WO2013100140A1 - Induction de cellules souches pluripotentes à partir de cellules germinales - Google Patents

Induction de cellules souches pluripotentes à partir de cellules germinales Download PDF

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WO2013100140A1
WO2013100140A1 PCT/JP2012/084138 JP2012084138W WO2013100140A1 WO 2013100140 A1 WO2013100140 A1 WO 2013100140A1 JP 2012084138 W JP2012084138 W JP 2012084138W WO 2013100140 A1 WO2013100140 A1 WO 2013100140A1
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cells
gene
cell
germ
pluripotent stem
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PCT/JP2012/084138
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Japanese (ja)
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隆司 篠原
誠司 高島
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国立大学法人 京都大学
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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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/04—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from germ cells

Definitions

  • the present invention relates to germline stem cell-derived pluripotent stem cell-like cells. More specifically, the present invention relates to a method for effectively inducing pluripotent stem cell-like cells from germ stem cells.
  • Germ cells are unique in that they have the ability to transmit genes to their offspring. Although this cell is highly specialized to produce gametes for reproduction, much evidence suggests the cell's pluripotency. For example, teratoma occurs almost always in the gonad and includes a large variety of cells and tissues of various maturation stages. Furthermore, it is known that fetal germ cells give rise to pluripotent cells when cultured under special conditions. These embryonic germ cells (embryonic cell germ: EG cells) have differentiation characteristics similar to embryonic stem cells (embryonic cell stem cells: ES cells) isolated from the inner cell mass (inner cell).
  • embryonic germ cells embryonic cell germ: EG cells
  • ES cells embryonic stem cells isolated from the inner cell mass (inner cell).
  • Non-patent Document 1 Biol. Reprod., .Vol. 69, p612-616, 2003.
  • newborn testicular cells are transformed into glial cell-line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF),
  • GDNF glial cell-line-derived neurotrophic factor
  • LIF leukemia inhibitory factor
  • EGF epidermal growth factor
  • GDNF glial cell-line-derived neurotrophic factor
  • LIF leukemia inhibitory factor
  • EGF epidermal growth factor
  • the reporters named germline stem cells (hereinafter also referred to as “GS cells”) to distinguish colonized cells from ES cells and EG cells.
  • GS cells are clearly different from ES / EG cells in their ability to differentiate.
  • Trp53 cell mutation-related protein 53 ⁇ ⁇ kDa: synonymous with tumor suppressor p53
  • ES-like pluripotent stem cells hereinafter also referred to as “pluripotent stem cell-like cells”.
  • pluripotent stem cell-like cells are selectively proliferated under ES cell culture conditions, and when the pluripotent stem cell-like cells are transplanted subcutaneously into nude mice, teratomas are generated and varied in vitro. It has been confirmed that differentiation is induced into various functional cells. It has been confirmed that normal embryogenesis occurs by microinjecting the pluripotent stem cell-like cells into blastcysts to form extremely diverse tissues including germ cells.
  • An object of the present invention is to provide a method for effectively inducing pluripotent stem cell-like cells from germ cells.
  • the present inventors have used a germ cell in which Sox2 and / or Oct4 are overexpressed, thereby enabling pluripotent stem cell-like efficiency more efficiently than conventional methods.
  • the present invention was completed by finding that cells can be induced.
  • this invention consists of the following. 1.
  • a method for inducing pluripotent stem cell-like cells comprising culturing germ cells in which Sox2 and / or Oct4 are overexpressed. 2.
  • a germ cell in which Sox2 and / or Oct4 is overexpressed is a germ cell in which one or more genes selected from the Dmrt gene, Dnmt (DNA methyltransferase) gene, Dmap gene and Oct1 gene are suppressed.
  • the method for inducing pluripotent stem cell-like cells according to item 1 above. 3. 3. 3.
  • the method for inducing pluripotent stem cell-like cells according to 8 above, wherein the testis-derived cells are spermatogonial stem cells.
  • 10. 10 The method for inducing pluripotent stem cell-like cells according to item 9 above, wherein the spermatogonial stem cells are germ stem cells (GS cells). 11.
  • a method for producing pluripotent stem cell-like cells comprising the following steps: 1) overexpression of Sox2 and / or Oct4 in germ cells; 2) A step of culturing germ cells overexpressing the above Sox2 and / or Oct4. 12 12.
  • the step of overexpressing Sox2 and / or Oct4 in germ cells by suppressing any one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene Of producing pluripotent stem cell-like cells.
  • Method. 14 14.
  • the pluripotent stem cell-like cell induction method of the present invention can induce pluripotent stem cell-like cells more efficiently.
  • the obtained pluripotent stem cell-like cells expressed a Nanog gene that is specifically expressed in pluripotent stem cells such as ES cells and iPS cells (induced Pluripotent Stem cells).
  • pluripotent stem cell-like cells having pluripotency prepared by the method of the present invention are transplanted into an animal, a pathological section of teratoma is confirmed, and the transplanted cell-derived neuroepithelium (neuroepithelium), endoderm epithelium ( endoderm) and chondrocytes were confirmed.
  • the pluripotent stem cell-like cells contribute to ontogeny and have differentiated into various organ cells of the born mouse. It was. Thereby, it was confirmed that the pluripotent stem cell-like cell of the present invention has pluripotency.
  • FIG. 3 is a photograph showing pluripotent germline stem cells (mGS cells) induced by infecting Trp53-KO (Trp53 knockout) GS cells with Dnmt1-KD (Dnmt1 knockdown) virus.
  • mGS cells pluripotent germline stem cells
  • Trp53-KO Trp53 knockout
  • Dnmt1-KD Dnmt1 knockdown virus.
  • A Photographs showing the morphology of wild-type and Trp53-KO GS cells on day 11 after infection with Dnmt1-KD virus.
  • B Photograph showing the proliferation curve of GS cells.
  • Dnmt1-KD cells gradually die. Trp53-KO GS cells are slower to kill than wild type.
  • C A photograph showing ES cells.
  • D A photograph showing mGS cells induced by infecting Trp53-KO GS cells with Dnmt1-KD virus.
  • FIG. 3 is a photograph showing mGS cells induced by infecting wild-type GS cells with Trp53-KD virus and Dnmt1-KD virus.
  • Example 1 It is the photograph which confirmed the expression of Nanog used as the parameter
  • Trp53-KO GS cells do not express Nanog, mGS cells induced by Dnmt1-KD virus infection expressed Nanog like ES cells.
  • FIG. 3 is a photograph showing mGS cells induced by infecting wild-type GS cells with Trp53-KD virus and Dmrt1-KD virus.
  • Example 1-2 It is a figure which shows the slice image of the teratoma formed from the mGS cell which infected the Trp53-KO * GS cell with the Dnmt1-KD virus, and was induced
  • Transplanted cell-derived neuroepithelium (NE: neuroepithelium), endoderm epithelium (Endo: endoderm) and cartilage tissue (Ch: chondrocyte) were confirmed.
  • Example 1-3 It is a figure which shows the slice image of the teratoma formed from the mGS cell induced by infecting wild type GS cells with Trp53-KD virus and Dnmt1-KD virus.
  • Example 1-3 It is a photograph figure which shows the section image of the teratoma formed from the mGS cell which infected the wild-type GS cell and infected with Trp53-KD virus and Dmrt1-KD virus. Endodermal epithelium (Endo: endoderm) and cartilage tissue (Ch: chondrocyte) were confirmed.
  • FIG. 1-3 FIG.
  • Example 2 is a photographic diagram showing mGS cells induced 2 months after the introduction of siRNAs against Trp53 gene and Dmrt1 gene into DBA-GFP mouse-derived GS cells using a transfection reagent.
  • Example 2 This is a photograph showing a chimeric mouse prepared from mGS cells induced 2 months after the introduction of siRNA for Trp53 gene and Dmrt1 gene into GS cells derived from DBA-GFP mice using transfection reagent. is there. Since it is a chimeric mouse in which the above cells derived from a cinnamon hair color individual are injected into a black hair mouse blastocyst, the cinnamon hair color portion is mixed in the black hair color.
  • Example 2 It is a figure which shows the result that suppression of Dmrt1 gene by infection of Dnmt1-KD virus induces the expression of Sox2 about Trp53-KO GS cells.
  • Example 3-1 It is a photograph figure which shows the mGS cell which infected the DBA-GFP mouse origin GS cell and infected with Trp53-KD virus and Sox2-OE virus.
  • FIG. 3 is a photographic diagram showing mGS cells induced by infecting DBA-GFP mouse-derived GS cells with Trp53-KD virus and Oct4-OE virus.
  • FIG. 3 It is a figure which shows the expression of Oct1, Oct4, and Oct6 genes in GS cells.
  • iPS cells and mGS cells, Oct4 which is essential for maintaining the totipotency of cells, is strongly expressed, but the expression of Oct1 ubiquitously expressed in somatic cells is low.
  • Example 4 It is the photograph of the cell which infected the AxCANCre adenovirus to the mGS cell induced by infecting the DBA-GFP mouse-derived GS cell with the Trp53-KD virus and the Oct4-OE virus to stop the overexpression of the Oct4 gene. The expression of Oct4 is stopped and the red fluorescent protein DsRed is expressed.
  • Example 4 It is a photograph figure which shows the chimeric mouse
  • Example 4 It is a photograph figure which shows the chimeric mouse
  • Example 4 It is a photograph figure which shows the chimeric mouse
  • Example 4 It is a photograph figure which shows the chimeric mouse
  • An object of the present invention is to provide a method for effectively inducing pluripotent stem cell-like cells from germ stem cells.
  • germ cells specifically refer to testis cells, and include all cells constituting testis, such as spermatogonial stem cells, spermatogonia, sperm cells, spermatogonia, spermatocytes, spermatocytes, Examples include daughter cells, sperm, Leydig cells, Sertoli cells, intercellular cells, male germ cells and the like.
  • a spermatogonial stem cell is capable of self-renewal and differentiation into a sperm or a progenitor cell thereof (for example, a spermatogonia cell, a sperm cell, a spermatogonia cell, a spermatocyte, a spermatogonia cell)
  • Germline cells having Examples of spermatogonial stem cells include primordial germ cells, male germ cells, gonocytes, germline stem cells (GS cells), and the like.
  • the spermatogonial stem cells are preferably male germ cells and GS cells.
  • the target product pluripotent stem cell-like cell is sometimes called ES-like stem cell or pluripotent germline stem cell (multipotent germline stem cells: mGS cell), so-called ES cell or EG cell. Or used separately from GS cells.
  • a pluripotent stem cell-like cell can be cultured in vitro, can proliferate over a long period of time, has a self-replicating ability, and can differentiate into all cells constituting the living body and its precursor cells. Refers to a cell with capacity.
  • the pluripotent stem cell-like cell of the present invention is derived from germ cells in which Sox2 and / or Oct4 are overexpressed.
  • the pluripotent stem cell-like cell of the present invention is also derived from germ cells in which one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene are suppressed.
  • to suppress a gene means to suppress the function and / or expression of the gene.
  • DNA information is not simply a complete genetic base sequence. It can be said that the cell is creating epigenetic information that adjusts whether to use a specific gene by adding control to the base sequence information. This control may be performed by a nucleosome, or may be performed by methylating a DNA base and correcting the way the base sequence is read during protein synthesis. By methylating the base of this DNA, a methyl group is added to a gene and the activity of the gene is controlled.
  • DNMT is also referred to as a DNA methyltransferase (methyltransferase). Dnmt1, Dnmt2, Dnmt3a, Dnmt3b, Dnmt3, etc.
  • DMAP is also called a DNA methyltransferase-related protein
  • Dmap gene is a gene encoding DMAP.
  • Dmrt gene is involved in genital differentiation and maintenance.
  • Oct4 and Sox2 are essential for the totipotency of embryonic stem cells (ES cells) and are genes included in the Yamanaka factor that induces iPS cells.
  • the Octamer transcriprion factor family, which includes Oct4 is composed of Oct1, Oct2, 4Oct4, 6Oct6, 7Oct7, 8Oct8, 9Oct9, and Oct11.
  • Oct1 and Oct6 are known to be ubiquitously expressed in various somatic cells.
  • Oct1 is a gene that is ubiquitously expressed in various somatic cells, but its function and structure are similar to Oct4. Like Oct4, it binds to Sox2 and controls transcription of ES cell-specific genes.
  • germ cells in which Sox2 and / or Oct4 are overexpressed or one or more genes selected from Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene
  • the pluripotent stem cell-like cells of the present invention are effectively induced.
  • Germ cells in which Sox2 and / or Oct4 are overexpressed, or germ cells in which one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene are suppressed are further suppressed by Trp53 It is preferable that the cell is a cell in which the Trp53 gene is suppressed.
  • Trp53 Transformation-related protein 53: cell mutation-related protein 53 kDa
  • Trp53 Transformation-related protein 53: cell mutation-related protein 53 kDa
  • Trp53 Transformation-related protein 53: cell mutation-related protein 53 kDa
  • a germ cell in which Sox2 is overexpressed refers to a state where the Sox2 gene is functionally excessive in the germ cell, or a state where Sox2 is contained in the germ cell culture system.
  • a state where the Sox2 gene is functionally excessive in the germ cell that is, a state where the effect of the Sox2 gene is exerted more than the original function can be mentioned.
  • a state in which the Dmrt gene is not expressed at all a state in which the expression level is reduced to such an extent that the normal function of the Dmrt gene cannot be exerted, or a function of the Dmrt gene product is completely lost, or the Dmrt gene Even when the function of the Dmrt gene product is reduced to such an extent that the normal function inherent in can not be exerted, Sox2 can be overexpressed.
  • a germ cell in which Oct4 is overexpressed means a state where the Oct4 gene is functionally excessive in the germ cell, or a state where Oct4 is contained in the germ cell culture system.
  • a state in which the Oct4 gene is functionally excessive in the germ cell that is, a state in which an effect exceeding the function inherent in the Oct4 gene is exhibited.
  • a state in which the Dmrt gene is not expressed at all a state in which the expression level is reduced to such an extent that the normal function of the Dmrt gene cannot be exerted, or a function of the Dmrt gene product is completely lost, or the Dmrt gene Even when the function of the Dmrt gene product is reduced to such an extent that normal functions inherent in can not be exhibited, Oct4 can be overexpressed.
  • an overexpression vector of each gene may be introduced into germ cells, or a protein may be added to the germ cell culture system.
  • Sox2 and / or Oct4 can be relatively overexpressed by regulating other genes.
  • regulating other genes for example, one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene, particularly preferably the Dmrt gene is suppressed.
  • the germ cell in which the Dmrt gene is suppressed refers to a state in which the Dmrt gene is functionally insufficient in the germ cell, that is, a state in which the normal function inherent to the Dmrt gene cannot be sufficiently exhibited.
  • a state in which the Dmrt gene is not expressed at all a state in which the expression level is reduced to such an extent that the normal function that the Dmrt gene originally has cannot be exhibited, a state in which the function of the Dmrt gene product is completely lost, or a Dmrt gene originally has A state in which the function of the Dmrt gene product is reduced to such an extent that normal functions cannot be exerted.
  • Dmrt is not particularly limited, but is preferably Dmrt1.
  • the germ cell in which the Dnmt gene is suppressed refers to a state in which the Dnmt gene is functionally insufficient in the germ cell, that is, a state in which the normal function inherent to the Dnmt gene cannot be sufficiently exhibited.
  • a state in which the Dnmt gene is not expressed at all a state in which the expression level is reduced to the extent that the normal function of the Dnmt gene cannot be exhibited, a state in which the function of the Dnmt gene product has been completely lost, or a Dnmt gene originally has A state in which the function of the Dnmt gene product is reduced to such an extent that normal functions cannot be exhibited.
  • Dnmt is not particularly limited, but is preferably Dnmt1.
  • the germ cell in which the Dmap gene is suppressed refers to a state in which the Dmap gene is functionally insufficient in the germ cell, that is, a state in which the normal function of the Dmap gene cannot be sufficiently exhibited.
  • a state in which the Dmap gene is not expressed at all a state in which the expression level is reduced to such an extent that the normal function inherent in the Dmap gene cannot be exerted, a state in which the function of the Dmap gene product is completely lost, or a function inherent in the Dmap gene
  • Dmap is not particularly limited, but is preferably Dmap1.
  • the germ cell in which the Oct1 gene is suppressed refers to a state in which the Oct1 gene is functionally insufficient in the germ cell, that is, a state in which the normal function inherent to the Oct1 gene cannot be sufficiently exhibited.
  • a state where it is not expressed at all a state where its expression level is reduced to the extent that the normal function inherent in the Oct1 gene cannot be exerted, a state where the function of the Oct1 gene product is completely lost, or a state where the Oct1 gene originally has A state in which the function of the Oct1 gene product is reduced to such an extent that normal functions cannot be exhibited.
  • the germ cell in which the Trp53 gene is suppressed refers to a state where the Trp53 gene is functionally insufficient in the germ cell, that is, a state where the normal function inherent to the Trp53 gene cannot be sufficiently exhibited.
  • a state in which the Trp53 gene is not expressed at all, a state in which the expression level is reduced to such an extent that the normal function inherent in the Trp53 gene cannot be exerted, a state in which the function of the Trp53 gene product is completely lost, or a Trp53 gene originally has Examples include a state in which the function of the Trp53 gene product has decreased to such an extent that normal functions cannot be exerted.
  • a targeting vector for any of the genes is introduced into the germ cell, and the homologous set It can also be produced by deleting one of the genes by replacement. In some cases, “deleting a gene” or “knocking out a gene (KO)”. In another embodiment, it is prepared by introducing a substance that suppresses the expression or function of any of the genes (eg, antisense nucleic acid, RNAi-inducible nucleic acid (siRNA, stRNA, miRNA, etc.)) into germ cells. can do.
  • a substance that suppresses the expression or function of any of the genes eg, antisense nucleic acid, RNAi-inducible nucleic acid (siRNA, stRNA, miRNA, etc.
  • a substance that suppresses the expression or function of any of the genes into germ cells can be performed by a method known per se.
  • the substance that suppresses the expression or function of each gene is a nucleic acid molecule such as an RNAi-inducible nucleic acid or an expression vector containing the same
  • the calcium phosphate method, lipofection method / liposome method, electroporation method, etc. can be used.
  • a germ cell in which the Trp53 gene is suppressed can also be prepared by the same method as that for a germ cell in which the expression of the Dmrt gene, Dnmt gene, Dmap gene, or Oct1 gene is suppressed.
  • the method disclosed in Patent Document 1 can be used.
  • Suppressing gene expression or function is sometimes referred to as “knocking down (KD) a gene”.
  • Germ cells that can be used in the method for inducing pluripotent stem cell-like cells of the present invention can be prepared by a method known per se.
  • testis cells can be dispersed and prepared by extracting the testis and digesting the extracted testis with a degrading enzyme such as collagenase, trypsin, or DNase.
  • a degrading enzyme such as collagenase, trypsin, or DNase.
  • GS cells refer to spermatogonial stem cells that are proliferated in vitro depending on a GDNF receptor agonist compound (GDNF or an equivalent thereof), for example, Biol.BioReprod., Vol. 69, p. This refers to spermatogonial stem cells grown by the method described in 612-616, 2003.
  • the germ cells of the present invention may be those obtained by selecting and concentrating a fraction having a high ability to produce pluripotent stem cell-like cells. Examples of the concentration method include a method using a cell sorter, an antibody magnetic microbead, or the like using an antibody that recognizes a cell surface antigen specifically expressed in cells of the fraction.
  • spermatogonial stem cells can be enriched with cell surface antigens such as ⁇ 6-integrin, c-kit, CD9 and the like as indicators.
  • Germ cells used in the present invention are not particularly limited as long as pluripotent stem cell-like cells can be produced by the method of the present invention, and may be derived from vertebrates or invertebrates, but preferably It is a vertebrate.
  • Vertebrate animals include, for example, mammals, birds, fish, amphibians and reptiles.
  • mammals include primates such as humans, monkeys, orangutans, chimpanzees, laboratory animals such as rodents such as mice, rats, hamsters, and guinea pigs, and rabbits, and domestic animals such as pigs, cows, goats, horses, and sheep. , Dogs, cats and other pets.
  • the birds include chickens, quails, ducks, geese, turkeys, ostriches, guinea fowls, and pigeons.
  • basal medium used in the present invention those known per se can be used. Although it is not particularly limited as long as pluripotent stem cell-like cells can be induced by the method of the present invention, for example, DMEM, EMEM, RPMI-1640, ⁇ -MEM, F-12, F-10, M-199, HAM, ATCC -CRCM30, DM-160, DM-201, BME, SFM-101, Fischer, McCoy's 5A, Le ibovitz's L-15, RITC80-7, HF-C1, MCDB107, NCTC135, Waymouth's MB 752/1, StemPro (R) 34 SFM and so on.
  • a medium modified for ES cell culture or the like may be used, or a mixture of the above basal medium may be used.
  • the medium can contain additives known per se.
  • the additive is not particularly limited as long as pluripotent stem cell-like cells can be produced by the method of the present invention.
  • growth factors eg, insulin
  • iron sources eg, transferrin
  • polyamines eg, putrescine, etc.
  • Minerals such as sodium selenate
  • saccharides such as glucose
  • organic acids such as pyruvic acid, lactic acid
  • serum proteins such as albumin
  • amino acids such as L-glutamine
  • reducing agents such as 2-mercaptoethanol, etc.
  • vitamins eg, ascorbic acid, etc.
  • steroids eg, ⁇ -estradiol, progesterone, etc.
  • antibiotics eg, streptomycin, penicillin, gentamicin, etc.
  • buffers eg, HEPES, etc.
  • nutritional additives for example, StemPro (R) Nutrient Supplement.
  • the medium can also contain serum.
  • the serum is not particularly limited as long as it is an animal-derived serum in the range in which pluripotent stem cell-like cells can be produced by the method of the present invention.
  • the above-mentioned mammal-derived serum for example, fetal bovine serum, human serum
  • serum substitute additives for example, Knockout Serum Replacement (KSR); manufactured by Invitrogen
  • KSR Knockout Serum Replacement
  • the concentration of serum is not particularly limited as long as pluripotent stem cell-like cells can be produced by the method of the present invention, but is usually in the range of 0.1 to 30 (v / v)%.
  • germ cells for inducing pluripotent stem cell-like cells may be cultured in the presence of feeder cells.
  • the feeder cells are not particularly limited as long as pluripotent stem cell-like cells can be induced by the method of the present invention, but when culturing pluripotent stem cells such as ES cells and EG cells while maintaining pluripotency.
  • the feeder cells known per se can be used, and examples thereof include fibroblasts (mouse fetal fibroblasts, mouse fibroblast cell line STO, etc.).
  • the feeder cells are preferably inactivated by a method known per se, for example, irradiation with radiation (eg gamma rays) or treatment with an anticancer agent (eg mitomycin C).
  • the culture temperature is usually in the range of about 30 to 40 ° C., preferably about 37 ° C.
  • the CO 2 concentration is usually in the range of about 1 to 10%, preferably about 5%.
  • the humidity is usually in the range of about 70 to 100%, preferably about 95 to 100%.
  • the pluripotent stem cell-like cell of the present invention can be cultured and induced by the following method. Germ cells prepared by the above-described method are suspended in the medium shown in the present specification, seeded in a cell culture vessel, and cultured (first culture). As the cell culture vessel, those used in normal cell culture can be used, but those coated with gelatin or the like are preferable. The same applies to the containers used for the following culture. Although it is possible to induce pluripotent stem cell-like cells only by continuing the first culture, a culture containing at least germ cells about 6 to 18 hours after the start of the first culture. Cells, preferably cultured cells that are floating, can be passaged to another cell culture vessel to form a second culture. Although the subcultured cultured cells differ depending on the culture conditions, colonies can be formed on the bottom surface of the cell culture container usually within one week after the subculture. Colony formation can be confirmed using a microscope or the like.
  • the cells in which colony formation has been observed can be detached from the culture vessel by trypsin treatment or the like, dispersed, suspended again in the medium, and further subcultured to a new culture plate (third culture). By repeating similar passages, somatic cells with a flat shape disappear. Therefore, after the second or third passage, the cells are preferably cultured in the presence of feeder cells.
  • the interval between passages and the dilution rate of the cells are appropriately determined depending on the culture conditions. Examples include an interval of 2 to 5 days and a dilution of 1 to 1/4 (preferably a dilution of 1 to 1/2 in the initial stage of culture).
  • the Further, examples of the passage interval and cell dilution rate of established pluripotent stem cell-like cell colonies include 2 to 5 day intervals and 1/4 to 1/10 dilution.
  • a medium having the same composition may be used throughout the process, but a medium having a plurality of compositions may be used separately over time.
  • the medium used for culturing can be changed from a medium for initial culture of germ cells (referred to as medium A) to a medium for long-term culture of pluripotent stem cell-like cells (referred to as medium B) during the culture. it can. That is, by culturing germ cells using medium A, obtaining cultured cells, and culturing the cultured cells using medium B, pluripotent stem cell-like cells can be efficiently obtained.
  • the concentration of the serum that can be contained in each of the media A and B is the same as described above, but the concentration of the serum that can be contained in the media A is preferably 0.1 to 5 (v / v)%, and more Preferably, it is 0.3 to 3 (v / v)%.
  • the concentration of serum that can be contained in the medium B is preferably 2 to 30 (v / v)%, more preferably 10 to 20 (v / v)%.
  • the basal media of each of the media A and B are the same as described above, but the basal media of the media A are basal media that are preferably used for culturing spermatogonial stem cells (eg, GS cells) (for example, StemPro® ). 34 SFM or the like), and the basal medium of the medium B can be a basal medium (for example, DMEM or the like) suitably used for culturing ES cells.
  • Additives that the media A and B can contain are the same as described above.
  • the timing for converting the medium from the medium A to the medium B varies depending on the culture conditions and the like, and thus it is difficult to uniformly define the medium. For example, in the case of mice, it is 10 to 120 days, preferably 14 to 40 days after the start of the first culture.
  • a medium having a composition in which GDNF or an equivalent thereof is added to the medium B at the above-mentioned concentration for about 4 to 40 days immediately after the conversion of the medium A to the medium B, it is possible to increase the efficiency.
  • Pluripotent stem cell-like cells can be induced.
  • culture of germ cells using such medium A and medium B may be performed in the presence of feeder cells as described above.
  • the pluripotent stem cell-like cells obtained by the method of the present invention can be proliferated while maintaining pluripotency for usually 2 months or more, preferably 5 months or more.
  • the above-mentioned medium B is preferably used.
  • the cultured cells can form two types of colonies by about 10 days to 6 weeks after the start of the culture.
  • One colony has a morphology characterized by intercellular bridges and morula-like structures, which are GS cell colonies.
  • the other colony is more tightly packed and has a morphology very similar to that of ES cell colonies.
  • Cells that form ES cell-like colony morphology are pluripotent stem cell-like cells according to the present invention. Therefore, a colony of GS cells and a colony of pluripotent stem cell-like cells according to the present invention can be clearly distinguished visually.
  • a pluripotent stem cell-like cell can be selected by selectively picking up a colony of pluripotent stem cell-like cells using a Pasteur pipette, a micromanipulator or the like under a microscope, or by limiting dilution. It can be isolated.
  • pluripotent stem cell-like cells can be isolated using a cell sorter or the like using a cell surface marker of the pluripotent stem cell-like cells as an index.
  • GS cells are obtained by culturing germ cells using a medium containing GDNF or an equivalent thereof under the same culture conditions as described above, and the GS cells are further cultured under the above-described culture conditions.
  • the pluripotent stem cell-like cells of the present invention can also be derived from GS cells and produced by culturing continuously using a medium containing an equivalent thereof. As described above, the GS cell colony morphology is clearly visually distinguishable from the pluripotent stem cell-like cell colony of the present invention.
  • the concentration of GDNF or an equivalent thereof contained in the medium when inducing pluripotent stem cell-like cells is not particularly limited as long as pluripotent stem cell-like cells can be induced by the method of the present invention.
  • usually 0.05 ng / ml to 100 mg / ml for example 0.5 ng / ml to 100 ⁇ g / ml, preferably 0.5 ng / ml to 10 ⁇ g / ml, more preferably 0.5 ng / ml to 1 ⁇ g / ml, still more preferably 0.5 to 200 ng / ml, even more preferably 0.5-50 ng / ml, most preferably 2-20 ng / ml.
  • the medium preferably further contains LIF.
  • LIF When LIF is contained in the medium, its concentration is not particularly limited as long as pluripotent stem cell-like cells can be induced by the method of the present invention, but usually 10 to 10 6 units / ml, for example 10 to 10 5 units / ml, preferably 10 2 to 10 4 units / ml, more preferably 3 ⁇ 10 2 to 5 ⁇ 10 3 units / ml.
  • the medium for inducing the pluripotent stem cell-like cell of the present invention can further contain at least one of EGF and bFGF, more preferably both.
  • EGF is contained in the medium, the concentration thereof is not particularly limited as long as pluripotent stem cell-like cells can be produced by the method of the present invention, but a normal concentration of 0.05 ng / ml to 100 mg / ml, for example 0.5 ng / ml to 100 ⁇ g / ml, preferably 0.5 ng / ml to 10 ⁇ g / ml, more preferably 0.5 ng / ml to 1 ⁇ g / ml, more preferably 0.5 to 200 ng / ml, even more preferably 0.5 to 50 ng / ml, Most preferably, it is 2 to 30 ng / ml.
  • the concentration thereof is not particularly limited as long as pluripotent stem cell-like cells can be produced by the method of the present invention, but a normal concentration of 0.05 ng / ml to 100 mg / ml, for example, 0.5 ng / ml to 100 ⁇ g / ml, preferably 0.5 ng / ml to 10 ⁇ g / ml, more preferably 0.5 ng / ml to 1 ⁇ g / ml, more preferably 0.5 to 200 ng / ml, even more preferably 0.5 to 50 ng / ml Most preferably, it is 2 to 20 ng / ml.
  • the present invention also extends to a method for producing pluripotent stem cell-like cells.
  • Pluripotent stem cell-like cells can be produced by a method comprising at least the following steps 1) and 2). 1) overexpression of Sox2 and / or Oct4 in germ cells; 2) A step of culturing germ cells overexpressing the above Sox2 and / or Oct4.
  • the step of overexpressing Sox2 and / or Oct4 in germ cells can also be achieved by suppressing one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene. can do. Furthermore, a step of suppressing the Trp53 gene of germ cells may be included.
  • the method for producing pluripotent stem cell-like cells can be achieved by a method for producing pluripotent stem cell-like cells comprising the following steps 1) to 4). Any of the steps 1), 2) and 3) may be performed first or simultaneously. Further, by performing the process 1), the process 2) can be achieved as a result. By performing the process 2), the process 1) can be achieved as a result. 1) overexpression of Sox2 and / or Oct4 in germ cells; 2) a step of suppressing one or more genes selected from germline Dmrt gene, Dnmt gene, Dmap gene and Oct1 gene; 3) repressing the Trp53 gene in germ cells; 4) A step of culturing cells in which each of the genes is manipulated.
  • the present invention further extends to cells for producing pluripotent stem cell-like cells.
  • Cells for producing pluripotent stem cell-like cells are cells in which Sox2 and / or Oct4 are overexpressed. It is preferable that one or more genes selected from the Dmrt gene, Dnmt gene, Dmap gene, and Oct1 gene are suppressed. Further, the Trp53 gene is preferably suppressed in the cell.
  • the pluripotent stem cell-like cell of the present invention is derived from, for example, a mouse, at least selected from the group consisting of SSEA-1, Forssman antigen, ⁇ 1- and ⁇ 6-integrin, EpCAM, CD9, EE2, and c-kit Either is positive, preferably all are positive. Forssman antigen and c-kit are preferably weakly positive.
  • GS cells are negative for SSEA-1 and Forsman antigen, pluripotent stem cell-like cells obtained by the induction method of the present invention are clearly distinguished from GS cells.
  • Whether or not the cells obtained by the production method of the present invention retain pluripotency is determined by, for example, synthesizing cDNA by reverse transcriptase (Reverse Transcriptase: RT) and using a reverse transcription polymerase chain using a primer for the target gene. It can be confirmed by analyzing the expression of a gene or the like specifically expressed in pluripotent stem cell-like cells by a reaction (RT-PCR) method or the like.
  • RT-PCR Reverse Transcriptase
  • genes such as Oct4, Rex-1, Nanog, Cripto, Eras, UTF1, ZFP57, Esg-1, etc. are specifically expressed in the pluripotent stem cell.
  • the pluripotent stem cell-like cell obtained by the method of the present invention expresses at least one gene selected from the group consisting of Oct4, Rex-1, Nanog, Cripto, Eras, UTF1, ZFP57 and Esg-1. Preferably, all genes are expressed.
  • the expression of these genes is generally weaker than that of pluripotent stem cell-like cells obtained by the method of the present invention, and in particular, expression of Nanog is hardly observed. This also clearly distinguishes the pluripotent stem cell-like cells from GS cells.
  • sulfite chromosome sequencing of DMR in chromosomal DNA (Development, vol.
  • Example 1 Induction of Pluripotent Germline Stem Cells (mGS Cells) from Germ Cells 1 1) Establishment of germline stem cells (GS cells) According to the description in Patent Document 1 (International Publication WO2005 / 100548 pamphlet), 7 to 10 days old wild type and GFP transgenic DBA / 2 mice or adult animals Testes were extracted from wild-type DBA / 2 mice, and testis cells were dispersed by trypsin collagenase treatment. GDNF (10 ng / ml), LIF (10 3 units / ml), and FGF-2 (10 ng / ml) are added to the mouse embryonic fibroblast (MEF) feeder. medium was (StemPro (R) 34 SFM medium; Invitrogen Corp.) were cultured using, was established GS cells. Establishing took about a month. The obtained GS cells were subcultured using the above medium.
  • GS cells germline stem cells
  • the culture supernatant from the 2nd to 5th day after gene transfer is collected, and 32 ml of the supernatant is ultracentrifuged at 19400 rpm and 4 ° C for 2 hours, and the resulting virus pellet is added to 100 ⁇ l medium (StemPro (R) 34 SFM medium ). Invitrogen) and stored frozen at ⁇ 80 ° C. until use.
  • a lentiviral vector that does not express shRNA against each gene is called a Mock virus and used as a control.
  • GS cells in which the above genes were suppressed were prepared by infection of GS cells with the Trp53-KD virus, Dnmt1-KD virus or Dmrt1-KD virus of 2) above.
  • the GS cells prepared in 1) above were seeded at 5 ⁇ 10 5 cells / 600 ⁇ l medium / well on a 12-well culture plate previously seeded with MEF feeders.
  • hexadimethrin bromide polybrene; Sigma Code H9268
  • Trp53-KD virus, Dnmt1-KD virus, Dmap1-KD virus, Dmrt1-KD virus were respectively infected, and the cells in which each gene was suppressed were respectively Trp53-KD GS cell, Dnmt1-KD GS cell, Dmap1- It is called KD GS cell, Dmrt1-KD GS cell.
  • a cell lacking the Trp53 gene by homologous recombination is also referred to as a Trp53-KO GS cell.
  • a GS cell in which each gene is not suppressed is called a wild-type GS cell.
  • the culture was continued.
  • the first passage was performed 3-5 days after infection.
  • 1 well of an infected GS cell 12 well culture plate was replated on 1 well of a 6 well culture plate on which the MEF feeder had been seeded.
  • 1-well cells from a 6-well culture plate were subcultured to 1-well of a new 6-well MEF plate (1 passage), and the culture was continued.
  • DNA methyltransferase type I (Dnmt1) is an enzyme that imparts a methyl group to DNA.
  • Dnmt1-KD GS cells gradually die (see FIGS. 1A and B).
  • Inhibiting the Trp53 gene at the same time when suppressing the Dnmt1 gene is known to prolong cell survival (Nat Genet 2001; 27: 31-39), so when Trp53-KO GS cells are infected with the Dnmt1-KD virus Compared with the case where wild type GS cells were infected with Dnmt1-KD virus, the rate of cell death was suppressed (see FIGS. 1A and 1B).
  • FIG. 1C shows ES cells
  • FIG. 1D shows cells in which Trp53-KO GS cells were infected with Dnmt1-KD virus.
  • Nanog could not be confirmed in (1) -Trp53-KO ⁇ GS cells, but the expression of Nanog was confirmed in cells induced by infection of (4) ⁇ ⁇ ⁇ ⁇ Trp53-KO GS cells with Dnmt1-KD virus. Therefore, it was confirmed that (4) mGS cells were induced in sputum (see FIG. 3). It confirmed also about the cell which suppressed Dnmt1 gene and Trp53 gene from the wild-type GS cell (double knockdown (KD)). As a result, when the Dnmt1 gene and the Trp53 gene were double knocked down, the expression of Nanog was confirmed and it was confirmed that mGS cells were induced (see FIG. 4).
  • Induced mGS cells (Dnmt1 KD-induced mGS cell), which were induced by suppressing Trp53 and Dnmt1 genes as described above for wild-type GS cells, and similarly induced mGS cells for wild-type GS cells by suppressing Trp53 and Dmrt1 genes
  • a transcriptome analysis using a DNA microarray was performed. As a result, it was confirmed that Dnmt1 KD-induced mGS cell and Dmrt1 KD-induced mGS cell showed gene expression patterns very similar to mGS cells that have been proven to be pluripotent. On the other hand, it was also confirmed that these cells showed a gene expression pattern quite different from that of wild-type GS cells (see FIG. 7).
  • the transplanted cells formed a tumor with a diameter of 2 to 3 cm in about one month, and were removed after dying due to cervical dislocation.
  • the obtained tissue was fixed with formalin, and a pathological section was prepared with a paraffin section, followed by HE (Hematoxylin-Eosin) staining and observation.
  • Trp53-KO GS cells infected with Dnmt1-KD virus, teratomas were confirmed in the pathological section, and the transplanted cell-derived neuroepithelium (NE: Neuroepithelium), endoderm epithelium (Endo: endoderm) ) And cartilage tissue (Ch: chondrocyte) were confirmed (see FIG. 8).
  • Example 2 Induction of pluripotent germline stem cells (mGS cells) from germ cells 2
  • Dnmt1 gene was suppressed and induced cells were prepared by a method not involving gene transfer using a viral vector.
  • siRNA Invitrogen, stealth RNAi
  • siRNA for each gene of Dmrt1 and Trp53 was introduced into the cells using siRNA and a transfection reagent for Stealth RNAi (Lipofectamine RNAi max reagent; Invitrogen).
  • SiRNA was introduced into cells every 3 days, and passage was performed every 6 days with 1-fold passage. When the morphology of the cells 1 to 2 months after the introduction of siRNA was observed, induction of mGS cells was observed (see FIG. 11).
  • GFP fluorescence was observed with a fluorescence stereomicroscope (see Table 1), and one of the two born animals showed part of the body showing GFP fluorescence (see FIG. 12) and was transplanted. It has been shown that the cells have the ability to form chimeras. This proved the pluripotency of induced mGS cells.
  • Example 3 Induction of pluripotent germline stem cells (mGS cells) from germ cells 3
  • mGS cells were induced by the same method as in Example 1.
  • suppression of Oct1 gene and overexpression of Sox2 and Oct4 were examined.
  • GS cells germline stem cells
  • GS cells The germline stem cells (GS cells) were established by the same method as in Example 1.
  • the lentiviral vector that expresses shRNA against the Oct1 gene is referred to as Oct1 gene knockdown virus (Oct1-KD virus).
  • lentiviral vectors that induce overexpression of Sox2 gene and Oct4 gene are referred to as Sox2 gene overexpression virus (Sox2-OE virus) and Oct4 gene overexpression virus (Oct4-OE virus), respectively.
  • Sox2-OE virus Sox2 gene overexpression virus
  • Oct4-OE virus Oct4 gene overexpression virus
  • a lentiviral vector that does not express shRNA against each gene is called a Mock virus and used as a control.
  • Trp53-KD GS cell Dnmt1-KD GS cell, Dmap1-KD GS cell, Dmrt1-KD GS cell, Sox2-OE GS, respectively.
  • Cells referred to as Oct4-OE GS cells.
  • a cell lacking the Trp53 gene by homologous recombination is also referred to as a Trp53-KO GS cell.
  • a GS cell in which each gene is not suppressed is called a wild-type GS cell.
  • Each produced cell was cultured by the same method as in Example 1.
  • Oct4 was weak in GS cells and expressed strongly in ES cells, iPS cells, and mGS cells. However, Oct1 was strongly expressed in fibroblasts and GS cells, but weakly expressed in ES cells, but not expressed in iPS cells and mGS cells (see FIG. 17). It is known that Oct4 protein produced from Oct4 gene exerts its function by directly interacting with Sox2 protein and maintains the totipotency of ES cells, but Oct1 protein is also Sox2 like Oct4 protein It is known to interact with proteins. These results suggest that Oct1 protein may competitively inhibit the interaction between Oct4 and Sox2 proteins in GS cells.
  • FIG. 18 shows mGS primary colonies that appeared on day 21 after infection of DBA-GFP mouse-derived GS cells with Trp53-KD virus and Oct1-KD virus. There was a large mass of undifferentiated cells in the center, and the development of slightly differentiated cells was seen around.
  • the transplanted cells formed a tumor with a diameter of 2 to 3 cm in about a month, and were removed after death by cervical dislocation.
  • the obtained tissue was observed after performing formalin fixation and preparing paraffin sections and staining with HE (Hematoxylin-Eosin).
  • Example 4 Induction of pluripotent germline stem cells (mGS cells) from germ cells 4
  • mGS cells pluripotent germline stem cells
  • the pluripotent stem cell-like cells obtained by overexpressing Sox2 and Oct4 were proved to be useful because of their proven pluripotency.
  • the pluripotent stem cell-like cells of the present invention are introduced into GS cells using a viral vector for overexpression of Sox2 gene and / or Oct4 gene, or these proteins are directly used, as in the conventional method of producing iPS cells. It can be induced by introduction into GS cells.
  • shRNA or siRNA for Dnmt1 gene, Dmrt1 gene, Dmap1 gene, Oct1 gene, etc., and Sox2 or Oct4 is excessive. It can also be expressed. It is possible to suppress the gene using a viral vector that expresses shRNA or siRNA of each factor, but it is also possible to suppress the gene by introducing siRNA itself using a transfection reagent. This is a safer and simpler method than methods using viral vectors and proteins.
  • the pluripotent stem cell-like cells produced by the induction method of the present invention can be used in the field of drug discovery / diagnosis. Specifically, it can be applied to pharmacological tests, toxicity tests, pharmaceutical screenings and the like. In addition, by producing these cells from a person suffering from a genetic disease, it can be applied to preparation of a disease model, analysis of the onset mechanism, development of a therapeutic method, and the like. Furthermore, it can be used for basic research of regenerative medicine and can also be used as a material for regenerative medicine.

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Abstract

La présente invention concerne un procédé qui permet d'induire efficacement des cellules souches pluripotentes à partir de cellules souches germinales. Les cellules souches pluripotentes peuvent être induites avec une efficacité supérieure à des procédés classiques par l'utilisation de cellules souches germinales dans lesquelles Sox2 et/ou Oct4 sont surexprimés. En ce qui concerne les cellules souches germinales, au moins une sorte de gène choisi parmi les gènes Dnmt1, Dmap1, Dmrt1 et Oct1 est inhibé, ou le gène Sox2 et/ou le gène Oct4 est surexprimé, ou autrement la protéine de Sox2 et/ou Oct4 est directement introduite. Ledit procédé permet à des cellules souches pluripotentes d'être plus efficacement induites à partir de cellules souches germinales que dans des procédés classiques.
PCT/JP2012/084138 2011-12-29 2012-12-28 Induction de cellules souches pluripotentes à partir de cellules germinales WO2013100140A1 (fr)

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WO2018116511A1 (fr) * 2016-12-19 2018-06-28 学校法人神戸女学院 Procédé de production de cellules souches pluripotentes
JPWO2018116511A1 (ja) * 2016-12-19 2019-10-24 学校法人神戸女学院 多能性幹細胞の製造方法
JP7033318B2 (ja) 2016-12-19 2022-03-10 学校法人神戸女学院 多能性幹細胞の製造方法

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