inhibitory factor (LIF), MEK inhibitor (PD0325901, AZD6244, CI-1040 (PD184352), RDEA119 (BAY869766), SL327, U0126, PD98059, U0124, U0125), GSK3 inhibitor (CHIR99021, SB216763). , CHIR98014, SB415286, etc.), TGFβ receptor inhibitors (SB431542, etc.), FGF receptor inhibitors (SU5402, PD1733074, etc.), ROCK inhibitors (GSK26962A, Y-27632, H-1152, etc.).
　さらに、１倍体胚性幹細胞の培養に用いられる培地には、アミノ酸（Ｌ－グルタミン、非必須アミノ酸等）、還元剤（２－メルカプトエタノール）、抗生物質（ペニシリン、ストレプトマイシン等）、脂肪酸又は脂質、糖類、ビタミン、増殖因子、サイトカイン、抗酸化剤、有機酸（ピルビン酸、乳酸等）、緩衝剤、無機塩類等を含有できる。 Furthermore, the medium used for the culture of haploid embryonic stem cells includes amino acids (L-glutamine, non-essential amino acids, etc.), reducing agents (2-mercaptoethanol), antibiotics (penicillin, streptomycin, etc.), fatty acids or lipids. , Sugars, vitamins, growth factors, cytokines, antioxidants, organic acids (pyruvic acid, lactic acid, etc.), buffers, inorganic salts, and the like.
　１倍体胚性幹細胞の培養に用いられる培養器には、胚性幹細胞等を培養するために用いられる公知のものを適宜利用することができ、細胞接着性の培養器（例えば、ＥＣＭ、マトリゲル、ゼラチン、コラーゲン等の細胞支持用基質にてコーティングされている培養器）であってもよく、細胞非接着性の培養器であってもよい。 As the incubator used for culturing haploid embryonic stem cells, known ones used for culturing embryonic stem cells and the like can be used as appropriate, and cell-adhesive incubators (for example, ECM, Matrigel) , A culture vessel coated with a cell support substrate such as gelatin or collagen) or a non-cell-adhesive culture vessel.
　１倍体胚性幹細胞のその他の培養条件は、公知の胚性幹細胞の培養条件に準じ、当業者であれば適宜選択、調整することができるが、例えば、培養温度は、特に限定されるものではないが、通常約３０～４０℃、好ましくは約３７℃である。ＣＯ２の濃度は、通常約１～１０％、好ましくは約２～５％である。酸素濃度は通常、１～１０％である。 Other culture conditions for haploid embryonic stem cells can be appropriately selected and adjusted by those skilled in the art in accordance with known culture conditions for embryonic stem cells. For example, the culture temperature is particularly limited. However, it is usually about 30 to 40 ° C, preferably about 37 ° C. The concentration of CO 2 is usually about 1-10%, preferably about 2-5%. The oxygen concentration is usually 1 to 10%.
　また、１倍体胚性幹細胞の培養は接着培養であってもよく、非接着培養であってもよい。接着培養の場合には、１倍体胚性幹細胞をフィーダー細胞の存在下で培養することもできる。フィーダー細胞としては特に制限はないが、例えば、放射線（ガンマ線等）の照射や抗生物質（マイトマイシンＣ等）処理により細胞分裂を停止させたマウス胎児繊維芽細胞（ＭＥＦ）、ＳＴＯ細胞、ＳＮＬ細胞が挙げられる。また、非接着培養としては、例えば、分散培養、凝集浮遊培養、担体上での浮遊培養が挙げられる。 The culture of haploid embryonic stem cells may be an adhesion culture or a non-adhesion culture. In the case of adhesion culture, haploid embryonic stem cells can also be cultured in the presence of feeder cells. The feeder cells are not particularly limited. For example, mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation (gamma rays or the like) or treatment with antibiotics (mitomycin C or the like) are used. Can be mentioned. Examples of non-adherent culture include dispersion culture, aggregated suspension culture, and suspension culture on a carrier.
　＜非ヒトキメラ動物を作製する方法＞
　後述の実施例において示す通り、上述の培養方法を用いて培養した１倍体胚性幹細胞は、１倍性の維持のみならず、ゲノムＤＮＡにおいて欠失等を生じさせることなく、分化多能性及び高い増殖能を保持しており、該１倍体胚性幹細胞からキメラ動物を得ることができる。 <Method for producing non-human chimeric animal>
As shown in the Examples below, haploid embryonic stem cells cultured using the above-described culture method are not only maintained monoploidy but also pluripotent without causing deletion or the like in genomic DNA. In addition, a chimeric animal can be obtained from the haploid embryonic stem cells.
　したがって、本発明は、非ヒトキメラ動物を作製する方法であって、
（ｉ）Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
（ｉｉ）該１倍体胚性幹細胞を初期胚に導入し、キメラ胚を作製する工程と、
（ｉｉｉ）該キメラ胚を非ヒト動物の母胎内に移植し、発生させ、非ヒトキメラ動物を得る工程とを含む方法をも提供する。 Accordingly, the present invention is a method for producing a non-human chimeric animal comprising:
(I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
(Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo;
(Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
　前記工程（ｉ）については、上述の＜１倍体胚性幹細胞の培養方法＞に記載の通りである。工程（ｉｉ）において、１倍体胚性幹細胞が導入される「初期胚」の由来としては、非ヒト動物であればよく、例えば、マウス及びラット等のげっ歯類、ウシ、ウマ、ブタ、ヒツジ、サル、イヌ、並びにネコ等の哺乳類、ニワトリ等の鳥類が挙げられる。また、初期胚の由来は、導入される１倍体胚性幹細胞の由来と同じ（同種）であることが好ましいが、異種の関係であってもよい。 The step (i) is as described in <Method for culturing haploid embryonic stem cells> above. In step (ii), the “early embryo” into which the haploid embryonic stem cells are introduced may be any non-human animal, such as rodents such as mice and rats, cows, horses, pigs, Examples include sheep, monkeys, dogs, mammals such as cats, and birds such as chickens. Moreover, the origin of the early embryo is preferably the same (same type) as that of the introduced haploid embryonic stem cell, but may be of a heterogeneous relationship.
　初期胚の発生段階としては、例えば、８細胞期、１６細胞期、桑実胚期又は胚盤胞期が挙げられるが、通常、胚盤胞期にある初期胚が、キメラ胚の作製には用いられる。さらに、本発明において、１倍体胚性幹細胞を導入する初期胚として、４倍体胚を用いてもよい。１倍体胚性幹細胞を導入した４倍体キメラ胚を発生させることにより、該１倍体胚性幹細胞に由来する動物を直接得ることができるからである（Ａ．Ｎａｇｙら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、１９９３年、９０巻、８４２４～８４２８ページ）。なお、４倍体胚は、胚盤胞を電気融合させること、またはマンニトール溶液中で電気パルスを適用して２細胞胚盤胞を電気融合させること等の当業者に公知の手法により調製することができる。 Examples of the early embryonic development stage include the 8-cell stage, the 16-cell stage, the morula stage, or the blastocyst stage. Usually, an early embryo in the blastocyst stage is used for producing a chimeric embryo. Used. Furthermore, in the present invention, a tetraploid embryo may be used as an early embryo into which a haploid embryonic stem cell is introduced. This is because an animal derived from the haploid embryonic stem cell can be directly obtained by generating a tetraploid chimeric embryo into which the haploid embryonic stem cell has been introduced (A. Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8424-8428). The tetraploid embryo should be prepared by a method known to those skilled in the art, such as electrofusion of blastocysts or electrofusion of 2-cell blastocysts by applying an electric pulse in a mannitol solution. Can do.
　１倍体胚性幹細胞から、前述の初期胚への「導入」は、マイクロインジュクション法や凝集法等の公知のキメラ胚作製法を用いて行うことができる。 The “introduction” from the haploid embryonic stem cells to the aforementioned early embryo can be performed using a known chimeric embryo production method such as a microinjection method or an aggregation method.
　工程（iii）においては、このようにして得られたキメラ胚を、非ヒト動物の母胎内（子宮又は卵管）に移植し、発生させることで、非ヒトキメラ動物を得ることができる。キメラ胚が移植される非ヒト動物は、好ましくは偽妊娠動物であり、また前記初期胚の由来と同じ、同種の動物であることが好ましい。 In step (iii), the chimeric embryo obtained as described above is transplanted into the maternal womb (uterus or fallopian tube) of the non-human animal and generated, whereby a non-human chimeric animal can be obtained. The non-human animal to which the chimeric embryo is transplanted is preferably a pseudopregnant animal, and is preferably the same animal as the origin of the early embryo.
　また、後述の実施例において示す通り、このようにして得られた非ヒトキメラ動物において、本発明の方法により培養された１倍体胚性幹細胞が、ヒト生殖系列に移行できることも明らかにしている。したがって、本発明は、前記工程（ｉ）～（ｉｉｉ）を含む、生殖細胞が１倍体胚性幹細胞に由来する、非ヒト生殖系列キメラ動物を作製する方法をも提供することができる。 In addition, as shown in the Examples described later, it has also been clarified that in the non-human chimeric animal obtained in this way, haploid embryonic stem cells cultured by the method of the present invention can be transferred to the human germline. Therefore, the present invention can also provide a method for producing a non-human germline chimeric animal, wherein the germ cells are derived from haploid embryonic stem cells, comprising the steps (i) to (iii).
　この方法により得られた非ヒト生殖系列キメラ動物を、正常動物（野生型動物）又は当該非ヒト生殖系列キメラ動物と交配し、その産仔の中から前記１倍体胚性幹細胞由来の遺伝子を保有する個体を選択すれば、前記１倍体胚性幹細胞由来の遺伝子を保有する動物（前記１倍体胚性幹細胞に由来する動物）を得ることもできる。前記１倍体胚性幹細胞由来の遺伝子を保有する個体の選択は、様々な形質を指標として用いることができるが、例えば体色等を指標として選択することができる。また、体の一部からＤＮＡを抽出し、サザンブロット解析やＰＣＲ解析を行うことにより、選択を行うことも可能である。 A non-human germline chimeric animal obtained by this method is mated with a normal animal (wild-type animal) or the non-human germline chimeric animal, and a gene derived from the haploid embryonic stem cell is generated from its offspring. If an individual to be possessed is selected, an animal having a gene derived from the haploid embryonic stem cell (an animal derived from the haploid embryonic stem cell) can also be obtained. For selection of an individual having a gene derived from the haploid embryonic stem cell, various traits can be used as an index. For example, body color or the like can be selected as an index. It is also possible to select by extracting DNA from a part of the body and performing Southern blot analysis or PCR analysis.
　また、本発明の方法において、初期胚に導入される前記１倍体胚性幹細胞として、上述の特定の外来遺伝子が導入された遺伝子改変細胞を用いれば、当該導入された外来遺伝子を保有する動物（トランスジェニック動物）を得ることができる。初期胚に導入される前記１倍体胚性幹細胞として、上述の特定の遺伝子の機能が抑制されている遺伝子改変細胞を用いれば、遺伝子欠損ヘテロ接合体動物を得ることができる。さらに得られた遺伝子欠損ヘテロ接合体動物同士を交配させることで、遺伝子欠損ホモ接合体動物（いわゆるノックアウト動物）を得ることもできる。 In the method of the present invention, if a genetically modified cell into which the above-mentioned specific foreign gene is introduced is used as the haploid embryonic stem cell introduced into the early embryo, an animal having the introduced foreign gene (Transgenic animals) can be obtained. As the haploid embryonic stem cell introduced into the early embryo, a gene-deficient heterozygous animal can be obtained by using a genetically modified cell in which the function of the specific gene is suppressed. Furthermore, gene-deficient homozygous animals (so-called knockout animals) can also be obtained by mating the obtained gene-deficient heterozygous animals.
　また、初期胚に導入される前記１倍体胚性幹細胞として、遺伝子の機能がランダムに抑制又は活性化されている遺伝子改変細胞を用いれば、得られた非ヒトキメラ動物又はその産仔の表現型に基づき、その原因となる遺伝子（機能が抑制又は活性化されている遺伝子）を探索することができる。すなわち、本発明の方法はフォワードジェネティクスに好適に用いることができる。 Further, as the haploid embryonic stem cell introduced into the early embryo, if a genetically modified cell whose gene function is randomly suppressed or activated is used, the phenotype of the obtained non-human chimeric animal or its offspring Based on the above, it is possible to search for a gene that causes the disease (a gene whose function is suppressed or activated). That is, the method of the present invention can be suitably used for forward genetics.
　＜１倍体分化細胞を製造する方法＞
　後述の実施例において示す通り、上述の培養方法により培養した１倍体胚性幹細胞を分化誘導することにより、１倍体の分化細胞を得ることに成功している。したがって、本発明は、１倍体分化細胞を製造する方法であって、
（i)Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
（ii)該１倍体胚性幹細胞を分化誘導する工程とを含む方法
をも提供する。 <Method for producing haploid differentiated cells>
As shown in the below-mentioned Examples, diploid differentiated cells have been successfully obtained by inducing differentiation of haploid embryonic stem cells cultured by the above-described culture method. Accordingly, the present invention is a method for producing haploid differentiated cells comprising:
(I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
And (ii) a step of inducing differentiation of the haploid embryonic stem cell.
　前記工程（ｉ）については、上述の＜１倍体胚性幹細胞の培養方法＞に記載の通りである。工程（ｉｉ）における分化誘導は、１倍体胚性幹細胞の分化を抑制しないという観点から、Ｃｄｃ２を恒常的に活性化する条件では行わないことが好ましい。また、分化誘導は、インビトロ培養にて行ってもよく、インビボ培養によって行ってもよく、当業者であれば、胚性幹細胞等の万能幹細胞から、目的とする分化細胞への公知の誘導法を適宜選択することにより行うことができる。 The step (i) is as described in <Method for culturing haploid embryonic stem cells> above. The differentiation induction in the step (ii) is preferably not performed under conditions that constitutively activate Cdc2 from the viewpoint of not suppressing the differentiation of haploid embryonic stem cells. Further, differentiation induction may be performed by in vitro culture or by in vivo culture. A person skilled in the art can perform a known induction method from a universal stem cell such as an embryonic stem cell to a target differentiated cell. It can be performed by selecting appropriately.
　例えば、インビトロ培養における分化誘導では、上述の１倍体胚性幹細胞の培地からＬＩＦ等の細胞の分化を抑制する因子を取り除き、非接着状態にて、前記１倍体胚性幹細胞を培養することにより、三胚葉性に分化した組織が含まれる胚様体（ＥＢ）を形成させることができる。そして、このＥＢを、ラミニンやフィブロネクチン等の基質上で、レチノイン酸やアクチビン等の分化誘導物質を添加した培地を用いて培養することにより、神経系や血球系の細胞等に分化した細胞を得ることができる。 For example, in differentiation induction in in vitro culture, the above-described haploid embryonic stem cells are cultured in a non-adhered state by removing factors that suppress the differentiation of cells such as LIF from the above-described haploid embryonic stem cell medium. By this, an embryoid body (EB) containing a tissue differentiated into three germ layers can be formed. Then, this EB is cultured on a substrate such as laminin or fibronectin using a medium supplemented with a differentiation inducer such as retinoic acid or activin to obtain cells differentiated into nervous system cells or blood cells. be able to.
　インビトロ培養においては、このようにＥＢを経由した分化誘導だけでなく、後述の実施例において示すような、サイトカイン、増殖因子等の組合せにより、前記１倍体胚性幹細胞から直接目的の分化細胞を得ることもできる。また、このようにして得られる分化細胞は、後述の実施例において示すような幹細胞（エピブラスト幹細胞等）であってもよく、当該幹細胞からさらに分化誘導して得られる細胞であってもよい。 In in vitro culture, in addition to differentiation induction via EB as described above, target differentiated cells can be directly obtained from the haploid embryonic stem cells by a combination of cytokines, growth factors and the like as described in Examples below. It can also be obtained. In addition, the differentiated cells obtained in this way may be stem cells (such as epiblast stem cells) as shown in Examples described later, or may be cells obtained by further differentiation induction from the stem cells.
　一方、インビボ培養における分化誘導としては、後述の実施例において示すような、テラトーマ形成法を利用することもできる。すなわち、前記１倍体胚性幹細胞を免疫不全動物に移植することにより、該動物内にてテラトーマを形成させ、当該テラトーマから多種多様な分化細胞を単離することもできる。 On the other hand, for differentiation induction in in vivo culture, a teratoma formation method as shown in Examples described later can also be used. That is, by transplanting the haploid embryonic stem cells into an immunodeficient animal, a teratoma can be formed in the animal, and a wide variety of differentiated cells can be isolated from the teratoma.
　そして、このようにして得られた分化細胞を、Ｃｄｃ２を恒常的に活性化する条件下にて培養することにより、該分化細胞の１倍性を維持することができる、なお、Ｃｄｃ２を恒常的に活性化する条件については上述の通りである。また、かかる培養は、当業者であれば、培養する分化細胞に適した培地、温度、ＣＯ２濃度等を適宜選択して行うことができる。 The differentiated cells thus obtained can be cultured under conditions that constantly activate Cdc2, whereby the monoploidy of the differentiated cells can be maintained. The conditions for activation are as described above. Such culture can be performed by those skilled in the art by appropriately selecting a medium, temperature, CO 2 concentration and the like suitable for the differentiated cells to be cultured.
　＜１倍性を維持するための薬剤＞
　後述の実施例において示す通り、Ｗｅｅ１及びＭｙｔ１に対する阻害剤の存在下にて、１倍体ＥＳ細胞又は１倍体分化細胞を培養することにより、これら細胞において、Ｃｄｃ２が恒常的に活性化され、１倍性を維持したまま長期に培養することができる。 <Drug to maintain monoploidy>
As shown in Examples below, by culturing haploid ES cells or haploid differentiated cells in the presence of inhibitors against Wee1 and Myt1, Cdc2 is constitutively activated in these cells, It can be cultured for a long time while maintaining monoploidy.
　したがって、本発明は、Ｃｄｃ２活性化剤を有効成分とする、１倍体胚性幹細胞又は１倍体分化細胞の１倍性を維持するための薬剤を提供するものである。 Therefore, the present invention provides a drug for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells containing a Cdc2 activator as an active ingredient.
　本発明の「Ｃｄｃ２活性化剤」としては、後述の実施例において示すような、Ｃｄｃ２活性化を阻害する因子であるＷｅｅ１又はＭｙｔ１の活性を抑制する作用を有する化合物、より具体的には、Ｗｅｅ１又はＭｙｔ１に結合する低分子化合物、Ｗｅｅ１又はＭｙｔ１をコードする遺伝子の転写産物に結合するＲＮＡが挙げられる。細胞に作用させるために、ウィルスベクターを用いた遺伝子導入、エレクトロポレーションによる導入、トランスフェクション試薬を用いた導入等の、煩雑かつ細胞に傷害を与えるような作業を必要とせず、培地に添加するだけで該細胞のＣｄｃ２を恒常的に活性化することができるという観点から、Ｗｅｅ１又はＭｙｔ１に結合する低分子化合物が、本発明のＣｄｃ２活性化剤として好ましい。 The “Cdc2 activator” of the present invention is a compound having an action of suppressing the activity of Wee1 or Myt1, which is a factor inhibiting Cdc2 activation, and more specifically, Wee1 as shown in Examples described later. Alternatively, a low molecular weight compound that binds to Myt1, and RNA that binds to a transcription product of a gene encoding Wee1 or Myt1. In order to act on cells, it does not require complicated and damaging work such as gene introduction using viral vectors, introduction by electroporation, introduction using transfection reagents, etc., and it is added to the medium From the standpoint that Cdc2 of the cell can be constitutively activated alone, a low molecular weight compound that binds to Wee1 or Myt1 is preferred as the Cdc2 activator of the present invention.
　「Ｗｅｅ１又はＭｙｔ１の活性を抑制する作用を有する低分子化合物」としては、例えば、ＰＤ１６６２８５（２－［［４－［２－（ジエチルアミノ）エトキシ］フェニル］アミノ］－６－（２，６－ジクロロフェニル）－８－メチルピリド［２，３－ｄ］ピリミジン－７（８Ｈ）－オン））、ＭＫ１７７５（２－アリル－１－（６－（２－ヒドロキシプロパン－２－イル）ピリジン－２－イル）－６－（（４－（４－メチルピぺラジン－１－イル）フェニル）アミノ）－１Ｈ－ピラゾロ［３，４－ｄ］ピリミジン－３（２Ｈ）－オン）等の市販の低分子化合物、国際公開第２０１０／０９８３６７号、国際公開第２０１０／０６７８８６号、国際公開第２００８／１１５７４２号、国際公開第２００８／１１５７３８号、国際公開第２００７／１２６１２２号、国際公開第２００７／１２６１２８号、国際公開第２００４／００７４９９号又は米国特許出願公開第２００５／００３７４７６号明細書に記載のＷｅｅ１の活性を抑制する作用を有する低分子化合物、国際公開第２０００／３３８４２号に記載のＭｙｔ１の活性を抑制する作用を有する低分子化合物、及びこれら化合物の生理学的に許容される塩又は溶媒和物が挙げられるが、Ｗｅｅ１及びＭｙｔ１の活性を共に抑制できるという観点から、ＰＤ１６６２８５が好ましく、ＰＤ１６６２８５二塩酸塩がより好ましい。 Examples of the “low molecular compound having an action of suppressing the activity of Wee1 or Myt1” include PD166285 (2-[[4- [2- (diethylamino) ethoxy] phenyl] amino] -6- (2,6-dichlorophenyl). ) -8-methylpyrido [2,3-d] pyrimidin-7 (8H) -one)), MK1775 (2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) Commercially available low molecular weight compounds such as -6-((4- (4-methylpiperazin-1-yl) phenyl) amino) -1H-pyrazolo [3,4-d] pyrimidin-3 (2H) -one), International Publication No. 2010/098367, International Publication No. 2010/067876, International Publication No. 2008/115742, International Publication No. 2008/115738, International Publication No. 20 7/126122, International Publication No. 2007/126128, International Publication No. 2004/007499 or US Patent Application Publication No. 2005/0037476, a low molecular weight compound having an action of suppressing the activity of Wee1, International Publication The low molecular weight compounds having the action of suppressing the activity of Myt1 described in 2000/33842 and physiologically acceptable salts or solvates of these compounds can be mentioned, but both the activities of Wee1 and Myt1 can be suppressed. In view of the above, PD166285 is preferable, and PD166285 dihydrochloride is more preferable.
　「Ｗｅｅ１又はＭｙｔ１をコードする遺伝子の転写産物に結合するＲＮＡ」としては、Ｗｅｅ１又はＭｙｔ１を標的とし、その発現の抑制を介してこれらタンパク質の活性を抑制できるものであればよく、例えば、Ｗｅｅ１又はＭｙｔ１をコードする遺伝子の転写産物と相補的な、ｓｉＲＮＡ、ｓｈＲＮＡ（ｓｈｏｒｔ　ｈａｉｐｉｎ　ＲＮＡ）等のｄｓＲＮＡ（二重鎖ＲＮＡ）が挙げられる。ｄｓＲＮＡの鎖長としては、標的遺伝子の発現を抑制することができ、かつ、毒性を示さなければ特に制限はなく、例えば、１５～４９塩基対であり、好適には１５～３５塩基対であり、さらに好適には２１～３０塩基対である。ｄｓＲＮＡは、標的遺伝子の塩基配列と完全に同一である必要はないが、少なくとも７０％以上、好ましくは８０％以上、さらに好ましくは９０％以上の配列の同一性を有する。配列の同一性は、ＢＬＡＳＴプログラムにより決定することができる。「Ｗｅｅ１又はＭｙｔ１をコードする遺伝子の転写産物に結合するＲＮＡ」の他の態様としては、Ｗｅｅ１又はＭｙｔ１をコードする遺伝子の転写産物と相補的なアンチセンスＲＮＡや該転写産物を特異的に開裂するリボザイム活性を有するＲＮＡ（リボザイム）も挙げられる。さらに、これらＲＮＡにおいては、その一部又は全部において、ＰＮＡ、ＬＮＡ、ＥＮＡ等の人工核酸によって、ＲＮＡが置換されているものであってもよい。また、これらＲＮＡに関しては、細胞内において発現させるべく、当該ＲＮＡをコードするＤＮＡを保持する発現ベクターの態様であってもよい。また、このようなＲＮＡは、当業者であれば、市販の合成機等を用いて化学合成することにより調製することができる。 The “RNA that binds to the transcript of the gene encoding Wee1 or Myt1” may be any RNA that targets Wee1 or Myt1 and can suppress the activity of these proteins through suppression of its expression. Examples include dsRNA (double stranded RNA) such as siRNA and shRNA (short haipin RNA) complementary to a transcription product of a gene encoding Myt1. The dsRNA chain length is not particularly limited as long as it can suppress the expression of the target gene and does not exhibit toxicity, and is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs. More preferably, it is 21 to 30 base pairs. The dsRNA need not be completely identical to the base sequence of the target gene, but has at least 70% or more, preferably 80% or more, more preferably 90% or more of sequence identity. Sequence identity can be determined by the BLAST program. As another embodiment of the “RNA that binds to the transcript of the gene encoding Wee1 or Myt1,” an antisense RNA complementary to the transcript of the gene encoding Wee1 or Myt1 or the transcript is specifically cleaved. An RNA having ribozyme activity (ribozyme) is also included. Furthermore, in these RNAs, RNA may be substituted in part or all by artificial nucleic acids such as PNA, LNA, ENA and the like. In addition, these RNAs may be in the form of an expression vector that holds DNA encoding the RNA for expression in cells. In addition, those skilled in the art can prepare such RNA by chemical synthesis using a commercially available synthesizer or the like.
　また、上述の通り、Ｗｅｅ１又はＭｙt１の活性を抑制することにより、Ｃｄｃ２の１４番目のスレオニン又は１５番目のチロシンのリン酸化が抑制され、Ｃｄｃ２は活性化される。したがって、本発明における「Ｃｄｃ２活性化剤」としては、これら被リン酸化部位におけるＣｄｃ２のリン酸化を抑制する作用を有する化合物であってもよく、かかる化合物としては、例えば、Ｃｄｃ２のデコイ、Ｃｄｃ２に対する抗体が挙げられる。 Also, as described above, by suppressing the activity of Wee1 or Myt1, phosphorylation of the 14th threonine or 15th tyrosine of Cdc2 is suppressed, and Cdc2 is activated. Therefore, the “Cdc2 activator” in the present invention may be a compound having an action of suppressing phosphorylation of Cdc2 at these phosphorylated sites. Examples of such a compound include Cdc2 decoy and Cdc2 An antibody is mentioned.
　Ｃｄｃ２のデコイとしては、例えば、Ｗｅｅ１又はＭｙt１によってリン酸化される部位（１４番目のスレオニン又は１５番目のチロシン）を含むＣｄｃ２の部分ポリペプチドが挙げられる。かかる部分ポリペプチドの鎖長としては特に制限はなく、例えば、５～３０アミノ酸が挙げられるが、Ｃｄｃ２を活性化させるために必要なリン酸化部位である１６１番目のチロシンは含まれていない鎖長であることが好ましい。また、このようなポリペプチドは、市販の合成機等を用いて化学合成することもでき、大腸菌等を宿主とする公知の組換えタンパク質製造法を利用しても調製することができる。さらに、細胞内への導入効率を高めるため、Ｃｄｃ２のデコイには細胞膜透過性ペプチドが付加されていてもよい。細胞膜透過性ペプチドとしては、例えば、ＶＰ２２、Ｋａｐｏｓｉ　ＦＧＦ、ＴＡＴ、ショウジョウバエ　アンテチィナペディア、Ｐｅｎｅｔｒａｔｉｎ、Ｍ９１８、Ｔｒａｎｓｐｏｒｔａｎ－１０、Ｐｏｌｙ－Ａｒｇｉｎｉｎｅ、及びこれらペプチドの派生物が挙げられる。また、細胞膜透過性ペプチドは、国際公開２００７／１３２５５号の記載等に基づき、適宜調製されるものであってもよい。 Examples of the Cdc2 decoy include a partial polypeptide of Cdc2 containing a site (14th threonine or 15th tyrosine) that is phosphorylated by Wee1 or Myt1. The chain length of such a partial polypeptide is not particularly limited and includes, for example, 5 to 30 amino acids, but does not include the 161st tyrosine, which is a phosphorylation site necessary for activating Cdc2. It is preferable that Such a polypeptide can also be chemically synthesized using a commercially available synthesizer or the like, and can also be prepared using a known recombinant protein production method using Escherichia coli or the like as a host. Furthermore, a cell membrane-permeable peptide may be added to the Cdc2 decoy to increase the efficiency of introduction into the cell. Examples of the cell membrane permeable peptide include VP22, Kaposi FGF, TAT, Drosophila Antitinapedia, Penetratin, M918, Transportan-10, Poly-Arginine, and derivatives of these peptides. In addition, the cell membrane permeable peptide may be appropriately prepared based on the description of International Publication No. 2007/13255.
　Ｃｄｃ２に対する抗体としては、例えば、Ｃｄｃ２の１４番目のスレオニン又は１５番目のチロシンの非リン酸化状態を特異的に認識し、結合することのできる抗体が挙げられる。抗体の態様としては、ポリクローナル抗体であってもよく、モノクローナル抗体であってもよく、これら抗体の機能的断片（ナノ抗体、１本鎖抗体）であってもよい。このような抗体は、当業者であれば、１４番目のスレオニン又は１５番目のチロシンを含むＣｄｃ２の部分ポリペプチドを利用して、公知の抗体作製方法（ハイブリドーマ法、ファージディスプレイ法等）により適宜調製することができる。 Examples of antibodies against Cdc2 include antibodies that can specifically recognize and bind to the non-phosphorylated state of the 14th threonine or 15th tyrosine of Cdc2. The form of the antibody may be a polyclonal antibody, a monoclonal antibody, or a functional fragment (nanoantibody, single chain antibody) of these antibodies. Those skilled in the art can appropriately prepare such an antibody by a known antibody production method (hybridoma method, phage display method, etc.) using a partial polypeptide of Cdc2 containing the 14th threonine or the 15th tyrosine. can do.
　また、Ｗｅｅ１又はＭｙｔ１によってリン酸化されたＣｄｃ２の１４番目のスレオニン又は１５番目のチロシンが、Ｃｄｃ２５により脱リン酸化されることによっても、Ｃｄｃ２は活性化される。したがって、本発明における「Ｃｄｃ２活性化剤」として、Ｃｄｃ２５の活性を亢進する作用を有する化合物も利用することができる。このような化合物としては、例えばＣｄｃ２５が挙げられる。すなわち、該タンパク質自体を細胞内に導入することにより、その発現量の増加を介して、該細胞におけるＣｄｃ２５の全体的な活性を亢進させることができる。また、Ｃｄｃ２５に関しては、タンパク質の態様のみならず、該タンパク質を細胞内において発現させるべく、Ｃｄｃ２５をコードするＤＮＡを保持する発現ベクターの態様であってもよい。このようなタンパク質や該タンパク質をコードするＤＮＡは、当業者であれば、市販の合成機を用いた化学合成法、組換えタンパク質製造法、遺伝子組み換え技術を利用することにより、適宜調製することができる。 Cdc2 is also activated by dephosphorylation of the 14th threonine or 15th tyrosine of Cdc2 phosphorylated by Wee1 or Myt1 by Cdc25. Therefore, a compound having an action of enhancing the activity of Cdc25 can also be used as the “Cdc2 activator” in the present invention. An example of such a compound is Cdc25. That is, by introducing the protein itself into the cell, the overall activity of Cdc25 in the cell can be enhanced through an increase in the expression level thereof. Moreover, regarding Cdc25, not only a protein aspect but also an expression vector aspect that holds a DNA encoding Cdc25 in order to express the protein in a cell. Those skilled in the art can appropriately prepare such a protein and DNA encoding the protein by using a chemical synthesis method, a recombinant protein production method, or a gene recombination technique using a commercially available synthesizer. it can.
　本発明の「１倍性を維持するための薬剤」においては、前述のＣｄｃ２活性化剤の他、生理学的に許容される担体を含むものであってもよい。生理学的に許容される担体としては、例えば、生理的な等張液（生理食塩水、培地、ブドウ糖やその他の補助薬（Ｄ－ソルビトール、Ｄ－マンニトール、塩化ナトリウム等）を含む等張液等）、賦形剤、防腐剤、安定剤（ヒト血清アルブミン、ポリエチレングリコール等）、結合剤、溶解補助剤、非イオン性界面活性剤、緩衝剤（リン酸塩緩衝液、酢酸ナトリウム緩衝液等）、保存剤、酸化防止剤が挙げられる。また、本発明の薬剤においては、Ｃｄｃ２活性化剤を細胞内に導入するために、適宜トランスフェクション試薬（例えば、ポリアミン系トランスフェクション試薬、カチオン脂質系トランスフェクション試薬）が添加されていてもよい。 The “drug for maintaining monoploidy” of the present invention may contain a physiologically acceptable carrier in addition to the aforementioned Cdc2 activator. Examples of physiologically acceptable carriers include isotonic solutions containing physiological isotonic solutions (physiological saline, medium, glucose and other adjuvants (D-sorbitol, D-mannitol, sodium chloride, etc.), etc. ), Excipients, preservatives, stabilizers (human serum albumin, polyethylene glycol, etc.), binders, solubilizers, nonionic surfactants, buffers (phosphate buffers, sodium acetate buffers, etc.) , Preservatives and antioxidants. In addition, in the drug of the present invention, a transfection reagent (for example, a polyamine-based transfection reagent or a cationic lipid-based transfection reagent) may be appropriately added in order to introduce the Cdc2 activator into cells.
　以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.
　（実施例１）
　＜雌性単為発生胚由来１倍体ＥＳ細胞（ｐｈＥＳ細胞、ｐｈＥＳＣ）の樹立、及びその培養＞
　Ｃ５７ＢＬ／６ＮＣｒマウス又はＣ５７ＢＬ／６Ｊ－ＣＡＧ－ＥＧＦＰマウスに、７．５ユニット／匹にてＰＭＳＧ（血清性性腺刺激ホルモン）、ｈＣＧ（ヒト絨毛性性腺刺激ホルモン）を投与することにより、これらマウスにおいて排卵を誘起し、未受精卵を回収した。回収した未受精卵は、塩化ストロンチウム（ＳｒＣｌ２）にて３０～６０分間処理し、その後、桑実胚（ｍｏｒｕｌａ）に発生するまでの間、ＫＳＯＭ培地にてｉｎ　ｖｉｔｒｏ培養した。発生した桑実胚は、１／２希釈酸性タイロードにて処理することにより、その透明帯を溶かし、そして、マイトマイシン処理をしたＩＣＲマウス由来フィーダー細胞上で培養することにより、純系マウスＣ５７ＢＬ／６の雌性単為発生胚に由来する１倍体ＥＳ細胞（ｐｈＥＳＣ－Ｂ６）を樹立した。 Example 1
<Establishment of female parthenogenetic embryo-derived haploid ES cells (phES cells, phESC) and culture thereof>
In these mice, PMSG (serum gonadotropin), hCG (human chorionic gonadotropin) was administered to C57BL / 6NCr mice or C57BL / 6J-CAG-EGFP mice at 7.5 units / mouse. Ovulation was induced and unfertilized eggs were collected. The collected unfertilized eggs were treated with strontium chloride (SrCl 2 ) for 30 to 60 minutes, and then cultured in vitro in KSOM medium until they developed in morula. The developed morulae are treated with 1/2 dilution acidic Tyrode to dissolve the zona pellucida, and cultured on ICR mouse-derived feeder cells treated with mitomycin to obtain pure mouse C57BL / 6 Haploid ES cells (phESC-B6) derived from female parthenogenetic embryos were established.
　なお、このようにして調製したｐｈＥＳ細胞及び２倍体（ｄｉｐｌｏｉｄ）ＥＳ細胞の混合群は、ノックアウトＤＭＥＭに２０％　ＫＳＯＭ、２ｍＭ　Ｌ－グルタミン、１ｘ非必須アミノ酸（ｎｏｎ　ｅｓｓｅｎｔｉａｌ　ａｍｉｎｏ　ａｃｉｄ）、１００ｕＭ　メルカプトエタノール、０．１％　ペニシリン／ストレプトマイシン、１０００ユニット　ＬＩＦ（ＥＳＧＲＯ、登録商標）に、さらに２ｉと呼ばれる３μＭ　ＣＨＩＲ９９０２１と１μＭ　ＰＤ０３２５９０１とを添加した培地を用いて培養した。そして、ＦＡＣＳにより１Ｎ細胞を回収した後は、前記培地に３００μＭ　ＰＤ１６６２８５を添加して培養した。また、前記混合群は、フィーダー細胞非存在下でも培養した。フィーダー細胞非存在下の培養は、培養器に０.１％ゼラチン溶液を入れて３７℃にて３０分間処理した後、ゼラチン溶液を捨て、その培養器を使用して行った。なお、他の培養条件（ＰＤ1６６２８５の添加濃度等）は、前述のフィーダー細胞存在下における培養同様に行った。 The mixed group of phES cells and diploid ES cells prepared in this manner is 20% KSOM, 2 mM L-glutamine, 1 × non-essential amino acid, 100 uM mercaptoethanol in knockout DMEM. Further, the cells were cultured in a medium in which 3 μM CHIR99021 and 1 μM PD0325901 called 2i were further added to 0.1% penicillin / streptomycin, 1000 unit LIF (ESGRO, registered trademark). After 1N cells were collected by FACS, 300 μM PD166285 was added to the medium and cultured. The mixed group was also cultured in the absence of feeder cells. Cultivation in the absence of feeder cells was performed using a 0.1% gelatin solution in an incubator and treating at 37 ° C. for 30 minutes, then discarding the gelatin solution and using the incubator. Other culture conditions (addition concentration of PD166285, etc.) were the same as those in the presence of the feeder cells described above.
　また、ＦＡＣＳによる１Ｎ細胞の回収は、ヘキスト３３３４２染色を３０分行い、ＰＢＳ（－）ｗｉｔｈ４％ＦＢＳで細胞を洗った後、ＦＡＣＳ　ＡｒｉａII（日本ベクトン・ディッキンソン社製）を用いて行った。 In addition, 1N cells were collected by FACS by staining with Hoechst 33342 for 30 minutes, washing the cells with PBS (−) with 4% FBS, and then using FACS Aria II (manufactured by Nippon Becton Dickinson).
　なお、１Ｎ細胞率の確認実験は、細胞を７０％エタノールにて４℃、８時間以上処理することにより固定し、ＰＩ染色を行った後にＦＡＣＳ　Ｃａｌｉｂｕｒ（日本ベクトン・ディッキンソン社製）で解析した。 In addition, in the confirmation experiment of the 1N cell rate, the cells were fixed by treating with 70% ethanol at 4 ° C. for 8 hours or more and analyzed by FACS Calibur (manufactured by Becton Dickinson, Japan) after PI staining.
　（実施例２）
　＜雄性単為発生胚由来１倍体ＥＳ細胞（ａｈＥＳ細胞、ａｈＥＳＣ）の樹立、及びその培養＞
　ａｈＥＳＣの樹立において、胚の作製は理研バイオリソースセンターに依頼した。マウス未受精卵を除核し、雄の円形精子細胞を注入した後、卵をＳｒＣｌ２を用いて活性化することによって、雄性単為発生桑実胚又は雄性単為発生胚盤胞（ｂｌａｓｔｏｃｙｓｔ）を得た。これらの胚をフィーダー細胞上におき、その後の培養はｐｈＥＳＣと同様の方法で行った。 (Example 2)
<Establishment of male parthenogenetic embryo-derived haploid ES cells (ahES cells, ahESC) and culture thereof>
In the establishment of ahESC, the production of embryos was requested from the RIKEN BioResource Center. After enucleating a mouse unfertilized egg and injecting male circular sperm cells, the male parthenogenetic morula or male parthenogenetic blastocyst is activated by activating the egg with SrCl2. Obtained. These embryos were placed on feeder cells, and the subsequent culture was performed in the same manner as phESC.
　（実施例３）
　＜雌性単為発生胚由来エピブラスト幹細胞（ｐｈＥｐｉＳＣ、ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃ　ｈａｐｌｏｉｄ　ｅｐｉｂｌａｓｔ　ｓｔｅｍ　ｃｅｌｌ）の樹立、及びその培養方法＞
　前述の通りに樹立したｐｈＥＳＣからｐｈＥｐｉＳＣへの分化誘導は以下のようにして行った。すなわち、ｐｈＥＳＣを、Ｎ２、Ｂ２７、ＮＥＡＡ、ＧｌｕｔａＭＡＸＩ、β－メルカプトエタノールを添加したＤＭＥＭ／Ｆ１２培地にて７日以上培養することにより、ｐｈＥｐｉＳＣへの分化誘導を行った。 Example 3
<Establishment of female parthenogenetic embryo-derived epiblast stem cells (phEpiSC, parthenogenetic haploid epiblast stem cell) and culture method thereof>
Differentiation induction from phESC established as described above to phEpiSC was performed as follows. That is, differentiation induction into phEpiSC was performed by culturing phESC in DMEM / F12 medium supplemented with N2, B27, NEAA, GlutaMAXI, and β-mercaptoethanol for 7 days or more.
　そして、ｐｈＥｐｉＳＣに分化誘導した後の細胞については、ＦＡＣＳを用いて１倍体細胞を選別した後、２０ｎｇ／ｍｌ　アクチビンＡ、２０ｎｇ／ｍｌ　ｂＦＧＦ、１０ｎＭ　ＸＡＶ９３９及び３００ｎＭ　ＰＤ１６６２８５を更に添加した培地にて培養した（該培養における培地に関し、ＰＤ１６６２８５以外の成分については、Ｓｕｍｉ，Ｔ．ら、ＰＬｏＳ　Ｏｎｅ、２０１３年、８、ｅ６３３７８　参照のこと）。また、ｐｈＥｐｉＳＣに分化誘導した後の細胞は、マイトマイシンＣ処理したフィーダー細胞上にて培養した。さらに、ＰＤ１６６２８５存在下における当該培養においては、７日毎にＦＡＣＳを用いて１倍体細胞を精製した。 The cells after differentiation induction into phEpiSC are selected from haploid cells using FACS, and then cultured in a medium further supplemented with 20 ng / ml activin A, 20 ng / ml bFGF, 10 nM XAV939 and 300 nM PD166285. (See Sumi, T. et al., PLoS One, 2013, 8, e63378 for components other than PD166285 regarding the medium in the culture). The cells after differentiation induction into phEpiSC were cultured on feeder cells treated with mitomycin C. Furthermore, in this culture in the presence of PD166285, haploid cells were purified using FACS every 7 days.
　なお、１倍体ｐｈＥｐｉＳＣの精製は、ヘキストにて３７℃、３０分間処理することにより染色した後、ＦＡＣＳ　ＡｒｉａIIにて、１倍体細胞を含むＧ１区画のみを選別することにより行った。また、ｐｈＥＳＣからｐｈＥｐｉＳＣへの分化誘導、該分化誘導後の培養において、細胞の継代は、コンプリートアキュターゼ（登録商標、ｃｏｍｐｌｅｔｅ　ａｃｃｕｔａｓｅ）にて、３７℃で２分間処理することにより行い、２日に１回のペースで継代した。 The haploid phEpiSC was purified by treating with Hoechst at 37 ° C. for 30 minutes, and then selecting only the G1 compartment containing the haploid cells with FACS Aria II. Further, in the differentiation induction from phESC to phEpiSC and the culture after the differentiation induction, the cells are subcultured by treating with complete acactase (registered trademark, complete accurate) at 37 ° C. for 2 minutes. Passage was once at a pace.
　上述の通り調製したｐｈＥＳＣ等については、以下に示す方法にてその性状等を解析した。 About the phESC etc. which were prepared as mentioned above, the property etc. were analyzed by the method shown below.
　（試験例１）
　＜アレイＣＧＨ解析、及び確認のためのＰＣＲ反応＞
　本発明の方法により得られたｐｈＥＳＣにおいて、ゲノムの欠失等が生じていないことを解析した。すなわち先ず、ｐｈＥＳＣの１Ｎ細胞をＦＡＣＳにて２ｘ１０６個回収した。細胞からのＤＮＡ抽出はプロテイナーゼＫ処理とフェノール／クロロホルム処理により行い、抽出したゲノムＤＮＡをＲＮａｓｅ　Ｇ、Ｓにて処理することによりＲＮＡを除去した。また、レファレンスゲノム及び実験コントロール用ゲノムとして、Ｃ５７ＢＬ／６ＮＣｒメスの肝臓から前記同様の方法にてゲノムＤＮＡを抽出した。 (Test Example 1)
<Array CGH analysis and PCR reaction for confirmation>
In the phESC obtained by the method of the present invention, it was analyzed that no genomic deletion occurred. That is, first, 2 × 10 6 phESC 1N cells were collected by FACS. DNA extraction from the cells was performed by proteinase K treatment and phenol / chloroform treatment, and RNA was removed by treating the extracted genomic DNA with RNase G and S. Further, genomic DNA was extracted from the liver of a C57BL / 6NCr female as a reference genome and an experimental control genome by the same method as described above.
　そして、このようにして調製したゲノムＤＮＡをＩｎ－ｓｉｔｕオリゴＤＮＡマイクロアレイキット（Ａｇｉｌｅｎｔ社製）にて処理し、４ｘ１８０Ｌスライドを用いたアレイＣＧＨにより解析した。なお、レファレンスゲノムはＣｙ３にて標識し、ｐｈＥＳＣ由来のゲノムはＣｙ５にて標識した。また、このようにして得られたアレイＣＧＨの結果を再確認するために、前記ゲノムＤＮＡを鋳型とし、ＰＣＲ解析も行った。 The genomic DNA thus prepared was treated with an In-situ oligo DNA microarray kit (manufactured by Agilent) and analyzed by array CGH using 4 × 180 L slides. The reference genome was labeled with Cy3, and the phESC-derived genome was labeled with Cy5. In addition, in order to reconfirm the results of the array CGH thus obtained, PCR analysis was also performed using the genomic DNA as a template.
　（試験例２）
　＜幹細胞マーカー発現の検出、並びにテラトーマ形成＞
　本発明の方法により得られたｐｈＥＳＣは、未分化性を維持していること及び多分化能を保持していることを確認するため、該細胞における幹細胞マーカーの発現を検出した。また、該細胞からテラトーマが形成されるかどうかも解析した。すなわち先ず、ｐｈＥＳＣを４％ＰＦＡにて４℃、１５分間処理することにより固定した。次いで、１％ＢＳＡによるブロッキング反応を室温にて１時間行った。そして、抗Ｏｃｔ３／４抗体（ヤギ由来抗マウスＯｃｔ３／４抗体）、抗Ｎａｎｏｇ抗体（ウサギ由来抗マウスＮａｎｏｇ抗体）及び抗ＳＳＥＡ１抗体（ロバ由来抗マウスＳＳＥＡ１抗体）を、各々１次抗体として用い、１／１００の濃度にて、４℃で１晩中ｐｈＥＳＣと反応させた。抗体反応後に、アレクサフルオロ５６８結合ヤギ由来抗ウサギＩｇＧ抗体、アレクサフルオロ４８８結合ヤギ由来抗マウスＩｇＭ抗体及びアレクサフルオロ４８８結合ロバ由来抗ヤギＩｇＧ抗体を、各々２次抗体として用い、１／５００の濃度にて、室温で１時間ｐｈＥＳＣと反応させた。これらの細胞は、二次抗体反応の後にＤＡＰＩ染色を室温にて５分間行った。アルカリフォスファターゼ染色は、ＳＫ－５３００キット（ベクター社製）を使用して行った。 (Test Example 2)
<Detection of stem cell marker expression and teratoma formation>
In order to confirm that the phESC obtained by the method of the present invention maintains undifferentiation and retains pluripotency, expression of a stem cell marker in the cells was detected. It was also analyzed whether teratoma was formed from the cells. That is, first, phESC was fixed by treatment with 4% PFA at 4 ° C. for 15 minutes. Next, a blocking reaction with 1% BSA was performed at room temperature for 1 hour. And, using anti-Oct3 / 4 antibody (goat-derived anti-mouse Oct3 / 4 antibody), anti-Nanog antibody (rabbit-derived anti-mouse Nanog antibody) and anti-SSEA1 antibody (donkey-derived anti-mouse SSEA1 antibody), respectively, as primary antibodies, Reacted with phESC overnight at 4 ° C. at a concentration of 1/100. After the antibody reaction, Alexafluoro 568-conjugated goat-derived anti-rabbit IgG antibody, Alexafluoro 488-conjugated goat-derived anti-mouse IgM antibody and Alexafluoro 488-conjugated donkey-derived anti-goat IgG antibody were each used as secondary antibodies, and the concentration was 1/500. And reacted with phESC at room temperature for 1 hour. These cells were DAPI stained for 5 minutes at room temperature after the secondary antibody reaction. Alkaline phosphatase staining was performed using the SK-5300 kit (manufactured by Vector).
　また、テラトーマ形成は、一倍体ＥＳ細胞の１Ｎ細胞をＦＡＣＳにて回収した後、その７日以内に２ｘ１０６個／匹にて、これら細胞をＫＳＮ雄マウスの皮下に投与した。そして、該細胞投与後３０日後にテラトーマの解析を行った。なお、解析の際のＨＥ染色は定法に従って行った。 For teratoma formation, 1N cells of haploid ES cells were collected by FACS, and within 7 days, these cells were administered subcutaneously to KSN male mice at 2 × 10 6 cells / mouse. Then, teratoma was analyzed 30 days after the administration of the cells. The HE staining at the time of analysis was performed according to a conventional method.
　（試験例３）
　＜マイクロアレイを用いた遺伝子発現解析＞
　本発明の方法により得られたｐｈＥＳＣにおける遺伝子発現量を解析した。すなわち先ず、ｐｈＥＳＣの１Ｎ細胞及びｐｈＥＳＣ由来倍数性ＥＳ細胞の２Ｎ細胞を、各２ｘ１０５個ずつＦＡＣＳにて回収し、トータルＲＮＡをトリゾールにて抽出した。自然交配によって得られた胚から樹立したＥＳ細胞は、ＦＡＣＳによる２Ｎ細胞の回収を行わずにＲＮＡ抽出を行った。このようにして抽出したＲＮＡを全マウスゲノムＤＮＡオリゴマイクロアレイキット（Ａｇｉｌｅｎｔ社製）にて処理し、４ｘ４４Ｋスライドを用いたマイクロアレイにて解析した。 (Test Example 3)
<Gene expression analysis using microarray>
The gene expression level in phESC obtained by the method of the present invention was analyzed. Specifically, first, 2 × 10 5 phESC 1N cells and 2N cells of phESC-derived ploidy ES cells were collected by FACS, and total RNA was extracted with Trizol. ES cells established from embryos obtained by natural mating were subjected to RNA extraction without collecting 2N cells by FACS. The RNA thus extracted was treated with a whole mouse genomic DNA oligo microarray kit (manufactured by Agilent) and analyzed with a microarray using 4 × 44K slides.
　（試験例４）
　＜キメラマウスの作製及びキメラ寄与率の確認＞
　本発明の方法により得られたｐｈＥＳＣの多分化能の保持を確認するため、該細胞を用いてキメラマウスの作製を試みた。また、ｐｈＥＳＣの生殖細胞系列移行能についても解析した。すなわち先ず、前述の通り樹立したｐｈＥＳＣ－Ｂ６ＧＦＰの１Ｎ細胞をＦＡＣＳにて回収した。そして、その当日に、Ｃ５７ＢＬ／６Ｎの胚盤胞にこれら１Ｎ細胞を注入した。ｐｈＥＳＣを注入した胚盤胞はＩＣＲ偽妊娠マウスへ移植した。その後、ｄ１４．５（１４．５日後）で解剖するか、自然出産により胎児を得てＧＦＰ蛍光を確認した。ｄ１４．５で解剖したマウスについては、ＦＡＣＳ　ＡｒｉａIIにてＧＦＰ陽性細胞の割合を臓器ごとに確認した。また、キメラマウスにおける生殖系列移行の確認は、キメラマウスを自然交配させることで得られた胎児において、ＧＦＰの発現を検出することにより確認した。 (Test Example 4)
<Production of chimeric mouse and confirmation of chimera contribution>
In order to confirm the retention of the pluripotency of the phESC obtained by the method of the present invention, an attempt was made to produce a chimeric mouse using the cells. We also analyzed the ability of phESCs to migrate to germline. That is, first, 1N cells of phESC-B6GFP established as described above were collected by FACS. On the same day, these 1N cells were injected into C57BL / 6N blastocysts. Blastocysts injected with phESC were transplanted into ICR pseudopregnant mice. Then, it was dissected at d14.5 (after 14.5 days), or a fetus was obtained by spontaneous delivery and GFP fluorescence was confirmed. For mice dissected at d14.5, the proportion of GFP-positive cells was confirmed for each organ by FACS Aria II. Confirmation of germline migration in chimeric mice was confirmed by detecting GFP expression in fetuses obtained by natural mating of chimeric mice.
　以上の試験例に基づき、本発明の方法により得られたｐｈＥＳＣ等を解析した結果を以下に示す。 Based on the above test examples, the results of analyzing phESC and the like obtained by the method of the present invention are shown below.
　＜ＰＤ１６６２８５を用いた１倍体ＥＳ細胞の培養＞
　マウス未受精卵に、ストロンチウム（Ｓｒ）刺激を与えて単為発生させることにより、母性（ｍａｔｅｒｎａｌ）ゲノムを１組持つ１倍体胚を作製できることが知られている（非特許文献１～４参照）。本発明者らは、Ｃ５７ＢＬ／６の雌マウスの卵を単為発生させ、インビトロ培養にて桑実胚になるまで培養し、フィーダー細胞上に乗せることでＥＳ細胞（雌性単為発生胚由来１倍体ＥＳ細胞（ｐｈＥＳ細胞、ｐｈＥＳＣ））を樹立することに成功している。 <Cultivation of haploid ES cells using PD166285>
It is known that haploid embryos having one set of maternal genome can be produced by generating parthenogenesis by giving strontium (Sr) stimulation to mouse unfertilized eggs (see Non-Patent Documents 1 to 4). ). The present inventors have partially generated eggs of C57BL / 6 female mice, cultured until in vitro culture until they became morulae, and placed on feeder cells to obtain ES cells (female parthenogenetic embryo-derived 1). It has succeeded in establishing polyploid ES cells (phES cells, phESC).
　なお、純系マウスＣ５７ＢＬ／６の卵は発生率が悪いこともあり、１倍体ＥＳ細胞の樹立は困難である。しかしながら、本発明者らは、Ｃ５７ＢＬ／６の胚において、胚盤胞よりも桑実胚の方が細胞の状態が良いということを見出し、ＥＳ細胞作製において通常用いられる胚盤胞ではなく、桑実胚を用いることにより、ｐｈＥＳＣ－Ｂ６の樹立に成功している（表１　参照）。 It should be noted that it is difficult to establish haploid ES cells because the eggs of pure mouse C57BL / 6 have a low incidence. However, the present inventors have found that in C57BL / 6 embryos, morulas are in a better cell state than blastocysts and are not blastocysts normally used in ES cell production, but mulberry. By using a real embryo, phESC-B6 has been successfully established (see Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　しかしながら、ｐｈＥＳＣ－Ｂ６においても、従前の報告にあるＦ１マウス由来の細胞同様に、細胞の２倍体化が進行し、ＦＡＣＳによる１Ｎ細胞の回収を行わなければ１倍体細胞は自然に消滅してしまう。そこで、本発明者らは、ｐｈＥＳＣ－Ｂ６の細胞周期を制御し、細胞の２倍体化を防ぐことを試みた。 However, even in phESC-B6, diploidization of cells progresses in the same manner as the cells derived from F1 mice reported in the previous report, and haploid cells naturally disappear unless 1N cells are collected by FACS. End up. Therefore, the present inventors tried to control the cell cycle of phESC-B6 and prevent cell diploidization.
　すなわち、１倍体細胞が２倍体化するメカニズムは不明であるが、２倍体化する際にはＧ１期、Ｓ期、Ｇ２期、Ｇ１期、Ｓ期、Ｇ２期、Ｍ期といった細胞周期が破綻していることが予想され、このようにＭ期に入る前に２度のＳ期を経過することによって、細胞分裂を介さずにＤＮＡ合成が生じ、１倍体細胞におけるゲノムが２倍体化していることが想定される（図１　参照）。 That is, the mechanism of diploidization of haploid cells is unknown, but when doubling, the cell cycle such as G1 phase, S phase, G2 phase, G1 phase, S phase, G2 phase, M phase In this way, by passing the S phase twice before entering the M phase, DNA synthesis occurs without cell division, and the genome in the haploid cell is doubled. It is assumed that it has become a body (see Fig. 1).
　そこで、かかる想定に基づき、細胞周期においてＧ２期からＭ期への移行を阻害する因子（Ｗｅｅ１及びＭｙｔ1）に対する阻害剤　ＰＤ１６６２８５を、１倍体ＥＳ細胞株の培養において使用することにより、該細胞株の２倍体化を防ぎ、１倍体の状態を維持したまま長期に培養することを試みた。 Therefore, based on this assumption, PD166285, an inhibitor against factors (Wee1 and Myt1) that inhibit the transition from G2 phase to M phase in the cell cycle, is used in the culture of haploid ES cell lines. In order to prevent the diploid formation, the culturing was continued for a long time while maintaining the haploid state.
　実施例１に記載の通り、２ｉ等を添加したノックアウトＤＭＥＭにて、ｐｈＥＳ細胞を２～４回継代した後に、１倍体ＥＳ細胞株（１Ｎ細胞）をＦＡＣＳにて回収し、その後の培養からＷｅｅ１及びＭｙｔ１に対する阻害剤　ＰＤ１６６２８５の存在下（３００ｎＭ、５００ｎＭ若しくは１０００ｎＭ）又は非存在下にて培養した。培養工程については、図２参照のこと、また、かかる培養工程におけるＦＡＣＳ解析の結果を図３～６に示す。さらに、このように培養して得られた細胞の形状及びそれらの増殖速度ついても解析した。得られた結果を図７及び８に示す。 As described in Example 1, phES cells were subcultured 2 to 4 times in a knockout DMEM supplemented with 2i and the like, and then a haploid ES cell line (1N cell) was recovered by FACS and then cultured. Cultivated in the presence (300 nM, 500 nM or 1000 nM) or absence of PD166285, an inhibitor against Wee1 and Myt1. See FIG. 2 for the culturing process, and FIGS. 3 to 6 show the results of FACS analysis in the culturing process. Furthermore, the shape of the cells obtained by culturing in this way and their growth rate were also analyzed. The obtained results are shown in FIGS.
　図３～６に示す通り、１倍体ＥＳ細胞株をＦＡＣＳにて回収し、その後の培養からＰＤ１６６２８５の存在下にて培養することにより、高い１Ｎ細胞率を維持できることが明らかになった。また、図７及び８に示した結果から明らかな通り、３００ｎＭのＰＤ１６６２８５を培地に添加した時の細胞は、添加しないものと比較してより立体的なコロニーを形成した。なお、図には示さないが、細胞の増殖速度は阻害剤の有無では変わらなかった。 As shown in FIGS. 3 to 6, it was revealed that a high 1N cell rate can be maintained by recovering the haploid ES cell line by FACS and culturing in the presence of PD166285 from the subsequent culture. Further, as is apparent from the results shown in FIGS. 7 and 8, the cells when 300 nM PD166285 was added to the medium formed more three-dimensional colonies than those not added. Although not shown in the figure, the cell growth rate did not change with or without the inhibitor.
　次に、ＰＤ１６６２８５を添加して樹立した細胞から、ＰＤ１６６２８５を抜いて培養したものと、ＰＤ１６６２８５を添加したまま培養したもので、減少していく１Ｎ細胞率に差があるのかを調べた。得られた結果を図９に示す。 Next, it was examined whether there was a difference in the decreasing 1N cell rate between the cells established by adding PD166285 and those cultured without PD166285 and those cultured with PD166285 added. The obtained results are shown in FIG.
　図９に示す通り、特に培養３３日目のＤＮＡ量解析において、ＰＤ１６６２８５を添加して培養した際には、大部分の細胞が１倍体状態を維持し続けられることが明らかになった。一方、ＰＤ１６６２８５を添加せずに培養した場合には、１Ｎ細胞率は低下しており、２倍体化した細胞が大部分を占めていた。 As shown in FIG. 9, in the analysis of the amount of DNA on the 33rd day of culturing, it was revealed that most cells can maintain the haploid state when PD166285 is added and cultured. On the other hand, when cultured without adding PD166285, the 1N cell rate decreased, and diploid cells accounted for the majority.
　通常、１Ｎ細胞率が１０％以上であればＦＡＣＳによって再度１Ｎ細胞を集めやすく、１倍体細胞株を再び安定に培養することが可能となるため、Ｃｄｃ２を恒常的に活性化させることを特徴とする、本発明の１倍体ＥＳ細胞の培養方法は、該細胞を長期かつ安定的に維持する上で極めて有効であることが明らかになった。 Usually, if the 1N cell rate is 10% or more, it is easy to collect 1N cells again by FACS, and it becomes possible to stably culture a haploid cell line again, so that Cdc2 is constantly activated. It has been clarified that the haploid ES cell culture method of the present invention is extremely effective in maintaining the cells for a long time and stably.
　また、図には示さないが、上述のｐｈＥＳＣ（雌性単為発生胚由来１倍体ＥＳ細胞）同様に、実施例２に記載の方法にて調製した雄性単為発生胚由来１倍体ＥＳ細胞（ａｈＥＳＣ）も、本発明の方法により、その１倍体の状態を安定的に維持できることは確認している。 Further, although not shown in the figure, male parthenogenetic embryo-derived haploid ES cells prepared by the method described in Example 2 in the same manner as the above-described phESC (female parthenogenetic embryo-derived haploid ES cells). (AhESC) has also confirmed that the haploid state can be stably maintained by the method of the present invention.
　次に、実施例１に記載の方法により調製したｐｈＥＳＣ－Ｂ６の核型を定法に従って解析した結果、７０％以上の細胞で染色体本数が２０本であることが確認できた。また、Ｇ－ｂａｎｄレベルでは染色体異常は発見されなかった（図１０　参照）。さらに細かく染色体の欠失や増幅の有無を検出するため、試験例１に記載の通り、アレイＣＧＨ解析を行った。その結果、ｐｈＥＳＣ－Ｂ６ＧＦＰにおいて特に大きな異常は見当たらなかった（図１１　参照）。なお、１４番染色体に欠失があるように見える部分についてはＰＣＲレベルでの詳細な解析を行った結果、図には示さないが、本当にゲノムが欠失しているわけではないということを確認している。 Next, as a result of analyzing the phESC-B6 karyotype prepared by the method described in Example 1 according to a conventional method, it was confirmed that the number of chromosomes was 20 in 70% or more of the cells. In addition, no chromosomal abnormality was found at the G-band level (see FIG. 10). In order to further detect the presence or absence of chromosome deletion or amplification, array CGH analysis was performed as described in Test Example 1. As a result, no particularly large abnormality was found in phESC-B6GFP (see FIG. 11). As a result of detailed analysis at the PCR level for the part that appears to have a deletion on chromosome 14, it was not shown in the figure, but it was confirmed that the genome was not really deleted is doing.
　＜ＰＤ１６６２８５存在下にて培養したｐｈＥＳＣ－Ｂ６における幹細胞マーカー発現の検出、並びにテラトーマ形成＞
　試験例２に記載の通り、ＰＤ１６６２８５存在下にて培養して樹立したｐｈＥＳＣ－Ｂ６の、未分化性を確認するために、ＦＡＣＳによる１Ｎ細胞回収を行ってから７日以内に、ｐｈＥＳＣ－Ｂ６における未分化マーカー（Ｏｃｔ４、Ｎａｎｏｇ及びＳＳＥＡ１）の発現を免疫染色にて検出した。また、アルカリフォスファターゼ染色により、未分化マーカー（ＡＬＰ）の発現を検出した。その結果、図１２及び１３に示す通り、検出したいずれの未分化マーカーについて、高い発現が認められた。 <Detection of stem cell marker expression and teratoma formation in phESC-B6 cultured in the presence of PD166285>
As described in Test Example 2, in order to confirm the undifferentiation of phESC-B6 established by culturing in the presence of PD166285, within 7 days after 1N cell recovery by FACS, The expression of undifferentiated markers (Oct4, Nanog and SSEA1) was detected by immunostaining. Moreover, the expression of the undifferentiation marker (ALP) was detected by alkaline phosphatase staining. As a result, as shown in FIGS. 12 and 13, high expression was observed for any detected undifferentiated marker.
　また、ＰＤ１６６２８５存在下にて培養して樹立したｐｈＥＳＣ－Ｂ６のｉｎ　ｖｉｖｏにおける分化能を確認するために、免疫不全マウスにｐｈＥＳＣ－Ｂ６ＧＦＰをインジェクションし、テラトーマの形成を試みた。得られた結果を図１４に示す。 In addition, in order to confirm the in vivo differentiation ability of phESC-B6 established by culturing in the presence of PD166285, phESC-B6GFP was injected into immunodeficient mice to try to form teratomas. The obtained result is shown in FIG.
　図１４に示す通り、得られたテラトーマを組織学的に解析することによって、インジェクションしたｐｈＥＳＣ－Ｂ６ＧＦＰに由来する３胚葉が形成されていることが明らかになった。したがって、ＰＤ１６６２８５存在下にて培養して樹立したｐｈＥＳＣ－Ｂ６は、未分化性を維持しており、さらにｉｎ　ｖｉｖｏにおける分化能を持ち合わせていることが明らかになった。 As shown in FIG. 14, histological analysis of the obtained teratomas revealed that three germ layers derived from the injected phESC-B6GFP were formed. Therefore, it was revealed that phESC-B6 established by culturing in the presence of PD166285 maintains undifferentiation and further has differentiation ability in vivo.
　＜ＰＤ１６６２８５存在下にて培養したｐｈＥＳＣ－Ｂ６の遺伝子発現解析＞
　試験例３に記載の通り、ｐｈＥＳＣ－Ｂ６の１倍体ＥＳ細胞と２倍体ＥＳ細胞とにおいて、遺伝子発現に差があるのかを調べるため、マイクロアレイ解析を行った。解析に使用した１倍体ＥＳ細胞はｐｈＥＳＣ－Ｂ６のＧ１期をＦＡＣＳで回収したものであり、２倍体ＥＳ細胞はｐｈＥＳＣ－Ｂ６が２倍体化した細胞のＧ１期をＦＡＣＳで回収したものである。得られた結果を図１５及び１６に示す。 <Gene expression analysis of phESC-B6 cultured in the presence of PD166285>
As described in Test Example 3, microarray analysis was performed to examine whether there was a difference in gene expression between haploid ES cells and diploid ES cells of phESC-B6. The haploid ES cells used in the analysis were those obtained by collecting the G1 phase of phESC-B6 by FACS, and the diploid ES cells were those obtained by collecting the G1 phase of cells doubling phESC-B6 by FACS. It is. The obtained results are shown in FIGS.
　図１５に示した結果から明らかなように、ほとんどの遺伝子においてライン間の差も少なく、１倍体ＥＳ細胞と２倍体ＥＳ細胞の発現量に差のある遺伝子も少なかった。また、図１６に示した結果から明らかなように、代表的な未分化マーカーとして利用される遺伝子や、分化した細胞でのみ発現する遺伝子の発現量についても、１倍体ＥＳ細胞と、２倍体ＥＳ細胞（前記１倍体ＥＳ細胞が２倍体化したもの、自然交配により得られた胚から樹立したＥＳ細胞）との間で、ほとんど差が見られなかった。 As is clear from the results shown in FIG. 15, there were few differences between the lines in most genes, and there were few genes that differed in the expression levels of haploid ES cells and diploid ES cells. As is clear from the results shown in FIG. 16, the expression levels of genes used as typical undifferentiated markers and genes that are expressed only in differentiated cells are also doubled with haploid ES cells. There was almost no difference between somatic ES cells (the haploid ES cells were doubling, ES cells established from embryos obtained by natural mating).
　＜ＰＤ１６６２８５存在下にて培養したｐｈＥＳＣ－Ｂ６のキメラマウス形成能と生殖細胞系列移行能についての解析＞
　試験例４に記載の通り、ＰＤ１６６２８５を使用して培養したｐｈＥＳＣ－Ｂ６ＧＦＰに由来する核が、個体になり得る能力があるかを確認するために、キメラマウス作製を行った。得られた結果を図１７～１９に示す。 <Analysis of the ability of phESC-B6 cultured in the presence of PD166285 to form chimeric mice and migrate to germline>
As described in Test Example 4, chimera mice were produced in order to confirm whether nuclei derived from phESC-B6GFP cultured using PD166285 have the ability to become individuals. The obtained results are shown in FIGS.
　図１７～１９に示した結果から明らかな通り、キメラマウスを妊娠１４日目で解剖したものでは、ｐｈＥＳＣ－Ｂ６ＧＦＰに由来する細胞の率（キメラ寄与率）は全身の５０％程度にて確認された。また、臓器ごとの寄与率を解析したところ、脳にて特に高い寄与率が示された。 As is clear from the results shown in FIGS. 17 to 19, when the chimeric mice were dissected on day 14 of gestation, the rate of cells derived from phESC-B6GFP (chimera contribution rate) was confirmed at about 50% of the whole body. It was. Moreover, when the contribution rate for each organ was analyzed, a particularly high contribution rate was shown in the brain.
　また、出産させたキメラにおいても、ＧＦＰの発現が認められる個体を得ることができ、またｐｈＥＳＣ－Ｂ６ＧＦＰは、生殖細胞系列に移行できることも確認された（表２　参照）。 In addition, it was also confirmed that even in the chimera that was born, individuals capable of expressing GFP could be obtained, and that phESC-B6GFP could be transferred to the germline (see Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　ＥＳ細胞等の樹立が困難であるとされている純系マウス　ｐｈＥＳＣ－Ｂ６ＧＦＰでも生殖細胞系列に移行することができる１倍体ＥＳ細胞株を樹立できたことから、本発明の方法によれば、非常に質の高い該細胞株を安定的に維持できることが明らかになった。 According to the method of the present invention, it was possible to establish a haploid ES cell line that can be transferred to the germline even with the pure mouse phESC-B6GFP, which is considered to be difficult to establish ES cells and the like. It was revealed that the high quality cell line can be stably maintained.
　＜ＰＤ１６６２８５存在下にて培養したｐｈＥＳＣ－Ｂ６における分化誘導＞
　本発明の方法により調製したｐｈＥＳＣ－Ｂ６から、１倍体のまま分化した細胞が調製できるかどうかを解析した。すなわち、実施例３及び図２０に記載の通り、１週間かけてｐｈＥＳＣ－Ｂ６においてエピブラスト幹細胞（ＥｐｉＳＣ）への分化を誘導した。そして、分化誘導後、１倍体細胞を選別し、ＰＤ１６６２８５存在下にて１４日間培養した細胞について、ｐｈＥＳＣ－Ｂ６同様に、ＦＡＣＳを用いて１Ｎ細胞率を解析した。得られた結果を図２１に示す。さらに該ＦＡＣＳにて単離した１倍体細胞における幹細胞マーカー遺伝子の発現をＰＣＲ法により解析した。得られた結果を図２２に示す。 <Induction of differentiation in phESC-B6 cultured in the presence of PD166285>
It was analyzed whether cells differentiated as haploid could be prepared from phESC-B6 prepared by the method of the present invention. That is, as described in Example 3 and FIG. 20, differentiation into epiblast stem cells (EpiSC) was induced in phESC-B6 over 1 week. Then, after differentiation induction, haploid cells were selected, and 1N cell rate was analyzed for cells cultured for 14 days in the presence of PD166285 using FACS in the same manner as phESC-B6. The obtained results are shown in FIG. Furthermore, the expression of the stem cell marker gene in the haploid cells isolated by the FACS was analyzed by the PCR method. The obtained result is shown in FIG.
　図２１に示した結果から明らかなように、実施例３及び図２０に記載の方法にて、ｐｈＥＳＣから分化した１倍体細胞を高効率にて得ることができた。さらに、これら１倍体分化細胞における幹細胞マーカー遺伝子の発現を検出した結果、図２２に示す通り、ＥｐｉＳＣ特有の幹細胞マーカー遺伝子であるＦｇｆ５の発現が、当該１倍体分化細胞において認められた。その一方で、ＥＳ細胞特有の幹細胞マーカー遺伝子であるＲｅｘ１の発現は、これら細胞において検出されなかった。 As is clear from the results shown in FIG. 21, haploid cells differentiated from phESC could be obtained with high efficiency by the method described in Example 3 and FIG. Furthermore, as a result of detecting the expression of the stem cell marker gene in these haploid differentiated cells, as shown in FIG. 22, the expression of Fgf5, a stem cell marker gene unique to EpiSC, was observed in the haploid differentiated cells. On the other hand, the expression of Rex1, a stem cell marker gene unique to ES cells, was not detected in these cells.
　したがって、本発明の方法によれば、１倍体ＥＳ細胞から、その１倍体状態を維持させたまま分化した細胞を得ることができ、さらには該分化細胞においてもその１倍性を維持させることができることも明らかになった。 Therefore, according to the method of the present invention, a differentiated cell can be obtained from a haploid ES cell while maintaining its haploid state, and the diploidy is also maintained in the differentiated cell. It became clear that it was possible.
　以上説明したように、本発明によれば、１倍体ＥＳ細胞を長期に渡って安定的に培養することが可能となる。すなわち、１倍性を維持しつつ、ゲノムＤＮＡにおいて欠失等を生じさせることなく、分化多能性及び高い増殖能を保持したまま、１倍体ＥＳ細胞を培養することが可能となる。 As described above, according to the present invention, haploid ES cells can be stably cultured over a long period of time. That is, it is possible to culture haploid ES cells while maintaining pluripotency and without causing deletion or the like in genomic DNA while maintaining differentiation pluripotency and high proliferation ability.
　マウスゲノムを用いたフォワードジェネティクス研究を行う際には、純系マウスを用いた方が解析をしやすい。それは、Ｆ１ハイブリッドマウスから１倍体ＥＳ細胞株を樹立した場合、作られるＥＳ細胞株はＦ２となり、その表現型を解析する場合には遺伝的背景（ｇｅｎｅｔｉｃ　ｂａｃｋｇｒｏｕｎｄ）による影響を考慮しなければならなくなるためである。しかしながら、従前の雌性半数体胚性幹細胞及び雄性半数体胚性幹細胞の報告例のほとんどがＦ１ハイブリッドマウスに由来するものであることからも明らかなように、Ｃ５７ＢＬ／６等の純系マウスから１倍体ＥＳ細胞株を樹立し、そして安定的に培養することは、Ｆ１ハイブリッドマウス由来のそれよりもさらに困難である。 When conducting forward genetics research using the mouse genome, it is easier to analyze using pure mice. That is, when a haploid ES cell line is established from an F1 hybrid mouse, the ES cell line produced is F2, and when analyzing its phenotype, the influence of genetic background must be considered. This is because it disappears. However, as is clear from the fact that most of the previous reports of female haploid embryonic stem cells and male haploid embryonic stem cells are derived from F1 hybrid mice, it is one time from pure mice such as C57BL / 6. Establishing and stably culturing somatic ES cell lines is even more difficult than that from F1 hybrid mice.
　一方、上述の通り、本発明によれば、Ｃ５７ＢＬ／６由来の１倍体ＥＳ細胞もその１倍性等を維持しながら培養することが可能である。したがって、本発明は、フォワードジェネティックス研究等において極めて有用である。 On the other hand, as described above, according to the present invention, it is possible to culture C57BL / 6-derived haploid ES cells while maintaining the haploidity thereof. Therefore, the present invention is extremely useful in forward genetics research and the like.
　さらに、上述の通り、本発明によれば、１倍体ＥＳ細胞からその１倍性を維持したまま分化した細胞を得ることができ、さらには該分化細胞においてもその１倍性を維持することができる。したがって、本発明は、多種多様な細胞、組織、臓器等の提供が強く求められている再生医療や創薬開発等においても有用である。 Furthermore, as described above, according to the present invention, a differentiated cell can be obtained from a haploid ES cell while maintaining its haploidity, and further, the uniploidy can be maintained in the differentiated cell. Can do. Therefore, the present invention is also useful in regenerative medicine, drug discovery development, and the like where there is a strong demand for providing a wide variety of cells, tissues, organs, and the like.

Claims (5)

  1. 　１倍体胚性幹細胞の培養方法であって、Ｃｄｃ２を恒常的に活性化する条件下にて該細胞を培養する工程を含む方法。 A method for culturing haploid embryonic stem cells, the method comprising culturing the cells under conditions that constantly activate Cdc2.
  2. 　非ヒトキメラ動物を作製する方法であって、
    （i)Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
    （ii)該１倍体胚性幹細胞を初期胚に導入し、キメラ胚を作製する工程と、
    （iii)該キメラ胚を非ヒト動物の母胎内に移植し、発生させ、非ヒトキメラ動物を得る工程とを含む方法。     A method for producing a non-human chimeric animal comprising:
    (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
    (Ii) introducing the haploid embryonic stem cells into an early embryo to produce a chimeric embryo;
    (Iii) transplanting the chimeric embryo into the maternal womb of a non-human animal and generating it to obtain a non-human chimeric animal.
  3. 　前記非ヒトキメラ動物が、生殖細胞が前記１倍体胚性幹細胞に由来する非ヒト生殖系列キメラ動物である、請求項２に記載の方法。 The method according to claim 2, wherein the non-human chimeric animal is a non-human germ line chimeric animal in which germ cells are derived from the haploid embryonic stem cells.
  4. 　１倍体分化細胞を作製する方法であって、
    （i)Ｃｄｃ２を恒常的に活性化する条件下にて１倍体胚性幹細胞を培養する工程と、
    （ii)該１倍体胚性幹細胞を分化誘導する工程とを含む方法。     A method for producing a haploid differentiated cell comprising:
    (I) culturing haploid embryonic stem cells under conditions that permanently activate Cdc2;
    (Ii) a step of inducing differentiation of the haploid embryonic stem cells.
  5. 　Ｃｄｃ２活性化剤を有効成分とする、１倍体胚性幹細胞又は１倍体分化細胞の１倍性を維持するための薬剤。 An agent for maintaining the haploidity of haploid embryonic stem cells or haploid differentiated cells, comprising a Cdc2 activator as an active ingredient.
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