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WO2017175876A1 - Méthode de rétablissement de cellules souches - Google Patents

Méthode de rétablissement de cellules souches Download PDF

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WO2017175876A1
WO2017175876A1 PCT/JP2017/014883 JP2017014883W WO2017175876A1 WO 2017175876 A1 WO2017175876 A1 WO 2017175876A1 JP 2017014883 W JP2017014883 W JP 2017014883W WO 2017175876 A1 WO2017175876 A1 WO 2017175876A1
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cells
mammalian species
cell
culture medium
stem cells
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Japanese (ja)
Inventor
遠藤　仁司
恭光 長尾
豊 花園
薫 冨永
司 大森
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Jichi Medical University
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Jichi Medical University
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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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a method for reestablishing stem cells by a multi-step process.
  • organ primordia or organ stem cells from iPS cells (artificial pluripotent stem cells) / ES cells (embryonic stem cells).
  • iPS cells artificial pluripotent stem cells
  • ES cells embryonic stem cells
  • Mouse and rat ES cells are generally established as naive pluripotent stem cells, and there are several reports on the production of chimeric animals by blastocyst complementation.
  • iPS / ES cells of animals such as monkeys and humans have a prime-type feature in which colonies are flat, and if this is transplanted into a heterologous blastocyst, a chimera can be formed, Even if it can be formed, it is a very small contribution rate.
  • the importance of the method for re-establishing stem cells having the ability to form chimeras is further increased from the viewpoint of increasing the success rate of production of chimeric embryos / chimeric animals.
  • Non-patent Document 1 An example of establishing high-quality iPS cells / ES cells by improving the medium such as addition of environmental factors has been reported (Non-patent Document 2).
  • Non-patent Document 2 an example of establishing high-quality iPS cells / ES cells by improving the medium such as addition of environmental factors has been reported (Non-patent Document 2).
  • these reported examples are techniques for producing chimera-forming iPS cells / ES cells, the produced iPS cells / ES cells are heterogeneous cell populations, and cells having no chimera-forming ability are mixed. It has not been evaluated enough to deny the possibility of being.
  • These techniques can be said to be techniques for increasing the ratio of cells capable of chimera formation among iPS cells / ES cells.
  • Stem cells such as iPS cells and ES cells are generally formed from a cell population having a characteristic with a certain degree of differentiation. For example, it has been reported that cells having totipotency are contained in a very small fraction of ES cells as a whole (Non-patent Document 13).
  • Patent Document 1 include two types including pluripotent cells in which a specific gene is mutated or deleted and the function involved in the gene is lost, and other pluripotent cells other than the pluripotent cell.
  • a method for producing a chimeric animal comprising injecting the above-described cells into an animal host embryo is disclosed.
  • Patent Document 1 improves the proliferative ability of other mouse ES cells by co-culturing ES cells established from mouse embryos that cannot form germ cells with other mouse ES cells, for example, ES cells that have been genetically modified. It states that we have found that we can do it.
  • the present invention relates to a method for re-establishing stem cells, re-established stem cells, and utilization of re-established stem cells.
  • the present invention includes the following aspects.
  • a method for re-establishing a stem cell having the ability to form a chimera by a multi-step process comprising co-culturing a first type of pluripotent stem cell or a multipotent stem cell with a host embryo of a second type of cell having different culture conditions from the first stem cell At least twice, where the culture medium is suitable for the first type of stem cells and the second type of cells in the step of co-culturing the first type of stem cells with the host embryo of the second type of cells.
  • a mixed medium of different culture media and Re-establishing a stem cell having the ability to form a chimera, wherein the re-established stem cell having the ability to form a chimera is a culture medium suitable for the second type of cell, a culture medium suitable for the first type of stem cell, or
  • the culture medium suitable for the first type of stem cells and the mixed medium of the culture medium suitable for the second type of cells can be cultured in any culture medium. Said method.
  • a method for re-establishing a stem cell capable of forming a chimera between different species by a multi-step process Performing at least two re-establishment steps comprising co-culturing pluripotent stem cells or multipotent stem cells from a first mammalian species with a host embryo from a second mammalian species;
  • the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species.
  • a mixed medium of culture media suitable for the species and Re-establishing a stem cell capable of forming a chimera between different species, wherein the stem cell having the ability to form a chimera between different species is a culture medium suitable for the second mammalian species, the first mammalian species Or a culture medium suitable for the first mammalian species and a mixed culture medium suitable for the second mammalian species can be used. Said method.
  • step (ii) and / or step (v) with a host embryo derived from a second mammalian species after co-culturing in a mixed medium, the medium is a culture medium suitable for the first mammalian species. 4. The method according to aspect 3, further comprising performing co-culture after changing to
  • the pluripotent stem cells are selected from the group consisting of: ES cells, induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), and germ cells (EG cells), and multipotent
  • the stem cell is selected from the group consisting of: trophoblast stem cell (TS cell), epiblast stem cell (EpiS cell), pluripotent germ cell (mGS cell), hematopoietic stem cell, neural stem cell and mesenchymal stem cell
  • TS cell trophoblast stem cell
  • EpiS cell epiblast stem cell
  • mGS cell pluripotent germ cell
  • hematopoietic stem cell neural stem cell and mesenchymal stem cell
  • step (ii) and step (v) the cells of step (i) and step (iv) are microinjected or aggregated into fertilized egg-derived embryos or tetraploid embryos derived from the second mammalian species, respectively.
  • a culture medium suitable for the first mammalian species comprises an inhibitor selected from the group consisting of glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin dependent kinase inhibitor.
  • a culture medium suitable for the second mammalian species is free of inhibitors selected from the group consisting of glycogen synthase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin dependent kinase inhibitors
  • Embodiment 19 capable of being cultured in a medium suitable for a second mammalian species; and, having one or more characteristics selected from the group consisting of being easily synchronized with a host embryo derived from a second mammalian species A cell according to 1.
  • a method for evaluating efficacy or pathophysiology using cells comprising the following: (I) Step of obtaining cells:
  • the cells are (A) stem cells having the ability to form chimera re-established by the method according to any one of embodiments 1 to 17, or (B) embodiments 1 to A somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating a stem cell having the ability to form a chimera re-established by the method according to any one of claims 17, wherein the somatic stem cell, organ Progenitor cells or somatic cells are obtained by any of the following methods (a) to (c): (A) A chimeric embryo or a chimeric fetus / fetus is produced from a stem cell having the ability to form a chimera re-established by the method according to any one of Embodiments 1 to 17, and a body derived from the chimeric embryo or chimeric fetus / fetus Obtaining
  • the multistage re-establishment method of the present invention has made it possible to efficiently obtain high-quality stem cells having the ability to form chimeras.
  • the present invention makes it possible to efficiently obtain high-quality stem cells that can form chimeras between different species.
  • the method of the present invention can be widely applied to different stem cells of the same species with different culture conditions.
  • FIG. 1 shows a schematic diagram of one embodiment of the multi-stage re-establishment method of the present invention.
  • the medium is appropriately changed step by step.
  • the embryo culture medium in the host embryo is optimized.
  • FIG. 2 is a photomicrograph of monkey ES cells before and after multi-stage re-establishment.
  • Monkey ES cells subcultured in monkey ES medium were observed with a fluorescence microscope (Axio Observer D1 system, Carl Zeiss) using an X10 or X20 objective lens.
  • ES cells after two-stage re-establishment showed green fluorescence and formed colonies of good shape and size.
  • FIG. 4 is a result of examining a HE-stained tissue image of teratoma formed by cell transplantation of monkey ES cells.
  • FIG. 5 shows that the teratoma is derived from monkey ES cells that have been re-established in multiple stages. It is the result of detecting the presence of the gene ⁇ 2 microglobulin ( ⁇ 2MG) by PCR.
  • FIG. 7A is a graph showing the gene expression levels of 4 endogenous factors of Yamanaka and NANOG before and after re-establishment.
  • FIG. 7A shows the FPKM value of each gene.
  • FIG. 7B FIG.
  • FIG. 7B is a diagram showing the gene expression levels of 4 endogenous factors of Yamanaka and NANOG before and after re-establishment.
  • FIG. 7B shows changes in gene expression levels before and after re-establishment as log 2 values.
  • FIG. 7C is a graph showing the expression levels of endogenous genes of Yamanaka 4 factor and NANOG before and after re-establishment.
  • FIG. 7C summarizes each FPKM value, log 2 value, and presence / absence of significance.
  • FIG. 8 FIG.
  • stem cells are cells having self-renewal ability and differentiation ability.
  • the self-replicating ability means the ability to replicate cells having the same ability as the self, and means the ability to differentiate into a plurality of cells having functions different from the differentiation ability.
  • Pluripotent stem cell is a stem cell that has the ability to differentiate into all cell types that form an individual.
  • Pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells) and the like.
  • multipotent stem cell is a stem cell that has the ability to differentiate into a plurality of cell types.
  • Pluripotent stem cells include trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, mesenchymal stem cells, etc. .
  • pluripotent stem cells and the like are described, the expression is understood to include pluripotent stem cells and multipotent stem cells.
  • Chimera embryos / chimera animals are produced by transplanting pluripotent stem cells or the like into embryos derived from fertilized eggs of a host (another individual) or artificially engineered embryos (tetraploid embryos, etc.) prepared by developmental engineering. Is done.
  • a chimeric embryo is produced from all or part of a host embryo body in which transplanted pluripotent stem cells and the like are produced.
  • a chimeric animal is produced by transplanted pluripotent stem cells and the like becoming part of an born individual.
  • a stem cell has “chimera-forming ability” or “capable of chimera formation” means that the stem cell is transplanted into a fertilized egg-derived embryo of a host, an artificially engineered embryo produced by developmental engineering, or the like. Means that the stem cells have the ability to become all or part of the resulting host embryo body, or part of various organs in the born individual.
  • the term “a chimera-forming ability” or “capable of chimera” is used when the stem cell is transplanted into a host fertilized egg-derived embryo or an artificially manipulated embryo.
  • a stem cell has the ability of a pluripotent stem cell or the like to form a part of an inner cell mass (ICM), that is, the ability to contribute to ICM.
  • ICM inner cell mass
  • chimera-forming ability does not mean only the ability to form a “individual” chimera.
  • species from which the transplanted stem cells are derived and the species of the host are different, it is particularly said that “chimera can be formed between different species”.
  • heterologous or “different species” means different animal species. Unless otherwise stated, “heterologous” or “different species” refers to different animal species at the genus level or higher.
  • “same species” means belonging to the same animal species. Unless otherwise stated, the range of “same species” includes not only those belonging to the same animal species at the species level but also “same species heterologous” belonging to the same animal species at the genus level.
  • pluripotent stem cell or the like is “naive type”.
  • a pluripotent stem cell or the like is “naive type” means that a pluripotent stem cell or the like forms a dome-shaped colony, has a chimera-forming ability, and can differentiate into a germ line such as a sperm or egg. It means having one or more, preferably two or more, more preferably all three properties selected from the group consisting of.
  • host embryo means an embryo of a host animal into which a pluripotent stem cell is transplanted when producing a chimeric embryo / chimeric animal.
  • the present invention provides a method for re-establishing stem cells having the ability to form chimeras by a multi-step process.
  • a re-establishing step comprising co-culturing a first type of pluripotent stem cell or a multipotent stem cell with a host embryo of a second type of cell having different culture conditions from the first stem cell At least twice, where the culture medium is suitable for the first type of stem cells and the second type of cells in the step of co-culturing the first type of stem cells with the host embryo of the second type of cells.
  • a mixed medium of different culture media and Re-establishing a stem cell having the ability to form a chimera, wherein the re-established stem cell having the ability to form a chimera is a culture medium suitable for the second type of cell, a culture medium suitable for the first type of stem cell, or
  • the culture medium suitable for the first type of stem cells and the mixed medium of the culture medium suitable for the second type of cells can be cultured in any culture medium. Including that.
  • the re-establishment step including co-culture with the second-type host embryo is performed at least twice.
  • the medium used for the re-establishment step is changed from the culture medium suitable for the first type of stem cells to the second type. It is characterized by changing the medium composition in a multistage manner to a culture medium suitable for cells.
  • the culture medium is composed of a culture medium suitable for the first type stem cells and a culture medium suitable for the second type cells.
  • Use mixed media The mixed medium to be used is appropriately optimized depending on the stage of re-establishment.
  • the obtained re-established cells have a chimera-forming ability, and are suitable for the second type of cells, the first type of stem cells, or the first type of stem cells. It is characterized in that it can be cultured in any one of a culture medium and a mixed culture medium suitable for the second type of cells. Preferably, a culture medium suitable for the second type of cells is used.
  • the first-type cell and the second-type cell have substantially different cell culture conditions.
  • a case where the species from which the first type cell and the second type cell are derived is different from each other is included.
  • embryonic stem cells and induced pluripotent stem cells (iPS cells) of “pluripotent stem cells”, cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells), etc.
  • multipotent stem cells trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, and Mesenchymal stem cells and the like have different culture conditions depending on the type of each cell.
  • ES cells and iPS cells, EG cells, TS cells, EpiS cells, and mGS cells have different culture conditions depending on the type of each cell.
  • the present invention can be provided.
  • the present invention provides a method for re-establishing stem cells having the ability to form chimera between different species by a multi-step process.
  • the method of the present invention comprises: Performing at least two re-establishment steps comprising co-culturing pluripotent stem cells or multipotent stem cells from a first mammalian species with a host embryo from a second mammalian species;
  • the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species.
  • a mixed medium of culture media suitable for the species and re-establishing a stem cell capable of forming a chimera between different species wherein the stem cell capable of forming a chimera between different species is
  • a culture medium suitable for a second mammalian species, a culture medium suitable for a first mammalian species, or a culture medium suitable for a first mammalian species and a culture suitable for a second mammalian species Culture medium Can be cultured in any culture medium of Including that.
  • the re-establishment step including co-culture with the second-type host embryo is performed at least twice.
  • the medium used for the re-establishment step is changed from the culture medium suitable for the first mammalian species to the second. It is characterized in that the medium composition is changed in a multistage manner to a culture medium suitable for the mammalian species.
  • the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Use a mixed culture medium suitable for the species.
  • the mixed medium to be used is appropriately optimized depending on the stage of re-establishment.
  • the obtained re-established cells have the ability to form chimeras between different species, and the culture medium suitable for the second mammalian species, the culture medium suitable for the first mammalian species, or the culture medium It is characterized in that it can be cultured in any one of a culture medium suitable for one mammalian species and a mixed culture medium suitable for a second mammalian species.
  • a culture medium suitable for the second mammalian species is used.
  • the method of the invention comprises, in one aspect, the following steps.
  • (Ia) co-culturing a pluripotent stem cell from a first mammalian species or a multipotent stem cell with a high quality pluripotent stem cell from a second mammalian species Obtaining a population of cells, wherein the culture medium is a culture medium suitable for the first mammalian species; or
  • (ib) Pluripotent stem cells or multipotent from the first mammalian species Combining multipotent stem cells with high quality pluripotent stem cells from a second mammalian species to obtain a population of cells;
  • a cell comprising a pluripotent or multipotent stem cell derived from a first mammalian species and a high quality pluripotent stem cell derived from a second mammalian species Get a group.
  • Pluripotent stem cells or multipotent from the first mammalian species A multipotent stem cell is combined with a high quality pluripotent stem cell derived from a second mammalian species to obtain a cell population.
  • the pluripotent stem cells or multipotent stem cells derived from the first mammalian species are co-cultured with high quality pluripotent stem cells derived from the second mammalian species (step (ia)).
  • Step (ib) comprises combining pluripotent stem cells or multipotent stem cells from a first mammalian species with high quality pluripotent stem cells from a second mammalian species, in particular co-culture. Without being done, it is an embodiment in which it is directly subjected to co-culture with the host embryo in step (ii).
  • the type of mammal is not particularly limited.
  • the first mammalian species is selected from the group consisting of dogs, cats, horses, cows, goats, sheep, monkeys and humans. Preferably, they are monkeys or humans.
  • the first mammalian species is a species belonging to the primate.
  • Primarymates are also called monkeys in the eyes of the mammals, and the nasal monkeys (original monkeys) including lemurs and loris lower eyes, and other straight-nosed monkeys (true monkeys) ).
  • humans are one of the primates (monkeys), but generally a general term that excludes humans from monkeys is called “monkey”. The same applies to the present specification.
  • the medium composition used in the re-establishment process is changed in multiple stages from a culture medium suitable for the first mammalian species to a culture medium suitable for the second mammalian species. It is characterized by.
  • the second mammalian species it is desirable for the second mammalian species to have a culture medium that is substantially different from a culture medium suitable for the first mammalian species.
  • the second mammalian species is preferably selected from the group consisting of mice, rats, rabbits and pigs.
  • the first mammalian species is a monkey and the second mammalian species is a mouse.
  • the pluripotent stem cell derived from the first mammalian species is not a human ES cell and the second mammalian species is not a human.
  • the “pluripotent stem cell” is selected from the group consisting of ES cells and induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), and germ cells (EG cells).
  • iPS cells induced pluripotent stem cells
  • ntES cells cloned embryo-derived ES cells
  • EG cells germ cells
  • “Multipotent stem cells” include trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, and mesenchymal stem cells, Selected from the group consisting of
  • the “pluripotent or multipotent stem cell” from the first mammalian species is an ES cell or iPS cell.
  • High-quality pluripotent stem cells derived from the second mammalian species include ES cells, iPS cells, or the inner cell mass of these cells. “High quality pluripotent stem cells” are preferably ES cells. The high-quality pluripotent stem cells are preferably naive pluripotent stem cells.
  • a cell aggregate is formed from the obtained cell population, and the first mammalian species
  • the cell group containing stem cells derived from derived pluripotent stem cells or multipotent stem cells may be selected.
  • This step is derived from a pluripotent or multipotent stem cell from a first mammalian species that colonizes (especially na ⁇ ve colony formation) with a high quality pluripotent stem cell from a second mammalian species
  • the stem cell may be expressed as a “stem cell contributing to colony formation”.
  • Pluripotent stem cells derived from the first mammalian species can be labeled with an appropriate selection marker before selection, and colonies containing the selection marker can be selected.
  • a high-quality pluripotent stem cell derived from a second mammalian species is labeled with an appropriate selection marker, and colonies that do not contain the selection marker are identified as pluripotent stem cells derived from the first mammalian species. It can also be identified as a cell group containing.
  • selection markers include: wedge orange (huKO), green fluorescent protein (GFP), Clover, DsRed, mCherry, luciferase, LacZ, neomycin resistance gene, puromycin resistance gene, hygromycin B resistance gene, blasticidin Resistance genes, zeocin resistance genes, DT-A genes, HSV-TK genes, and the like.
  • the identification can be performed by identification by fluorescence, luminescence, staining, or the like, or by drug selection by a drug resistance gene or the like.
  • step (ii) of the above method the cell group of step (i) is co-cultured with a host embryo derived from a second mammalian species.
  • a chimeric embryo is produced by co-culturing the cell population of step (ii) with a host embryo derived from a second mammalian species.
  • the chimeric embryo is cultured until the initial blastocyst stage where an internal cell mass (ICM) is obtained.
  • the culture medium is a culture medium suitable for the first mammalian species. And using a mixed medium of culture medium suitable for the second mammalian species.
  • the cocultivation in this step is performed by microinjecting or aggregating the cell group of step (i) into a host embryo derived from a second mammalian species and coculturing.
  • Microinjection is a method for producing a chimeric embryo by transplanting cells (such as stem cells) into a host embryo.
  • Aggregation is a method of producing a chimeric embryo by using an early embryo up to the morula stage as a host embryo and contacting and assembling stem cells and the like.
  • the co-culture of step (ii) is performed by microinjecting the cell population of step (ii) into a host embryo derived from a second mammalian species and co-culturing.
  • the host embryo is not particularly limited. Artificially manipulated embryos such as embryos derived from fertilized eggs and tetraploid embryos (tetraploid embryos) are included. In a preferred embodiment, the host embryo is a fertilized egg-derived embryo or a tetraploid embryo early embryo.
  • Early embryo means an embryo from a two-cell stage embryo to a blastocyst stage embryo.
  • the tetraploid embryo is an embryo prepared by electrically fusing wild type 2-cell blastomeres.
  • a chimeric embryo is produced by co-culturing with a pluripotent stem cell using a tetraploid embryo, the tetraploid cell cannot contribute to the embryo body itself, but can contribute to extraembryonic tissues such as placenta. Therefore, the embryo body or individual obtained is almost 100% derived from the pluripotent stem cells.
  • step (ii) of the above method comprises co-culturing the cell population of step (i) by microinjection or aggregation into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species. Done. More preferably, step (ii) of the above method is performed by microinjecting the cell population of step (i) into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species and co-culturing. Is called.
  • step (iii) the inner cell mass is separated from the host embryo co-cultured in step (ii). Separation of the inner cell mass can be performed using a technique known to those skilled in the art. Preferably, the inner cell mass is separated by microscopic surgery or immunosurgery. Microsurgery is an excellent way to remove the inner cell mass while observing the state of the embryo. On the other hand, immunosurgery (Solter, D. and Knowless, BB, Proc. Nat. Acad. Sci. USA, 72 (12): 5099-5102 (1975)) is more mechanically damaged than microscopic surgery. Less, it is excellent for isolating the inner cell mass of blastocysts. Either may be used.
  • step (iv)-(vi) the re-establishment step of step (i) (specifically, step (ia))-step (iii) is repeated to perform the second re-establishment step.
  • the re-establishment step including co-culture with the second-type host embryo is performed at least twice.
  • the medium used for the re-establishment step is changed from the culture medium suitable for the first mammalian species to the second. It is characterized in that the medium composition is changed in a multistage manner to a culture medium suitable for the mammalian species.
  • the mixed medium to be used is appropriately optimized depending on the stage of re-establishment.
  • the culture medium is a culture medium suitable for the second mammalian species, a culture medium suitable for the first mammalian species, or a culture medium suitable for the first mammalian species and the second culture species.
  • the mixed culture media suitable for the mammalian species Preferably, it is a culture medium suitable for the second mammalian species.
  • steps (v) to (vii) may be repeated for the stem cells re-established in step (vii).
  • step (iii) and step (vi) When the inner cell mass separated from the host embryo in step (iii) and step (vi) is cultured in step (iv) and step (vii), the inner cell mass obtained in step (vi) Cloning of pluripotent stem cells derived from mammalian species or stem cells derived from multipotent stem cells may be performed.
  • Cloning of pluripotent stem cells and the like from the inner cell mass can be performed using techniques known to those skilled in the art. That the cloned cell is a pluripotent stem cell derived from the first mammalian species or a stem cell derived from a multipotent stem cell is, for example, labeled with an appropriate selection marker before step (ii). Can be identified. Alternatively, before performing step (ii), the host embryo is labeled with an appropriate selection marker, and cells that do not contain the selection marker are identified as pluripotent or multipotent stem cells derived from the first mammalian species. And can be cloned.
  • selection markers include: wedge orange (huKO), green fluorescent protein (GFP), Clover, DsRed, mCherry, luciferase, LacZ, neomycin resistance gene, puromycin resistance gene, hygromycin B resistance gene, blasticidin Resistance genes, zeocin resistance genes, DT-A genes, HSV-TK genes, and the like.
  • the identification can be performed by identification by fluorescence, luminescence, staining, or the like, or by drug selection by a drug resistance gene or the like.
  • pluripotent stem cells labeled with wedge orange it can be carried out as follows. Remove the blastocyst together with the blastocyst under a microscope, or remove the part by microscopic surgery. Alternatively, only the inner cell mass is taken out by microscopic surgery or immunosurgery, and transferred and cultured separately on feeder cells in a 4-well plate. From the inner cell mass that has proliferated after seeding, only those in the mosaic state that have been confirmed to have red fluorescence with a fluorescence microscope are visually selected and picked up using the proliferation ability and morphology as indicators. In addition, colonies and cells showing the form of trophoblast-like cells, epithelium-like cells, endoderm-like cells, etc.
  • the cell mass is dispersed by trypsin treatment, decomposed into several new cell masses, transferred to newly prepared feeder cells, and cultured separately. After sowing, colonies having a good state are selected from newly formed colonies by confirming that they have red fluorescence with a fluorescence microscope. Next, when the cells are dispersed, the cells are further dispersed into a cell cluster having a small number of cells, and the process is repeated several times to obtain colonies containing only pluripotent cells.
  • step (iv) When re-establishing the second stage of step (iv), high-quality pluripotent stem cells derived from the second mammalian species may or may not be added, as in step (i). good. This is because mouse ES cells were established together from mouse ICM through the first-stage re-establishment of steps (i)-(iii) and already in a co-culture state.
  • the re-establishment step including co-culture with the second-type host embryo is performed at least twice.
  • the medium used for the re-establishment step is selected from a culture medium suitable for the first mammalian species. It is characterized by changing the medium composition in a multi-step manner to a culture medium suitable for the second mammalian species.
  • the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species.
  • the mixed medium to be used is appropriately optimized depending on the stage of re-establishment.
  • a suitable medium according to the species of the first mammal that is the donor species.
  • the first mammalian species is a human
  • primate ES / iPS cell culture medium RCHEMD001, RCHEMD001A, RCHEMD001B, etc. sold by REPROCELL without limitation. Yes (https://www.reprocell.com/products/research/hes-ipsc/medium).
  • the culture medium for human contains, for example, bFGF.
  • TeSR2 ST-CELL Technologies
  • ES / iPS cells sold by REPROCELL. 05860, ST-05880
  • TeSR2 is a medium for ES / iPS cells for primates including humans, characterized by feeder cell-free, non-human-derived component-free, and poisonous and deleterious substances-free.
  • the medium for monkeys and the medium for humans are not particularly distinguished, and a medium that can be used for either one can be used for the other.
  • the culture medium for monkeys and the culture medium for humans may be appropriately added with bFGF or TGF ⁇ , or BMPi, ROCKi, BRAFi, SRCi, activin or the like.
  • a medium having the following composition can be used when the first mammalian species is a monkey or a human.
  • DMEM / F12 Neurobasal (Invitrogen) N2 supplement (Invitrogen) B27 supplement (Invitrogen) GlutaMAX (Invitrogen) Non-essential amino acid (NEAA) solution (Invitrogen) 2-mercaptoethanol (2-ME) (Invitrogen) Penicillin / Streptomycin (Invitrogen) Bovine serum albumin (BSA) (Sigma) PD0325901 (Stemgent) MEK1 / 2 inhibitor CHIR99021 (Stemgent) GSK3 inhibitor Forskolin (Sigma) AC (adenylate cyclase) activator Human LIF (hLIF) (Millipore) Kenpaulone (Sigma) Cyclin-dependent kinase (CDK) inhibitor Go6983 (Sigma) PKC inhibitor
  • the culture medium of human or monkey iPS cells for example, those shown in Table 1 below can be used.
  • a suitable medium according to the species of the second mammal that is the donor species.
  • the second kind of mammal is a mouse ES cell or a naive pig iPS cell
  • a culture solution having the following composition can be used.
  • Example of mouse ES cell culture composition 80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM pyruvic acid solution, 0.1 mM 2-mercaptoethanol, 1 ⁇ MEM non-essential amino acid solution, 10 3 U / mL mLIF (Wako) or Medium containing 1xrhLIF (Wako)
  • Example of medium composition of naive porcine iPS cells 82% (v / v) D-MEM, 15% (v / v) FCS, 0.1 mM 2-mercaptoethanol, 1 ⁇ MEM non-essential amino acid solution, 1 ⁇ GlutaMAX TM -I (GIBCO), 1 ⁇ rhLIF (Wako), 10 ⁇ M Forskolin Medium containing
  • a suitable culture medium for the first mammalian species eg, primates such as humans, monkeys, etc.
  • the second mammal that provides the host embryo.
  • a culture medium suitable for various species pigs, mice, etc.
  • An example of the composition of the culture medium suitable for the first mammalian species and the example composition of the culture medium suitable for the second mammalian species were compared.
  • the culture medium suitable for the first mammalian species is preferably glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin. Inhibitors selected from the group consisting of dependentase inhibitors.
  • the culture medium suitable for the second mammalian species is selected from the group consisting of glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin dependent kinase inhibitor. Contains no inhibitor.
  • the medium composition used in the re-establishment process is changed in multiple stages from a culture medium suitable for the first mammalian species to a culture medium suitable for the second mammalian species.
  • the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species.
  • stem cells derived from the first mammalian species are difficult to efficiently co-culture only with a culture medium suitable for culturing the second mammalian species. Therefore, in the present invention, a mixed medium in which a culture medium suitable for the first mammalian species is appropriately added to a culture medium suitable for the second mammalian species is used (optimization of the embryo culture medium).
  • a mixed medium in which a culture medium suitable for the first mammalian species is appropriately added to a culture medium suitable for the second mammalian species is used (optimization of the embryo culture medium).
  • a culture medium suitable for the first mammalian species is used as the second mammalian species.
  • a suitable culture medium it is added, but not limited to, preferably in the range of about 10/1-1/10, more preferably in the range of about 2 / 1-1 / 2.
  • the ratio of adding a culture medium suitable for the first mammalian species may be further increased
  • a culture medium suitable for the first mammalian species is possible relative to a culture medium suitable for the second mammalian species, but is not limited, preferably in the range of about 100: 1-1: 100, Those added in a range of preferably about 10: 1-1: 10 can be used.
  • the culture medium is a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species, the presence or absence of serum or serum substitutes, the presence or absence of various inhibitors, the presence or absence of various growth factors, It can be appropriately prepared depending on the presence or absence of other trace components.
  • the medium is a culture medium suitable for the first mammalian species. Further, co-cultivation may be carried out.
  • step (iii) the inner cell mass separated from the host embryo is cultured (step (iv)).
  • the culture medium in step (iv) differs from step (i) in that it is not a culture medium suitable for the first mammalian species but a culture medium suitable for the first mammalian species and a second mammalian species.
  • the mixed culture medium can be used.
  • the culture medium suitable for the first mammalian species is non-limiting, preferably in the range of about 1: 3-3: 1, more preferably relative to the culture medium suitable for the second mammalian species. Can be added in the range of about 1: 2-2: 1.
  • the culture medium after re-establishment is a culture medium suitable for the second mammalian species, a culture medium suitable for the first mammalian species, or a culture medium
  • a culture medium suitable for the first mammalian species or a mixed medium of culture media suitable for the second mammalian species may be used.
  • it is a culture medium suitable for the second mammalian species. That is, the stem cells re-established by the method of the present invention can be adapted to the culture conditions of the recipient.
  • the ratio of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species in the mixed medium can also be appropriately selected.
  • the invention also provides primate animal stem cells re-established from pluripotent or multipotent stem cells.
  • the stem cells of the present invention are as follows: -Uniformized; -Chimera can be formed, especially between different species; • can form cell aggregates; and • has high affinity for the niche environment of the inner cell mass; Having one or more features selected from the group consisting of: Preferably, it has 2 or more and 3 or more of the above properties.
  • Uniform means that the original cell group is re-established by re-establishing it from a group of cells that have multiple properties ranging from a more undifferentiated state to a relatively advanced state. This means that the cell group is made up of cells with more similar properties.
  • a cell aggregate can be formed means that when cultured under the condition of co-culturing different cell groups, it has the ability to form a cell aggregate, or the cells adhere to each other, or the cells are the same. It means that it can exist in the place.
  • “High affinity to the niche environment of the inner cell mass” means that the ability of the stem cells to survive in the niche environment of the inner cell mass, that is, the situation in the vicinity of the periphery, is high.
  • the colony shape of the re-established stem cells showed a clean and large dome shape, and the teratoma formation experiment confirmed the diversity of trilobal differentiation. Furthermore, when the origin of teratomas was confirmed by PCR, the presence of a marker gene introduced into monkey ES cells (first mammalian ES cells) was confirmed.
  • the stem cell of the present invention further includes the following: Can be cultured in a medium suitable for a second mammalian species; and; has one or more characteristics selected from the group consisting of being easily synchronized with a host embryo derived from a second mammalian species. Preferably, it means both of the above two conditions.
  • “Can be cultured in a medium suitable for the second mammalian species” means, but is not limited to, for example, five passages when cultured in a medium suitable for the second mammalian species. It means that it has one of the requirements such as being able to survive, maintaining pluripotency as confirmed by, for example, a teratoma experiment, or maintaining the shape of a dome-shaped colony. To do.
  • “Easy to synchronize with a host embryo derived from a second mammalian species” means that the re-established stem cell of the present invention is a recipient species (second secondary species) that is different from the donor species (first mammalian species).
  • second secondary species that is different from the donor species (first mammalian species).
  • the stem cells having chimera-forming ability re-established by the method of the present invention are not particularly limited, but can be used for the following uses.
  • An organ / organ primordium derived from the stem cell can be formed by producing a chimeric animal by the blastocyst complementation method using the stem cell having the chimera-forming ability of the present invention.
  • Such organs / organ discs are useful in regenerative medicine.
  • a chimeric embryo or a chimeric fetus / fetus is produced by the blastocyst complementation method using the stem cell having the ability to form a chimera of the present invention, and the somatic stem cell, organ progenitor cell and body derived from the chimeric embryo or chimeric fetus / fetus Sex cells can be obtained. Also, organ progenitor cells and somatic cells can be obtained by differentiating somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus in vitro.
  • somatic stem cells, organ progenitor cells, and somatic cells can be obtained by differentiating stem cells having the ability to form chimeras of the present invention in vitro.
  • somatic stem cells, organ progenitor cells and somatic cells are useful for use in drug efficacy evaluation and pathological analysis. Since the stem cell having the chimera-forming ability of the present invention is a high-quality stem cell that maintains the chimera-forming ability, it can efficiently produce or differentiate a chimeric embryo / chimera animal using the cell as a starting cell. .
  • an animal to which the stem cells have been transmitted can be produced by the tetraployless cue method.
  • the tetraploid dress cue method utilizes the fact that, by injecting iPS / ES cells or the like into tetraploid embryos, tetraploid cells develop into the placenta and only iPS / ES cells etc. develop into the individual. (Nagy, A., et al., Development, 110, 815-821 (1990)).
  • Such an animal production method is effective for the preservation, reproduction, and / or maintenance of rare animals such as endangered species, pet mammals such as pets, and useful commercial animals.
  • the present application further provides a medicinal effect using a chimera-forming stem cell obtained by the re-establishment method, or a somatic stem cell, organ progenitor cell or somatic cell obtained by differentiating the chimera-forming stem cell.
  • a method for performing an evaluation or pathological analysis is provided.
  • the method of the present invention includes the following: (I) A step of obtaining a cell: wherein the cell is (A) a stem cell having the ability to form a chimera re-established by the method of the present invention, or (B) the cell according to any one of aspects 1 to 17 A somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating a stem cell having the ability to form a chimera re-established by the method, wherein the somatic stem cell, organ progenitor cell, or somatic cell is It is obtained by any of the following methods (a) to (c): (A) A chimeric embryo or a chimeric fetus / fetus is produced from a stem cell having the ability to form a chimera re-established by the method according to any one of Embodiments 1 to 17, and a body derived from the chimeric embryo or chimeric fetus / fetus Obtaining sex stem cells, organ progen
  • the “stem cell capable of forming a chimera” in (A) is regenerated from a pluripotent stem cell or a multipotent stem cell by the “method for reestablishing a stem cell by a multi-step process”. Can be established.
  • the “somatic stem cell, organ progenitor cell or somatic cell obtained by differentiating a stem cell having chimera-forming ability” in (B) Can be obtained by performing any one of the following methods (a) to (c).
  • the method of (a) is to produce a chimeric embryo or a chimeric fetus / fetus from a stem cell capable of forming a chimera, and obtain a somatic stem cell, organ progenitor cell, or somatic cell derived from the chimeric fetus / fetus. It is.
  • the method for producing a chimeric embryo or a chimeric fetus / fetus can be carried out by the method described for producing a chimeric embryo / chimera animal in the item “Definitions” above.
  • somatic stem cells, organ progenitor cells, or somatic cells from a chimeric embryo or a chimeric fetus / fetus the cells are sorted and established. Sorting and establishment of somatic stem cells, organ progenitor cells, or somatic cells from a chimeric embryo or chimeric fetus / fetus can be appropriately performed by methods commonly used by those skilled in the art.
  • the somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus obtained in (a) are differentiated in vitro to obtain organ progenitor cells or somatic cells.
  • a method for differentiating somatic stem cells in vitro to obtain organ progenitor cells or somatic stem cells can be appropriately performed by methods commonly used by those skilled in the art.
  • the method (c) is to obtain somatic stem cells, organ progenitor cells, or somatic cells by differentiating stem cells having the ability to form chimera in vitro.
  • a method of obtaining somatic stem cells, organ progenitor cells, or somatic cells by differentiating stem cells having the ability to form chimera in vitro is appropriately performed by a method commonly used by those skilled in the art to differentiate pluripotent stem cells in vitro. be able to.
  • the “somatic stem cell, organ progenitor cell, or somatic cell” obtained in step (i) of the above method is related to, for example, the heart, nerve, kidney, liver, pancreas, skeletal muscle, blood cell, etc. Although it is a cell, it is not specifically limited.
  • step (ii) of the above method drug efficacy evaluation or pathological condition analysis is performed using the cells obtained in step (i).
  • the efficacy evaluation or pathological analysis is not particularly limited as long as it is a test method using cells. This can be appropriately carried out by methods usually used by those skilled in the art.
  • Example 1 Multi-stage Re-establishment Method Using ES Cells This example describes a multi-stage re-establishment method using ES cells.
  • Monkey ES cells sESCs
  • sESCs are derived from cynomolgus monkeys (Macaca fascicularis) and are cell lines (CMK6G) (Non-Patent Documents 5 and 6) into which green fluorescent protein (GFP) and neomycin resistance gene (neo) are introduced as marker genes.
  • GFP green fluorescent protein
  • neo neomycin resistance gene
  • a multi-stage re-establishment method was carried out using a temperature-sensitive Sendai virus (SeV18 + hNANOG / TS7dF) (Non-patent Document 7) expressing human nanog.
  • SeV18 + hNANOG / TS7dF temperature-sensitive Sendai virus
  • the ES cells were treated with monkey ES medium [35.9% (v / v) DMEM / F12, 48% (v / v) Neurobasal, 1% (v / v) N2 supplement, 2% (V / v) B27 supplement, 1 mM GlutaMAX, 1% (v / v) non-essential amino acid (NEAA), 0.1 mM 2-mercaptoethanol (2-ME), 1X penicillin / streptomycin, 5 mg / mL bovine serum albumin ( BSA), 1 ⁇ M PD0325901, 1 ⁇ M CHIR99021, 10 ⁇ M forskolin, 20 ng / mL human LIF, 5 ⁇ M Kenpaulone, 5 ⁇ M Go6983] were co-cultured with mouse ES cells. Monkey ES cells that had aggregated together with the cell mass of mouse ES cells were selected (step (i)).
  • the obtained monkey ES cells were microinjected into 40 mouse tetraploid blastocysts (MCH), followed by mouse medium [80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM. pyruvic acid solution, 0.1mM 2-ME, 1xNEAA, 10 3 U / mL mLIF] in the monkeys for ES culture solution 1: 1 early mouse embryo culture conditions was added at a rate of (mwm broth, 5% CO 2 , 37 ° C.) for 1 day (step (ii)).
  • the abnormally shaped blastocysts with monkey ES vesicles other than ICM were removed under a microscope, and only good embryos were separately transferred onto 4-well plate feeder cells and cultured.
  • As the culture solution an ES culture solution for monkeys was used.
  • the cell mass was dispersed by treatment with 0.025% trypsin / 0.1 mM EDTA (step (iii)). Thereafter, the cells were separately transferred onto the newly prepared 4-well plate feeder cells and cultured.
  • the culture solution used was a monkey medium added to a mouse medium at a ratio of 2: 1.
  • the first-stage re-established monkey ES cells can be cultured in a mixed culture solution in which the monkey medium is mixed with the mouse medium, for example, 1: 1, so that the proliferative property and the colony can be improved.
  • the shape was maintained (step (iv)).
  • mice ICM-derived mouse 4NES cells and mouse ES cells contributed by mouse ICM was used for co-culture, and colonies with good growth ability and morphology were selected.
  • a mouse initial embryo culture condition in which a monkey medium was added to a mouse initial embryo culture medium at a ratio of 1:10.
  • Solution, 5% CO 2 , 37 ° C.) for 1 day step (v)).
  • Abnormally shaped blastocysts with monkey ES cells other than ICM are removed under a microscope, and only good embryos are transferred separately onto feeder cells on a 4-well plate, and monkey medium is used for mice. Culturing was performed using a mixed culture solution added to the medium at a ratio of 2: 1.
  • the monkey ES cells after the second stage re-establishment had a significantly increased contribution to ICM than before the re-establishment.
  • the cell mass was dispersed by treatment with 0.025% trypsin / 0.1 mM EDTA (step (vi)). Thereafter, the cells were separately transferred onto the newly prepared 4-well plate feeder cells and cultured.
  • the culture medium used was a monkey medium.
  • the monkey ES cells after the two-stage re-establishment were re-established in a mixed culture medium, but the modified mouse culture medium supplemented with 2 inhibitors [80% (v / v) D-MEM, 20% (v / V) FCS, 1 mM pyruvic acid solution, 0.1 mM 2-ME, 1 ⁇ NEAA, 10 3 U / mL hLIF, 1 ⁇ M PD0325901, 1 ⁇ M CHIR99021] have good growth ability, and have good shape and size of colonies Formation was possible (the upper part of FIG. 3).
  • Example 2 Examination of pluripotency of ES cells after two-stage re-establishment
  • pluripotency of monkey ES cells after two-stage re-establishment was examined in a teratoma formation experiment.
  • neuroepithelial tissue and glial tissue exhibiting Rosetta structure as ectoderm
  • neuroepithelial tissue containing melanin pigment lumen lined with pseudostratified columnar epithelium or single layer columnar epithelium as endoderm Structured epithelial tissue, fibrous connective tissue and muscle tissue producing collagen as mesoderm are observed (middle and bottom in FIG. 3).
  • ectoderm, mesoderm, and endoderm-derived tissues were formed from monkey ES cells after two-stage re-establishment.
  • Example 3 Confirmation of Teratoma Derived from Monkey ES Cells
  • the origin of teratoma formed in Example 2 was indeed monkey ES cells.
  • DNA was extracted from teratoma tissue, and detection of neo gene derived from monkey ES cells and ⁇ 2 microglobulin ( ⁇ 2MG), which is a monkey-derived gene, was attempted by PCR.
  • ⁇ 2MG ⁇ 2 microglobulin
  • neo neomycin resistance gene
  • PCR neomycin resistance gene
  • 35 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed using the following primer sets of SEQ ID NOS: 1 and 2 and rTaq.
  • Nested PCR was used for detection of the monkey ⁇ 2 microglobulin ( ⁇ 2MG) gene.
  • ⁇ 2MG microglobulin
  • 30 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed using the following primer sets of SEQ ID NOS: 3 and 4 and rTaq. 5′-CAG GTT TAC TCA CGT CAT CCA G-3 ′ (SEQ ID NO: 3) and 5′-GGT TCA CAC GGC AGG CAT ACT C-3 ′ (SEQ ID NO: 4)
  • telomeres The specificity of PCR was high, and the neo gene and monkey ⁇ 2MG gene were not detected in the DNAs of the negative control mouse liver and B6 mouse tail, but were detected only in teratoma tissues and monkey ES cells after re-establishment (FIG. 5). ). From the above, it was confirmed that teratomas are derived from monkey ES cells carrying the neo gene. That is, it was shown that the re-established monkey ES cells have pluripotency by forming teratomas differentiated into trilobes.
  • the multi-step re-establishment method is effective in forming a dome-shaped colony with a beautiful shape in primate pluripotent stem cells and maintaining pluripotency with trilobal differentiation ability. It was.
  • the fact that pluripotent stem cells after multi-stage re-establishment have acquired a trait that can be cultured in a medium for mice is advantageous in the formation of heterologous chimeric embryos such as mice, and the maintenance and maintenance of pluripotent stem cells. This is advantageous in terms of amplification and cost.
  • Example 4 Hierarchical clustering of gene expression profiles in cells before and after re-establishment Three culture dishes of monkey ES cells (sES cell) before re-establishment, monkey ES cells (Re-sES cell) after re-establishment RNA was extracted from each of three culture dishes.
  • RNA extracted from each cell was subjected to RNA-Seq using Illumina HiSeq 2000 and mapped in the rhesus monkey transcriptome pipeline. As a result, 18,339 genes were identified.
  • the expression level of the gene tested by the combination of comparison between samples is indicated by FPKM (Fragments Per Kilobase of exon per Million mapped fragments), clustered by the group average method, and the distance was measured by the canberra algorithm .
  • FIG. 6 shows the results of hierarchical clustering of gene expression profiles in each cell before and after re-establishment of monkey ES cells.
  • the gene expression profiles of cells before and after re-establishment were roughly grouped into two groups. That is, it was revealed that the gene expression profile of cells after re-establishment shows a specific gene expression pattern, unlike that before re-establishment.
  • Example 5 Expression of Yamanaka 4 Factor and NANOG in Re-Established Monkey ES Cells
  • Monkey ES cells before re-establishment obtained in Example 4 (sES1, sES2, sES3), and monkey ES cells after re-establishment (Re_sES1) , Re_sES2, Re_sES3) of monkey endogenous genes (PAU5F1 (Oct4), KLF4, SOX2, MYC) corresponding to Yamanaka 4 factors and NANOG genes important for maintaining pluripotency before and after re-establishment. Changes were examined based on changes in FPKM values. The results are shown in FIG. FIG. 7A shows FPKM values of each gene, and FIG.
  • FIG. 7B shows changes in gene expression levels before and after re-establishment as log 2 values.
  • FIG. 7C summarizes each FPKM value, log 2 value, and presence / absence of significance.
  • PAU5F1 Oct4
  • KLF4 KLF4
  • SOX2 SOX2
  • MYC MYC
  • Example 6 Gene Expression Pattern in Re-established Monkey ES Cells From the results of RNA-Seq obtained in Example 6, naive pluripotency, core pluripotency, prime pluripotency
  • FIG. 8 shows the results of comparing the expression of genes related to the characteristics of sex (primed primipotency) and mesoderm / primitive streak before and after the re-establishment of monkey ES cells.
  • the upper row shows the FPKM value of each gene, and the lower row shows the change in gene expression level before and after re-establishment as a log 2 value.
  • naive pluripotency such as KLF4, KLF5, DPPA3, ZFP42, TFCP2L1 and PRDM14, NANOG, SOX2, POU5F1, GDF3, SALL4, UTF4 core, etc.
  • the expression level of genes related to the characteristics of core pluripotency has increased.
  • the expression levels of LEF1 and CER1 related to the characteristics of primed pluripotency and the gene expression levels of EOMES and MSX1 related to the characteristics of mesoderm / primitive streak decreased. From the above, it is understood that monkey ES cells have become a higher quality pluripotent stem cell state by re-establishment.
  • PRDM14 methyltransferase activity
  • KLF4 Krueppel-like factor 4

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Abstract

L'invention concerne un procédé de rétablissement de cellules souches à capacité de formation de chimères, au moyen d'un processus à plusieurs étapes. Dans ce procédé, une étape de rétablissement est mise en oeuvre au moins deux fois, dont une étape dans laquelle un premier type de cellule souche pluripotente ou une cellule souche multipotente est co-cultivée avec un embryon hôte comportant un second type de cellule dont les conditions de culture sont différentes de celles des premières cellules souches. Au cours de ce processus, dans l'étape dans laquelle le premier type de cellule souche est co-cultivé avec l'embryon hôte comportant le second type de cellule, un milieu mixte, issu d'un milieu de culture approprié pour le premier type de cellule souche et d'un milieu de culture approprié pour le second type de cellules, est utilisé comme milieu de culture et des cellules souches à capacité de formation de chimères sont rétablies. Les cellules souches rétablies à capacité de formation de chimères peuvent être cultivées au moyen de l'un quelconque des milieux de culture suivants: le milieu de culture approprié pour le second type de cellules, le milieu de culture approprié pour le premier type de cellules souches; ou une culture mixte du milieu de culture approprié pour le premier type de cellules souches et du milieu de culture approprié pour le second type de cellules.
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