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WO2007108393A1 - Procédé permettant la prolifération in vitro de cellules souches germinales mâles - Google Patents

Procédé permettant la prolifération in vitro de cellules souches germinales mâles Download PDF

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Publication number
WO2007108393A1
WO2007108393A1 PCT/JP2007/055213 JP2007055213W WO2007108393A1 WO 2007108393 A1 WO2007108393 A1 WO 2007108393A1 JP 2007055213 W JP2007055213 W JP 2007055213W WO 2007108393 A1 WO2007108393 A1 WO 2007108393A1
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Prior art keywords
stem cells
germline stem
male germline
cells
fgf
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Japanese (ja)
Inventor
Mito Shinohara
Takashi Shinohara
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Kyoto University NUC
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Kyoto University NUC
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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
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-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
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10

Definitions

  • the present invention relates to a method for proliferating male germline stem cells using FGF, a male germline stem cell expanded by the method, an infertility treatment agent containing the cell, a male germline stem cell proliferation promoter, a male germline stem cell
  • the present invention relates to a method for producing pluripotent stem cells using FGF.
  • male testicular male germline stem cells such as spermatogonial stem cells, continue to proliferate indefinitely in adults and are the source of spermatogenesis through meiosis . Since male germline stem cells are the only stem cells in adults that are distributed to transfer genes to the next generation, they are useful for in vivo experiments, medical research, biotechnology, etc.
  • GDNF glial cell-derived neurotrophic factor
  • the present inventors have developed a method for in vitro long-term culture of male germline stem cells such as spermatogonial stem cells (WO 2004Z092357 pamphlet: Patent Document 1, Biology of Rep reduction, vol. 69, No. 2). , p.612-616, 2003: Non-patent document 4).
  • Newborn testicular cells When cultured in the presence of cell-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), etc., germ cells formed colonies with unique shapes, and stem cells proliferated over 5 months. When transplanted into the seminiferous tubule of the infertile mouse, the cultured cells formed spermatogenic colonies and produced normal sperm and offspring. This allowed male germline stem cells to be proliferated to a practical level, manipulated, and applied to biotechnology and the like.
  • GDNF cell-derived neurotrophic factor
  • LIF leukemia inhibitory factor
  • male germline stem cell growth methods were developed mainly using mouse cells, male germline stem cells derived from other animal species by the same method. In some cases, the growth speed was slow.
  • the induction efficiency of male germline stem cells varies depending on the age of the animal from which testis cells are derived. Especially when the testicular cells of prenatal animals are used, the induction efficiency decreases.
  • Non-Patent Document 6 includes the culture conditions disclosed in Patent Document 1 and Non-Patent Document 5 described above. A method for growing rat spermatogonial stem cells using similar conditions is described.
  • Proc. Natl. Acad. Sci. USA, vol.102, p.14302-14307, 2005 includes GDNF, soluble GDNF receptor a-1 (GFR a-1) and bFGF. It has been disclosed to support the growth of combined cut spermatogonial stem cells. However, even with these methods, it is not possible to achieve a sufficiently satisfactory proliferation efficiency of male germline stem cells.
  • the inventors of the present application are similar to the aforementioned in vitro long-term culture method for male germline stem cells. It was found that when testicular cells were cultured under similar conditions, colonies of pluripotent stem cells with a morphology that was indistinguishable from that of ES cells were derived in addition to GS cell colonies ( International Publication No. 2005Z100548 Pamphlet: Patent Document 4, Cell, vol.119, p.1001-1012, 2004: Non-patent Document 8).
  • This method is an epoch-making method that can produce pluripotent stem cells from an individual after birth, but the production efficiency depends on the age of the animal from which testis cells are derived, and in particular, testicular cells before birth (for example, When pluripotent stem cells were induced from primordial germ cells, etc., the production efficiency was sometimes reduced.
  • fibroblast growth factor is known as a polypeptide growth factor having various biological activities (TRENDS in Genetics, Vol. 20, No. ll, p.563-569). , 2004: Non-patent document 11). FGF1 to FGF23 have been confirmed in humans and mice. Since human FGF19 is an ortholog of mouse FGF15, the human and mouse FGF families are composed of 22 members (Genome Biol, vol. 2, 3005.1-3005.12, 2001: Non-patent document 12).
  • FGF1 subfamily FGF4 subfamily
  • FGF7 subfamily FGF8 subfamily one
  • FGF9 subfamily FGF11 subfamily
  • FGF19 subfamily The Human FGF is also about 150-300 amino acids in strength and contains a conserved core of about 120 amino acid residues with about 30-60% amino acid identity.
  • FGF has various biological activities and is reported to be useful for culturing various cells.
  • Patent Document 1 and Non-Patent Document 5 describe a method of proliferating spermatogonial stem cells using FGF2.
  • Cell, vol.104, p.875-889, 2001 (Non-patent Document 13) reported that FGF9-deficient mice undergo sex reversal to male and female, and that FGF9 is involved in testis development.
  • International Publication No. 96Z41523 Patent Document 2 discloses that FGF9 is a high affinity ligand for FGFR3 and is involved in bone and cartilage repair.
  • FGF R3 is a marker of mesenchymal skeletal progenitor cells and that FGF9, which is a ligand of FGFR3, can be used to isolate mesenchymal skeletal progenitor cells. ing.
  • Non-Patent Document 14 describes that FGF4 promotes proliferation of primordial germ cells (PGC).
  • Experimental Cell Research, vol.294, p.77-85, 2004 describes that FGF4 also protects male germ cells with apoptotic potential.
  • Development, vo 1.132, p.5399-5409, 2005 shows that FGF7 affects the number of germ cells.
  • Oncogene, vol.21, p.899-908, 2002 (Non-Patent Document 17) describes that overexpression of FGF4 in the testis enhances spermatogenesis.
  • Patent Document 1 International Publication No. 2004Z092357 Pamphlet
  • Patent Document 2 Pamphlet of International Publication No. 96Z41523
  • Patent Document 3 Pamphlet of International Publication No. 96Z41620
  • Patent Document 4 International Publication No. 2005Z100548 Pamphlet
  • Patent Document 5 Pamphlet of International Publication No. 2006Z028278
  • Patent Document 6 Japanese Unexamined Patent Application Publication No. 2006-115767
  • Non-patent literature l Proc. Natl. Acad. Sci. USA, vol. 91, No. 24, p. 11298-11302, 1994
  • Non-patent literature 2 Cellular and Molecular Life Sciences, vol. 58, No. 8, p. .1061- 1066, 200
  • Non-Patent Document 3 Science, vol.287, No.5457, p.1489-1493, 2000
  • Non-Patent Document 4 Biology of Reproduction, vol.69, No.2, p.612-616, 2003
  • Non-Patent Document 5 Biol. Reprod., Vol.72, p.985-991, 2005
  • Non-patent literature 6 Proc. Natl. Acad. Sci. USA, vol.102, p.17430-17435, 2005
  • Non-patent literature 7 Proc. Natl. Acad. Sci. USA, vol.102, p.14302-14307 , 2005
  • Non-Patent Document 8 Cell, vol.119, p.1001-1012, 2004
  • Non-Patent Document 9 Cell, vol.70, p.841-847, 1992
  • Non-Patent Document 11 TRENDS in Genetics, Vol.20, No.ll, p.563-569, 2004
  • Non-Patent Document 12 Genome Biol, vol.2, 3005.1-3005.12, 2001
  • Non-Patent Document 13 Cell, vol.104, p.875-889, 2001
  • Patent Document 14 Molecular Reproduction and Development, vol.68, p.5-16, 2004
  • Non-patent Document 15 Experimental Cell Research, vol.294, p.77-85, 2004
  • Non-Patent Document 16 Development, vol.132, p.5399-5409, 2005
  • Non-Patent Document 17 Oncogene, vol.21, p.899-908, 2002
  • Non-Patent Document 18 Nature, vol.359, 550-551, 1992
  • the first object of the present invention is to produce male germline stem cells stably and efficiently while minimizing the effects of differences in animal species and age differences in animals from which male germline stem cells are derived. And to provide a method that can be propagated.
  • the second object of the present invention is to stably and efficiently induce pluripotent stem cells from male germline stem cells while minimizing the effects of age differences in animals from which male germline stem cells are derived. Is to provide a possible way.
  • male germline stem cells can be induced with high efficiency by culturing mouse primordial germ cells in the presence of FGF9.
  • pluripotent stem cells can be induced even with mouse primordial germ cell power.
  • the present invention relates to the following.
  • a method for growing male germline stem cells comprising culturing male germline stem cells in a medium containing FGF (excluding FGF2).
  • FGF is a member of the FGF4 subfamily, FGF7 subfamily, FGF8 subfamily, FGF9 subfamily or FGF19 subfamily.
  • FGF is any one selected from the group consisting of FGF4, FGF5, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, and FGF20.
  • FGF is any one selected from the group consisting of FGF4, FGF5, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19 and FGF20.
  • a method for producing male germline stem cells comprising culturing male germline stem cells in a medium containing FGF (excluding FGF2).
  • a method for producing a fertility treatment comprising the following steps:
  • a method for producing a non-human animal that forms sperm derived from transplanted male germline stem cells comprising the following steps:
  • a method for producing sperm comprising the following steps:
  • a) proliferating male germline stem cells by culturing male germline stem cells in a medium containing FGF (excluding FGF2);
  • Method for producing embryos derived from male germline stem cells comprising the following steps: a) Proliferating male germline stem cells by culturing male germline stem cells in a medium containing FGF (excluding FGF2) The step of:
  • a method for producing non-human offspring derived from male germline stem cells comprising the following steps: a) Male germline stem cells are cultured by culturing male germline stem cells in a medium containing FGF (excluding FGF2). Proliferating;
  • a method for producing non-human progeny derived from male germline stem cells comprising the following steps: a) Male germline stem cells are cultured by culturing male germline stem cells in a medium containing FGF (excluding FGF2). Proliferating;
  • a method for producing male germline stem cells into which a foreign gene has been introduced including the following steps:
  • An agent for inducing differentiation of male germline stem cells including FGF (excluding FGF2).
  • a method for producing pluripotent stem cells comprising culturing a male germline stem cell precursor in a medium containing FGF (excluding FGF2) to obtain pluripotent stem cells.
  • Pluripotent stem cells derived from male germline stem cells including FGF (except FGF2) Composition for manufacture.
  • composition of [53], further comprising a GDNF receptor ligand [55] The composition of [53], further comprising a GDNF receptor ligand.
  • FGF excluding FGF2
  • male germline stem cells grown by the method of the present invention can be used to produce genetically modified animals (transgenic animals, gene-deficient animals, etc.), to treat male infertility, to develop therapeutic agents for male infertility, at the germ cell level.
  • the present invention is useful in the fields of biotechnology and medicine because it can be used for gene therapy research and drug development.
  • pluripotent stem cells from male germline stem cells stably and efficiently, regardless of the age difference of the animal from which the primitive germline stem cells are derived.
  • pluripotent stem cells obtained by the method of the present invention it is possible to construct various tissues having histocompatibility for autotransplantation, which is useful in the medical fields such as regenerative medicine and gene therapy. It is.
  • the pluripotent stem cells are useful in the biotechnology field because they can be used to produce genetically modified animals such as transgenic animals and knockout animals.
  • FIG. 1 is a graph showing the effect of FGF9 on the proliferation of rat spermatogonial stem cells.
  • FIG. 2 is a graph showing the effect of FGF9 on the proliferation of rat spermatogonial stem cells. The results of three independent trials are shown. The black mark indicates the FGF9 non-added group, and the white mark indicates the FGF9 added group.
  • FIG. 3 is a graph showing the effect of various combinations of site force-in on rat GS cell proliferation. All cultures are supplemented with FGF9.
  • E EGF
  • F FGF2
  • G GDN F 0
  • FIG. 4 is a photograph showing the result of observing with a microscope the effect of FGF9 on the establishment of GS cell colonies from rat testis cells.
  • FIG. 5 is a diagram showing the results of observing a recipient mouse testis transplanted with GFP-labeled rat GS cells under UV light. A vast GFP-positive spermatogenesis from the donor cell is observed.
  • FIG. 6 A photograph showing the results of observation of spermatogenic cells used for microinsemination under visible light and UV. In the center of the photo, donor cell-derived GFP-positive round spermatids are observed.
  • FIG. 7 is a photograph showing the results of observation of rat GS cell-derived progeny grown in a medium containing FGF9 under visible light and UV. Since GS cells are derived from GFP rats, their offspring (right rat) show fluorescence under UV.
  • FIG. 8 is a photograph showing the results of observation of rat GS cells 3 days after EGFP gene transfection under visible light and UV. Bar: 200 m
  • FIG. 9 is a photograph showing the results of observation of a stable EGFP gene-introduced GS cell established by G418 selection under UV. Shows the state of cells 99 days after transfusion
  • FIG. 10 is a photograph showing a male reproductive lineage stem cell obtained by culturing mouse primordial germ cells in a medium containing FGF9.
  • FIG. 11 is a photograph showing a colony of pluripotent stem cells induced by culturing 12.5 dpc mouse primordial germ cells in a medium containing FGF9.
  • the male germline stem cell growth method of the present invention is characterized in that male germline stem cells are cultured in a medium containing FGF (excluding FGF2). By using FGF, the proliferation efficiency of male germline stem cells is dramatically increased.
  • Method I of the present invention is a method for producing a new male germline stem cell by culturing male germline stem cells in a medium containing FGF (excluding FGF2). But there is.
  • male germline stem cells self-replicate and differentiate into sperm or a precursor cell thereof (for example, spermatogonia, sperm cells, spermatogonia, spermatocytes, sperm cells, etc.).
  • Germline cells that have the ability to obtain (ability as male germline stem cells).
  • male germline stem cells include spermatogonial stem cells and GS cells.
  • GS cells are spermatogonial stem cells proliferated in vitro dependently on GDNF receptor agonist compounds (GDNF, etc.), for example, Biol. Reprod., Vol.69, p612-616, 2003 It refers to spermatogonial stem cells grown by the method.
  • GDNF GDNF receptor agonist compounds
  • the cell is a male germline stem cell is described in, for example, Brinster, RL et al., Proc Natl Acad Sci USA, vol.91, No.24, p.11298-11302, 1994, etc. According to the method
  • cells having the ability to form spermatogenic colonies are determined to be male germline stem cells.
  • Whether or not a cell is a male germline stem cell can also be determined by analyzing the expression of a cell surface marker or the like using a flow cytometer or the like, for example.
  • a cell surface marker include j81-integrin, ⁇ 6-integrin, EE2, EpCAM, SSEA-1, c-kit, CD9, Forssman antigen and the like (for example, WO2004 / 092357 etc.)
  • the male germline stem cell used in Method I of the present invention is not particularly limited as long as it is a vertebrate cell.
  • the vertebrates include mammals, birds, fish, amphibians, and reptiles. Mammals include, for example, rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, domestic animals such as pigs, rabbits, goats, horses, hedges, minks, and dogs. And pets such as cats, primates such as humans, monkeys, monkeys, marmosets, orangutans and chimpanzees.
  • the vertebrate is preferably a mammal.
  • the mammal may be before or after birth as long as male germline stem cells can be grown by the method I of the present invention.
  • the age of the animal from which the cells are derived is not particularly limited as long as the male germline stem cells can be propagated by the method I of the present invention, and the newborn, infant, although it may be adult or senile, younger animals have a higher frequency of stem cells (spermatogonial stem cells, etc.) contained in the testis. Is preferably used.
  • the male germline stem cells used in the method I of the present invention may be isolated or purified, or may be used in the present invention as testis cells containing male germline stem cells.
  • Testicular cells can be prepared from the vertebrate testis by a method known per se. For example, the testis is removed, and the extracted testis is digested with a degrading enzyme such as collagenase, trypsin or DNase to disperse testis cells (see, for example, WO2004 / 09 2357). The dispersed testis cells are washed with a culture solution and collected.
  • Isolation and purification of male germline stem cells can be achieved, for example, by 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 male germline stem cells. It can be carried out.
  • spermatogonial stem cells can be enriched using cell surface antigens such as ⁇ 6-integrin, c kit, CD9, etc. (for example, Proc. Natl. Acad. Sci. USA, 97, 8346-8351, 2001). Etc.).
  • spermatogonial stem cells can be concentrated using d ye such as Hoechst (eg, Development, 131, 479 -487, 2004 etc.).
  • the male germline stem cells may be cultured in vitro before being used in Method I of the present invention.
  • the conditions for this preculture are not particularly limited. For example, as described in WO2004 / 092357 and Biol. Reprod., Vol.72, p.985-991, 2005, the pre-culture conditions were obtained by the enzyme treatment described above. By culturing testis cells in the presence of GDNF, LIF, EGF, FGF2, etc., male germline stem cells can be proliferated.
  • FGF is a known site force-in, and FGF1 to FGF23 have been confirmed in humans and mice. Since human FGF19 is an ortholog of mouse FGF15, one human and mouse FGF family consists of 22 members (TRENDS in Genetics, Vol. 20, No.ll, p.563-569, 2004, Genome Biol, vol.2, 3005.1—3005.12, 2001). In this specification, the name of FGF shall follow the human FGF nomenclature.
  • FGFs include human and mouse FGFs as shown in Table 1.
  • Table 1 shows the Genbank accession numbers of the amino acid sequences of human and mouse wild-type FGF.
  • FGF is classified into seven subfamilies (FGF1 subfamily, FGF4 subfamily, FGF7 subfamily, FGF8 subfamily, FGF9 subfamily, FGF11 subfamily and FGF19 subfamily) based on phylogenetic analysis.
  • the FGF1 subfamily consists of FGFl (aFGF) and FGF2 (bFGF) forces.
  • the first FGF4 subfamily consists of FG F4, FGF5 and FGF6 forces.
  • the FGF7 subfamily is FGF3, FGF7 (KGF), F It consists of GF10 and FGF22.
  • the FGF8 subfamily consists of FGF8, FGF17 and FGF18.
  • the FGF9 subfamily consists of FGF9, FGF16 and FGF20.
  • the FGF11 subfamily consists of FGF11, FGF12, FGF13 and FGF14.
  • the FGF19 subfamily consists of FGF19, FGF21 and FGF23 forces.
  • the FGF used in the method I of the present invention is preferably a member of the FGF4 subfamily, the FGF7 subfamily, the FGF8 subfamily, the FGF9 subfamily or the FGF19 subfamily, more preferably the FGF9 subfamily. Be a member.
  • the FGF used in the method I of the present invention is preferably FGF4, FGF5, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, 019 and. It is any one selected from the group consisting of 20, and more preferably FGF9.
  • the concentration of FGF contained in the medium is not particularly limited as long as it is a concentration that promotes the growth of male germline stem cells and can achieve the proliferation of the cells, but is usually 0.05 ngZml ⁇ : LOOmgZml, for example, 0. 5ngZml ⁇ : LOO / z gZml, preferably more preferably 0. 5ng / ml ⁇ 10 ⁇ gm 0. 511 8 / 1111 ⁇ 1 8/1111, more preferably 0. 5 ⁇ 200ng / ml, more /! More preferably, it is 0.5-50 ngZml, most preferably 2-20 ngZml.
  • the medium used in the method I of the present invention preferably further contains a GDNF receptor ligand.
  • the GDNF receptor ligand means a compound that can bind to a GDNF receptor or a co-receptor of the receptor and activate a cell or a living body through the receptor.
  • Examples of the GDNF receptor ligand include glial cell-derived neurotrophic factor (GDN F), northrin (Neurturin), persephin (Artemin), and the like.
  • GDN F glial cell-derived neurotrophic factor
  • Neuroturin northrin
  • Artemin persephin
  • the GDNF receptor ligand is preferably GDNF.
  • GDNF examples include GDNF such as human and rat HWO93Z06116 pamphlet) and mouse (see, for example, Gene 203, 2, 149-157, 1997).
  • the GDNF receptor means a compound that is a GDNF binding substance and can transmit a GDNF signal into a cell or a living body.
  • the GDNF receptor includes GDNF Mention may be made of the cR et receptor thycin synkinase which is a gnnal-mediated receptor.
  • the co-receptor for GDNF means a compound that does not directly transmit a GDNF receptor ligand signal into cells or living bodies, but activates a receptor that transmits the GDNF receptor ligand signal.
  • Such compounds are in particular receptors whose members are referred to as GDNF family receptor alphas (GFR a s). They are also related to the signaling receptor complex of GDNF, percefin, artemin and northrin. This family of receptors includes 4 members (GFR al-4) (Jing, S “et al” Cell, 85, 9-10 (1996); Jing, S. Q "et al” J. Biol.
  • the GDNF receptor ligand also includes an antibody that specifically recognizes the GDNF receptor or a neural co-receptor of the receptor, a binding fragment thereof, and the like.
  • the concentration thereof is particularly limited as long as it is a concentration that can promote the growth of male germline stem cells and achieve the proliferation of the cells. but are not usually 0. 05NgZml ⁇ 100mgZml, for example 0. 5ng / m 1 ⁇ : LOO gZml , preferably 0. 5ng / ml ⁇ 10 g / ml, more preferably 0. 5ng / ml ⁇ 1 8/1111 , further Preferably it is 0.5 to 200 ng / ml, more! /, More preferably 0.5 to 50 ng / m, most preferably 2 to 20 ng Zml.
  • the medium used in the method I of the present invention preferably further contains FGF2.
  • FG F2 is useful for assisting the proliferation of male germline stem cells, particularly for inducing or enhancing proliferation, and by adding FGF2 to the medium, male germline stem cells can be propagated more stably and efficiently. .
  • FGF2 examples include human FGF2 (see, for example, Endocrine Rev., 8, 95, 1987), ushi FGF2 (see, for example, Proc. Natl. Acad. Sci. USA, 81, 6963, 1984), mouse FGF2 (See, for example, Dev. Biol, 138, 454-463, 1990), rat FGF2 (eg Biochem. Biophys. Res Commun., 157, 256-263, 1988).
  • human FGF2 see, for example, Endocrine Rev., 8, 95, 1987
  • ushi FGF2 see, for example, Proc. Natl. Acad. Sci. USA, 81, 6963, 1984
  • mouse FGF2 See, for example, Dev. Biol, 138, 454-463, 1990
  • rat FGF2 eg Biochem. Biophys. Res Commun., 157, 256-263, 1988.
  • FGF2 when FGF2 is contained in the medium, its concentration is not particularly limited as long as it is a concentration that can support the growth of male germline stem cells and achieve the growth of the cells.
  • concentration S normal concentration 0.05 ng / ml ⁇ : LOOmg / ml, f row, 0.5 ng / ml ⁇ : LOO / zg Zml, preferably 0.5 ngZml ⁇ : L0 ⁇ g / mU, more preferably 0. 5 ng Zml to l ⁇ g / m 1, more preferably 0.5 to 200 ng / ml, more preferably 5 to 50 ng / ml, most preferably 2 to 20 ng Zml.
  • the medium used in the method I of the present invention may further contain epidermal growth factor (EGF).
  • EGF is useful for assisting the proliferation of male germline stem cells, particularly for inducing or enhancing proliferation, and by adding EGF to the medium, male germline stem cells can be more stably and efficiently proliferated.
  • EGF EGF such as mouse (see Nature, 257, 325-327, 1975), human (see Proc. Natl. Acad. Sci. USA, 88, 415, 1991), for example. Is done.
  • the concentration thereof is not particularly limited as long as it is a concentration that can support the proliferation of male germline stem cells and achieve the proliferation of the cells.
  • the medium used in Method I of the present invention may further contain leukemia inhibitory factor (LIF).
  • LIFs include humans (Japanese Patent Laid-Open No. 1-502985), mice (Japanese Patent Laid-Open No. 1-502985), hedges (Japanese Patent Laid-Open No. 4-502554), pigs (Japanese Patent Laid-Open No. 4-502554), ushi (Japanese Patent Laid-Open No. 8-154681). LI F etc. are exemplified.
  • LIF when contained in the medium, its concentration is usually 10 to: L0 6 units / ml, for example, 10 to: L0 5 units / ml, preferably 10 2 to 10 4 units / ml, More preferably, it is 3 ⁇ 10 2 to 5 ⁇ 10 3 units / ml.
  • the cyto force-in (FGF, GDNF receptor ligand, FGF2, EGF, LIF, etc.) that can be contained in the medium in the method I of the present invention is derived from an animal, preferably from the aforementioned mammals. If it is not particularly limited! ,.
  • the site force-in is purified as long as it has the same activity as that of the corresponding wild-type site force-in in promoting the growth of male germline stem cells. Natural, synthetic or recombinant proteins, mutant proteins (including insertion, substitution and deletion mutants), fragments, and chemically modified derivatives thereof.
  • Proliferation promoting activity of male germline stem cells of the same degree means that the growth promoting activity of male germline stem cells is within the range of about 0.1 to: LO times, preferably about 0.5 to 2 times. It is within the range.
  • the growth promotion activity of male germline stem cells is determined by determining the growth rate of male germline stem cells when male germline stem cells are cultured for a certain period of time in a medium containing the noted site force-in, and in a control medium that does not contain the site force-in. It can be determined by comparing with the case where male germline stem cells are cultured in the same period.
  • each site force-in has the same activity as that of the corresponding wild-type site force-in as long as it has the same activity as that of male germline stem cells. It also includes proteins that are substantially homologous to the wild type amino acid sequence.
  • substantially homologous means the degree of homology to the wild-type amino acid sequence, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. It means that.
  • Homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is used for optimal alignment). % Of the same amino acid and similar amino acid residues to all overlapping amino acid residues (which can be considered to introduce gaps into one or both of the sequences).
  • Similar amino acids '' mean amino acids that are similar in physical and physical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met) Amino acids that fall into the same group are listed. It is expected that such substitutions with similar amino acids will not change the phenotype of the polypeptide (ie, are conservative amino acid substitutions). Examples of conservative amino acid substitutions are well known in the art and are described in various literature (eg, Bowie et al. , Science, 247: 1306-1310 (1990)).
  • NCBI BLAST-2 National Center for Biotechnology Information Basic Local Alignment Search Tool
  • the basal medium of the medium used in the method I of the present invention may be any known per se, and is not particularly limited.
  • a mixture of the above basal medium which may be a medium modified for ES cell culture or the like, may be used.
  • the medium used in the method I of the present invention may contain an additive known per se.
  • the additive is not particularly limited, but for example, growth factors (such as insulin), iron sources (such as transferrin), polyamines (such as putrescine), minerals (such as sodium selenate), sugars (such as glucose) ), Organic acids (eg pyruvic acid, lactic acid) Etc.), serum proteins (eg albumin), amino acids (eg L-glutamine, etc.), reducing agents (eg 2-mercaptoethanol, etc.), vitamins (eg ascorbic acid, d biotin etc.), steroids (eg estradiol, progesterone etc.) ), Antibiotics (eg, streptomycin, penicillin, gentamicin, etc.), buffering agents (eg, HEPES, etc.), nutritional additives (eg, StemPro- Nutrient Supplement, etc.), and the like.
  • the additives are preferably contained within a concentration range known per se.
  • the medium used in the method I of the present invention may contain serum.
  • the serum is not particularly limited as long as it is an animal-derived serum, but is preferably the above-mentioned mammal-derived serum (for example, fetal bovine serum, human serum, etc.).
  • serum substitute additives for example, Knockout Serum Replacement (KSR) (manufactured by Invitrogen)
  • KSR Knockout Serum Replacement
  • the serum concentration is not particularly limited, but is usually in the range of 0.1 to 30 ( ⁇ )%.
  • the serum concentration is lowered (for example, 0.1 to 5). (vZv)%) May be set.
  • the medium used in the method I of the present invention is B27 (J. Neurosci. Res., Vol. 35, p. 567-576, 1993, Brain Res., Vol. 494, p. 65-74, 1989. ) May be included.
  • B27 can enhance the growth of male germline stem cells (Biol. Reprod., Vol.72, p.985-991, 2005).
  • B27 commercially available products such as B-27 Supplement ⁇ p ⁇ 3 ⁇ 4 J, B-27 Supplement Minus AU ⁇ p ⁇ 3 ⁇ 4 J, B-27 Supplement Minus Vitamin A (trade name) (above, manufactured by Invitrogen) It may be used.
  • the amount of B27 added to the medium is not particularly limited as long as proliferation of male germline stem cells can be enhanced in the method of the present invention.
  • B-27 Supplement Invitrogen
  • 1- 100 ⁇ l / m, preferably about 5 to 40 ⁇ l Zml is added to the medium.
  • male germline stem cells may be cultured in the presence of feeder cells.
  • a feeder cell is a non-male germline stem cell that, when cultured with a male germline stem cell, provides an environment that supports the growth of the male germline stem cell while maintaining its ability as a male germline stem cell.
  • the feeder cell is not particularly limited, but a per se known one can be used. Mammalian fetal fibroblasts, mouse fibroblast cell line STO, etc.). Feeder cells are preferably inactivated by methods known per se, such as irradiation with radiation (gamma rays, etc.) or treatment with anticancer agents (mitomycin c, etc.).
  • one feeder cell for example, about 10 5 to 10 6 cells per hole of a 6-well plate is usually used.
  • the male germline stem cell adheres to the feeder cell and can form a growing colony more efficiently.
  • male germline stem cells may be cultured while attached to an insoluble carrier via an extracellular matrix such as laminin (Biol. Reprod., Vol. 72, p.985-991, 2005).
  • an extracellular matrix such as laminin (Biol. Reprod., Vol. 72, p.985-991, 2005).
  • male germline stem cells adhere indirectly to the insoluble carrier by adhering to the extracellular matrix coated on the surface of the insoluble carrier.
  • the insoluble carrier is not particularly limited as long as it can achieve proliferation of male germline stem cells when used in the method I of the present invention.
  • a member having an affinity for can be used.
  • plastic or glass members are preferred.
  • the insoluble carrier may be metal or ceramic and is not limited to a certain material.
  • the insoluble carrier is usually formed into a culture device (equipment or material for use in culture) such as a petri dish, a plate, a flask, a bottle, a bead, or a hollow fiber, and provided to the culture.
  • the extracellular matrix is a biopolymer that constitutes the space outside the cell.
  • extracellular matrix include fibrous proteins such as collagen and elastin, cell adhesion proteins such as darcosaminodarlicans such as hyaluronic acid and chondroitin sulfate, proteoglycan, fibronectin, vitronectin and laminin.
  • cell adhesion proteins such as darcosaminodarlicans such as hyaluronic acid and chondroitin sulfate, proteoglycan, fibronectin, vitronectin and laminin.
  • it is not particularly limited as long as it can mediate the adhesion of lineage stem cells to an insoluble carrier and can achieve proliferation of male germline stem cells, it is preferably a cell adhesion protein, more preferably laminin.
  • a method for coating the extracellular matrix with an insoluble carrier generally, a method using a non-covalent bond (hydrogen bond, ionic bond, hydrophobic bond, etc.), a covalent bond, or the like can be used.
  • a suitable buffer containing an extracellular matrix eg phosphate buffer
  • the extracellular matrix can be bound to the insoluble carrier by noncovalent bonding.
  • the culture conditions in Method I of the present invention can be used.
  • the culture temperature is usually in the range of about 30-40 ° C, preferably about 37 ° C.
  • the CO concentration is usually in the range of about 1-10%,
  • the humidity is usually in the range of about 70 to 100%, preferably about 95 to 100%.
  • the passage interval and dilution rate of male germline stem cells are appropriately set depending on the culture conditions, but are usually 1Z2 to: LZ 10 times diluted at intervals of 2 to 10 days.
  • the cells are passaged.
  • the male germline stem cells grown by the method I of the present invention can be cryopreserved semipermanently, and can be used after thawing and asleep as necessary.
  • the male germline stem cells maintain their ability to self-replicate and differentiate into sperm or their progenitor cells (capability as male germline stem cells) even after cryopreservation / thawing.
  • cells are suspended in a composition for cell cryopreservation known per se such as a cell banker (manufactured by DIA-IA TRON) containing dimethyl sulfoxide and ushi fetal serum albumin. Store cells at -200 ° C, preferably -196 ° C (in liquid nitrogen).
  • the male germline stem cells proliferated by the method I of the present invention are allowed to sleep after cryopreservation, they are thawed and suspended in a solvent according to a conventional method to obtain a cell suspension.
  • the method of thawing is not particularly limited, but for example, it can be carried out using DMEM (DMEMZFCS) containing 10% fetal bovine serum in a 37 ° C constant temperature bath. Specifically, a freezing tube is floated on a thermostatic bath, and DMEMZFCS is dropped into the frozen cells to thaw. The cells are washed by centrifugation and then resuspended in the medium.
  • DMEM fetal bovine serum
  • the male germline stem cell expanded by the method I of the present invention maintains the ability as a male germline stem cell
  • the cell is transplanted into the seminiferous tubule of an infertile animal.
  • male germline stem cell transplantation methods include direct injection into the seminiferous tubule, injection from the export tube, and injection from the testis network. The type of animal to be injected, ease of operation, etc. Can be selected as appropriate. For example, in rodents such as mice and rats, injection from an export tube is preferably used, and in livestock such as eagle, injection of testicular force is preferably used.
  • the transplanted male germline stem cells proliferate in the seminiferous tubule and form spermatogenic colonies.
  • infertile animals include male animals that have been infertile by administration of an anticancer agent such as busulfan, or male animals that are genetically deficient in spermatogenic ability (eg, W mice). Natl. Acad. Sci. USA, vol.91, No.24, p.11298-11302, 1994, Cellular and Molecular Life Sciences, vol.58, No.8, p.1061-1066, 2001 etc.) .
  • the animal species of the infertile animal is preferably the same as the animal species of the male germline stem cell to be transplanted. Further, it is more preferable that the line of the infertile animal is the same as the line of the male germline stem cell to be transplanted.
  • the infertile animals should be used to reduce the possibility of transplanted cells being rejected.
  • an antibody that is genetically deficient in immune function eg, a nude mouse
  • an antibody against cell surface antigens eg, CD4
  • T cells tumor necrosis cells
  • the male force obtained by the above-described method to form spermatozoa derived from transplanted male germline stem cells is collected (isolated), so that the males propagated by the method of the present invention.
  • Sperm derived from germline stem cells can be produced.
  • embryos derived from male germline stem cells grown by the propagation method of the present invention by fertilizing the sperm obtained by the above-described method into an egg to obtain an embryo.
  • An egg is a female gamete that can be fertilized by a sperm.
  • eggs include egg cells and oocytes. Fertilization of sperm and eggs is performed by well-known methods such as micro-fertilization and IVF. It can be carried out.
  • the embryo obtained by the above-described method is transferred to the uterus or fallopian tube of the host female animal to obtain offspring, so that the offspring derived from male germline stem cells proliferated by Method I of the present invention Can be manufactured.
  • the host female is preferably a pseudopregnant animal.
  • Pseudopregnant animals can be obtained by mating a female animal with a normal cycle with a male animal castrated by vas deferens or the like.
  • the host female animal into which the embryo has been transferred becomes pregnant and gives off offspring derived from male reproductive lineage stem cells grown by Method I of the present invention.
  • the animal of the present invention obtained by the above-described method can be naturally mated with a spermatogenic animal derived from transplanted male germline stem cells to obtain offspring.
  • Progeny derived from male germline stem cells grown by Method I can be produced.
  • the female used for the natural mating is usually a wild type female animal.
  • the method I of the present invention By using the method I of the present invention, it is possible to proliferate male germline stem cells stably over a long period of time while maintaining the ability as male germline stem cells.
  • the gene of the male germline stem cell proliferated by the method I of the present invention is modified, for example, a male germline stem cell into which a specific foreign gene has been introduced, a male germline stem cell lacking a specific gene, etc. It is possible to produce genetically modified male germline stem cells.
  • a vector constructed so that a specific gene can be expressed functionally is introduced into male germline stem cells.
  • a vector a plasmid vector, a virus vector, or the like can be used.
  • virus vectors include retrovirus, adenovirus, lentivirus, herpes virus, adeno-associated virus, parvovirus, Semliki Forest virus, vaccinia virus, and the like.
  • Examples of a method for introducing a vector into a male germline stem cell include a general gene introduction method such as a calcium phosphate method, a DEAE dextran method, an electopore position method, or a lipofuxion method.
  • a virus When a virus is used as a vector, the virus genome may be introduced into the cell by the general gene transfer method described above, and the virus particle The virus genome can also be introduced into a cell by infecting the pup with cells.
  • a marker gene may be introduced into a cell simultaneously with a vector, and the cell may be cultured by a method according to the nature of the marker gene.
  • the marker gene is a gene that confers drug resistance to a selected drug exhibiting lethal activity on the host cell
  • the cell into which the vector has been introduced can be cultured using a medium containing the drug. Ryo.
  • Examples of combinations of a drug resistance-conferring gene and a selective drug include a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, and a combination of a blasticidin S resistance-conferring gene and blasticidin S. Combinations can be given.
  • male germline stem cells lacking a specific gene it is also possible to obtain male germline stem cells lacking a specific gene using the same method.
  • Examples of a method for obtaining male germline stem cells deficient in a specific gene include homologous thread reversion (gene targeting method) using a targeting vector.
  • a chromosomal DNA of a specific gene is isolated, and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (j8-galactosidase gene), cat (chloramphee-cholacetyltransferase)
  • a reporter gene such as a gene
  • a DNA sequence for example, polyA addition signal
  • a gene conferring drug resistance When a gene conferring drug resistance is used as the marker gene, it is sufficient to culture the cell into which the vector has been introduced using a medium containing the drug.
  • a drug resistance-conferring gene and a selective drug include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a nodylomycin resistance-conferring gene and hygromycin, and blasticidin S resistance. Examples include a combination of a sex-imparting gene and blastcidin s.
  • a marker gene such as a thymidine kinase (TK) gene that is toxic to cells can be placed outside the homologous recombination region in the targeting vector, and the marker gene can exert toxicity to the cell.
  • TK thymidine kinase
  • the DNA on the DNA of the specific gene is the Southern hybridization analysis using the DNA sequence in the vicinity as a probe, or the DNA sequence on the targeting vector and the specific DNA used for the preparation of the targeting vector. It can be obtained by PCR analysis using a DNA sequence in the vicinity region other than the DNA of the gene as a primer and selecting male germline stem cells lacking a specific gene.
  • a Cre— ⁇ system that deletes a specific gene in a tissue-specific or developmental stage-specific manner may be used (Marth, JD (1996) Clin. Invest. 97: 19 99-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 25: 4323-4330).
  • the gene trap method searches for an unknown gene by utilizing the fact that a trap vector containing a reporter gene is introduced into a cell and randomly incorporated into an endogenous locus on the chromosome, or an endogenous gene.
  • Trap vectors are usually a reporter gene (such as a lacZ gene), a plasmid sequence (a sequence necessary for recovery of an endogenous gene by the plasmid rescue method, including the replication origin), and a selectable marker gene (neo). Gene etc.).
  • the reporter gene usually does not have a promoter and has only a splice acceptor, the reporter gene is expressed only when it is incorporated downstream of the endogenous gene. Single integration of the trap vector most often results in the destruction of the endogenous gene. Therefore, endogenous genes are disrupted by introducing trap vectors into male germline stem cells. Male germline stem cells can be obtained.
  • a non-human animal that forms sperm derived from the genetically modified male germline stem cells and genetically modified male reproduction by the same method as described above.
  • Sperm derived from lineage stem cells (genetically modified sperm)
  • embryo derived from genetically modified male germline stem cells (genetically modified embryo)
  • non-human offspring derived from genetically modified male germline stem cells (genetically modified non-human offspring or genetically modified) Non-human animals)
  • Non-human animals can be manufactured.
  • the method I of the present invention and the male germline stem cells expanded by the method are useful for the treatment of male infertility.
  • male germline stem cells are collected by biopsy from the testes of infertile patients, and the cells are cultured in a test tube by the method I of the present invention. Then, male germline stem cells grown in the seminiferous tubule of the infertile patient are injected (microinduction) to cause spermatogenesis derived from cultured cells in the seminiferous tubule of the patient's testis. Infertility treatment can be implemented. This technique is particularly effective for infertility due to, for example, chemotherapy or radiotherapy.
  • the infertility treatment agent specifically for males containing the male germline stem cells is also provided by the present invention. Is within the range.
  • the infertility treatment agent of the present invention can be produced by mixing an effective amount of the male germline stem cells with a pharmaceutically acceptable carrier according to conventional means.
  • the fertility treatment agent of the present invention is usually produced as a parenteral preparation such as an injection, a suspension, or a drip.
  • Carriers that can be included in the parenteral preparation include, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose, and other adjuvants.
  • An aqueous liquid for injection can be mentioned.
  • the immunomodulating agent of the present invention includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, salt benzalcoum, hydrochloric acid hydrochloride, etc.), stable You may mix
  • a buffer for example, phosphate buffer, sodium acetate buffer
  • a soothing agent for example, salt benzalcoum, hydrochloric acid hydrochloride, etc.
  • blend with an agent for example, human serum albumin, polyethyleneglycol etc.
  • a preservative for example, human serum albumin, polyethyleneglycol etc.
  • the preparation thus obtained is safe and has low toxicity. It can be administered to dairy animals.
  • the male germline stem cells grown by the method are useful for gene therapy at the germline level.
  • a male germline stem cell is collected by biopsy from a patient having a mutation in a specific gene and cultured in a test tube by the method of the present invention. Then, a male germline stem cell in which the mutated gene is replaced with a normally functioning gene by the above-described gene modification method or the like is prepared, and the mutation is prevented from being transmitted to offspring.
  • a treatment method also requires the maintenance and proliferation of male germline stem cells. In this case, the method of the present invention can be used effectively.
  • the present invention also relates to a male germline stem cell proliferation promoter comprising FGF (excluding FGF2).
  • FGF excluding FGF2
  • culturing male germline stem cells by the method I of the present invention using a medium containing the agent of the present invention the proliferation efficiency of male germline stem cells is dramatically increased.
  • the growth efficiency of male germline stem cells is dramatically increased by supplementing the agent of the present invention with a conventional culture medium for male germline stem cells.
  • the FGF used in the agent of the present invention is preferably a member of the FGF4 subfamily, FGF7 subfamily, FGF8 subfamily, FGF9 subfamily or FGF19 subfamily, more preferably a member of the FGF9 subfamily. It is.
  • the FGF used in the agent of the present invention is preferably any one selected from the group consisting of FGF4, FGF5, FGF6, FGF8, F GF9, FGF10, FGF16, FGF17, FGF18, FGF19 and FGF20, More preferred is FGF9.
  • the agent of the present invention may further contain a GDNF receptor ligand.
  • the GDNF receptor ligand is preferably GDNF.
  • the agent of the present invention may further contain FGF2.
  • the agent of the present invention may further contain EGF.
  • the agent of the present invention may further contain LIF.
  • the agent of the present invention may further contain a physiologically acceptable carrier (for example, physiological isotonic solution (physiological saline, basal medium described above, glucose and other adjuvants (for example, D-sorbitol, D — Isotonic solutions including mantol, sodium chloride, etc.), excipients, preservatives, stabilizers ( (E.g., human serum albumin, polyethylene glycol, etc.), binders, solubilizers, nonionic surfactants, buffers (eg, phosphate buffer, sodium acetate buffer), preservatives, antioxidants, And the like).
  • physiological isotonic solution physiological saline, basal medium described above, glucose and other adjuvants (for example, D-sorbitol, D — Isotonic solutions including mantol, sodium chloride, etc.
  • excipients for example, preservatives, stabilizers (E.g., human serum albumin, polyethylene glycol, etc.), binders
  • the mixing ratio of each of the above-mentioned constituents contained in the agent of the present invention is such that when the agent of the present invention is added to the medium used in Method I of the present invention, It is preferable that the concentration in the medium is configured to fall within the above-described preferable range.
  • the agent of the present invention is used in the form of an isotonic aqueous solution or powder and added to the medium used for the method I of the present invention.
  • the agent of the present invention may be a medium used for the method I of the present invention.
  • the male germline stem cell production method of the present invention is characterized in that a male germline stem cell precursor is cultured in a medium containing FGF (excluding FGF2) to obtain a male germline stem cell.
  • FGF excluding FGF2
  • the activity as a male germline stem cell is induced, the precursor power of the male germline stem cell is strongly induced into the male germline stem cell, and the production efficiency of the male germline stem cell is dramatically increased.
  • FGF excluding FGF2
  • a male germline stem cell precursor refers to an undifferentiated cell having the ability to be separated into a male germline stem cell.
  • male germline stem cell precursors include epipiblast, primordial germ cells, and gonocytes.
  • the precursor is preferably a primordial germ cell.
  • the male germline stem cell precursor used in the method II of the present invention is not particularly limited as long as it is an animal derived from a vertebrate.
  • vertebrates include those similar to Method I above.
  • the vertebrate is preferably a mammal.
  • the mammal may be before or after birth as long as male germline stem cells can be produced by the method II of the present invention.
  • male germline stem cell progenitor When a prenatal male germline stem cell progenitor is used, the stage of development of the embryo (embryo) from which the cell is derived is special as long as male germline stem cells can be produced by Method II of the present invention.
  • male germline stem cells derived from embryos after the appearance of primordial germ cells in the embryo preferably after the formation of the male genital ridge, are used.
  • a male germline stem cell precursor of usually 8.5 dpc or later, preferably 11.5 dpc or later is used.
  • the age of the animal from which the cell is derived is not particularly limited as long as male germline stem cells can be produced by the method II of the present invention. Any of an infant, an adult, and an old body may be used. For example, an individual at a stage before gonocytes are separated into spermatogonial stem cells in the testis is used. For example, in mice, gonocytes are separated into spermatogonial stem cells by 5 days after birth, so male germline stem cell precursors are used younger than 5 days after birth.
  • the male germline stem cell precursor used in the method II of the present invention may be isolated or purified, or as a testis cell containing a male germline stem cell precursor in the present invention. May be used.
  • Testis cells can be prepared from vertebrate testis by a method known per se. For example, the testis is removed, and the extracted testis is digested with a degrading enzyme such as collagenase, trypsin, or DNase to disperse testis cells (see, for example, WO2004 / 092357). The dispersed testis cells are washed with a culture solution and collected.
  • the sex of the embryo can be determined by confirming the presence of the Y chromosome by, for example, dienoting based on the PCR method.
  • the embryo is a mouse, for example, the Ubel PCR method (Dev. Biol, vol. 229, p. 468-479, 2001) can be used.
  • Primordial germ cells are at various stages of embryonic development! Tissues rich in primordial germ cells (bottom of the urinary membrane, the rear end of the embryo, hindgut, mesentery, urogenital ridge, reproductive ridge, etc.) ) Can be collected by digestion with a degrading enzyme such as trypsin.
  • a degrading enzyme such as trypsin.
  • trypsin a degrading enzyme
  • the posterior third of the embryo in the 10.5dpc embryo, the mesentery and urogenital ridge, and in the 11.5-16.5dpc embryo, the genital ridge is rich in primordial germ cells.
  • the FGF used in the method II of the present invention is preferably a member of the FGF9 subfamily, more preferably FGF9.
  • the concentration of FGF contained in the medium is not particularly limited as long as male germline stem cells can be produced by the method II of the present invention, but is usually 0.05 ngZml to 100 mgZml, such as 0.5 ng / ml to 100 mu g / ml, preferably 0. 5ng / ml ⁇ : LO ⁇ g / ml, more rather preferably is 0.511 8 / 1111-1 8/1111, more preferably 0. 5 ⁇ 200ng / ml, more ! /, Preferably 0.5-50 ngZml, most preferably 2-20 ngZml.
  • the medium used in the method II of the present invention preferably further contains a GDNF receptor ligand.
  • the GDNF receptor ligand is preferably GDNF.
  • the GDNF receptor ligand when the GDNF receptor ligand is contained in the medium, its concentration is not particularly limited as long as male germline stem cells can be produced by the method of the present invention.
  • 05 ngZml to 100 mgZml for example 0.5 ngZml to 100 ⁇ g / mU, preferably 0.5 ng / ml to: LO ⁇ g / ml, more preferably 0.5 ngZml to l ⁇ g / ml, more preferably 0.5 to 200 ng / ml, more! /, More preferably 0.5-50 ng / ml, most preferably 2-20 ng Zml.
  • the medium used in the method II of the present invention preferably further contains FGF2.
  • the concentration thereof is not particularly limited as long as male germline stem cells can be produced by the method II of the present invention, but a normal concentration of 0.05 ng / ml ⁇ : L00mg / ml, f row, 0.5ng / ml to 100 ⁇ g / ml, preferably 0.5ngZml to 10 ⁇ g / ml, more preferably 0.5ngZml to l ⁇ g / ml, and more Preferably it is 0.5 to 200 ng / ml, more preferably 0.5 to 50 ng / ml, most preferably 2 to 20 ng Zml.
  • the medium used in the method II of the present invention may further contain epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • the concentration thereof is not particularly limited as long as male germline stem cells can be produced by the method II of the present invention, but the normal concentration is 0. 05ngZml ⁇ : LOOmgZml, eg 0.5ngZml ⁇ : LOO / z gZml, preferably 0.5ngZml ⁇ 10 ⁇ g / ml, more preferably 0.5ngZml ⁇ l ⁇ g / ml, more preferred Or 0.5 to 200 ng / ml, more preferably 0.5 to 50 ng / ml, and most preferably 2 to 30 ng Zml.
  • the medium used in the method II of the present invention may further contain LIF.
  • the concentration is usually 10 to 10 6 units
  • / ml for example 10 ⁇ : L0 5 units / ml, preferably 10 2 ⁇ 10 4 units / ml, more preferably 3
  • the site force-in (FGF, GDNF receptor ligand, FGF2, EGF, etc.) that can be contained in the medium in the method II of the present invention is particularly limited if it is derived from an animal, preferably from the aforementioned mammals. Do not be! ,.
  • mutant protein includes insertions, substitutions and deletion mutants, fragments, and chemically modified derivatives thereof.
  • Substantially homologous means that the degree of homology to the wild-type amino acid sequence is preferably
  • the basal medium of the medium used in Method II of the present invention the same medium as in Method I described above can be used.
  • the medium used in the method II of the present invention may contain an additive known per se.
  • the additive those similar to the above-mentioned method I can be used.
  • Each additive is preferably contained within a known concentration range.
  • the medium used in the method II of the present invention may contain serum.
  • serum those similar to the above-mentioned method I can be used.
  • the serum concentration range is the same as in Method I above.
  • male germline stem cell precursors are used as feeder cells. You may culture in the presence.
  • the feeder cell the same one as in the above method I can be used.
  • one feeder cell for example, about 10 5 to 10 6 cells per one hole of a 6-well plate are usually used.
  • male germline stem cell precursors may be cultured in an adherent to an insoluble carrier via an extracellular matrix such as laminin. Good.
  • the cell passage interval and dilution rate are appropriately set according to the culture conditions. Usually, cells are subcultured at an interval of 2 to 10 days at 1Z2 to LZ 10-fold dilution. Be born.
  • the cultured cells form colonies of two types of forms by about 3 to 6 weeks after the start of the culture.
  • One colony has a morphology characterized by an intercellular bridge and a morula-like structure, which is a male germline stem cell colony.
  • the other mouth is packed more tightly and has a morphology very similar to that of ES cell colonies, which are colonies of pluripotent stem cells (described below). Therefore, male germline stem cell colonies and pluripotent stem cell colonies can be clearly distinguished visually.
  • male germline stem cells can be obtained by, for example, limit dilution that selectively picks up male germline stem cell colonies using a Pasteur pipette, a micromanipulator or the like under a microscope. Isolation and purification.
  • Method II of the present invention Whether or not the cells obtained by Method II of the present invention are male germline stem cells is determined by the method described in the above Method I (for example, transferred to the seminiferous tubule and confirmed for the presence of spermatogenic colonies) Etc.).
  • the male germline stem cells obtained by the method II of the present invention can be proliferated over a long period of time by using the above-described method I of the present invention.
  • the male germline stem cells obtained by the method II of the present invention can be cryopreserved semipermanently, and can be used after thawing and asleep as necessary.
  • the male reproductive lineage stem cells are cryopreserved and maintain their ability as male germline stem cells even after thawing.
  • the cells are suspended in a composition for cell cryopreservation known per se, such as a cell banker (manufactured by DIA-IATRON) containing dimethyl sulfoxide and urinary fetal serum albumin.
  • the male germline stem cells obtained by the method II of the present invention are allowed to sleep after cryopreservation, they are thawed and suspended in a solvent according to a conventional method in the same manner as in the method I described above, and the cell suspension is suspended.
  • the male germline stem cells grown by the method I can be used for the following uses, for example:
  • the present invention also relates to an agent for inducing differentiation of male germline stem cells, comprising FGF (excluding FGF2).
  • agent ⁇ of the invention By culturing a male germline stem cell precursor by the above-mentioned method II using a medium containing the agent II of the present invention, the precursor force is induced to differentiate into a male germline stem cell, or the differentiation induction is performed. Promote and produce male germline stem cells.
  • the FGF used in the agent II of the present invention is preferably a member of the FGF9 subfamily, more preferably FGF9.
  • Agent II of the present invention may further contain a GDNF receptor ligand.
  • the GDNF receptor ligand is preferably GDNF.
  • the agent II of the present invention may further contain FGF2.
  • Agent II of the present invention may further contain EGF.
  • the agent II of the present invention further comprises a physiologically acceptable carrier (for example, physiological isotonic solution (physiological saline, the above-mentioned basic medium, glucose and other adjuvants (for example, D-sorbitol, Isotonic solutions including D-mannitol, sodium chloride, etc.), excipients, preservatives, stabilizers (eg, human serum albumin, polyethylene glycol, etc.), binders, solubilizers, nonionics Surfactants, buffers (eg, phosphate buffer, sodium acetate buffer), preservatives, antioxidants, additives described above, and the like.
  • physiological isotonic solution physiological saline, the above-mentioned basic medium, glucose and other adjuvants (for example, D-sorbitol, Isotonic solutions including D-mannitol, sodium chloride, etc.)
  • excipients for example, preservatives, stabilizers (eg, human serum albumin, poly
  • the mixing ratio of each of the above-mentioned constituents contained in the agent II of the present invention is as follows when the agent II of the present invention is added to the medium used for the method II of the present invention. Concentration force of the constituent components in the medium It is preferable that the constituent components are configured to fall within the above-described preferable range.
  • the agent II of the present invention is used in the form of an isotonic aqueous solution or powder and added to the medium used in the method II of the present invention.
  • the agent II of the present invention may be a medium used in the method II of the present invention.
  • the method for producing pluripotent stem cells of the present invention is characterized by culturing precursors of male germline stem cells in a medium containing FGF (excluding FGF2) to obtain pluripotent stem cells.
  • FGF excluding FGF2
  • Pluripotent stem cells can be cultured at the in vitro mouth, can proliferate over a long period of time, have the ability to self-replicate, and have the ability to differentiate into all the cells that constitute the body and their progenitor cells. Cell.
  • the same male germline stem cell precursor as in the above-mentioned method II can be used.
  • the developmental stage of the embryo from which the cell is derived is not particularly limited.
  • male reproduction is preferably performed.
  • Primordial reproduction derived from embryo after formation of genital ridge Cells are used.
  • primordial germ cells usually 8.5 dpc or more, preferably 11.5 dpc or more are used.
  • primordial germ cell force EG cells of a mouse of 14.5 dpc or later cannot be induced.
  • pluripotent stem cells can be induced even with primordial germ cell force of 14.5 dpc or later. Can be induced.
  • the FGF used in the method III of the present invention is preferably a member of the FGF9 subfamily, more preferably FGF9.
  • the concentration of FGF contained in the medium is not particularly limited as long as pluripotent stem cells can be produced by the method ⁇ of the present invention, but usually 0.05 ngZml to 100 mgZml, for example 0.5 ngZml to 100 gZml, preferably 0 0. 5ngZml ⁇ 10 gZml, more preferably. 511 8 / 1111-1 8/1111, further [this preferably ⁇ or 0. 5 ⁇ 200ng / ml, more! /, Tsuso preferably ⁇ or
  • ngZml 0.5 to 50 ngZml, most preferably 2 to 20 ngZml.
  • the medium used in Method III of the present invention preferably further contains a GDNF receptor ligand.
  • the GDNF receptor ligand is preferably GDNF.
  • the concentration thereof is not particularly limited as long as pluripotent stem cells can be produced by the method ⁇ of the present invention, but usually 0.05 ngZml ⁇ 100MgZml, for example, 0. 5 ⁇ 8 / ⁇ 1 ⁇ 100 ⁇ g Zml, preferably 0. 5ngZml ⁇ : LO ⁇ g / mU more preferably 0. 5ngZml ⁇ l ⁇ g / m
  • the medium used in the method III of the present invention preferably further contains FGF2.
  • the concentration thereof is not particularly limited as long as pluripotent stem cells can be produced by the method III of the present invention, but a normal concentration of 0.05 ngZml to : LOOmgZml, for example 0.5 ngZml ⁇ : LOO / z gZml, preferably 0.5 ngZml to 10 ⁇ g / ml, more preferably 0.5 ngZml to l ⁇ g / ml, more preferably 0.5 to 200 ngZml, more So preferably 0.5 to 50 ng Zml, most preferably 2 to 20 ng, ml.
  • the medium used in the method of the present invention further contains epidermal growth factor (EGF). It may be.
  • EGF epidermal growth factor
  • the concentration thereof is not particularly limited as long as pluripotent stem cells can be produced by the method III of the present invention, but a normal concentration of 0.05 ngZml to : LOOmgZml, for example 0.5 ngZml ⁇ : LOO / z gZml, preferably 0.5 ngZml to 10 ⁇ g / ml, more preferably 0.5 ngZml to l ⁇ g / ml, more preferably 0.5 to 200 ngZml, more It is preferably 0.5 to 50 ng Zml, most preferably 2 to 30 ng, ml.
  • the medium used in the method III of the present invention is a primordial germ cell in the conventional method.
  • SCF and LIF which were essential for inducing EG cells, do not have to be substantially contained, but inclusion of these factors has no adverse effect.
  • the cyto force-in (FGF, GDNF receptor ligand, FGF2, EGF, etc.) that can be contained in the medium in the method III of the present invention is particularly limited as long as it is derived from an animal, preferably from the above-mentioned mammals. Do not be! ,.
  • the purified natural, synthetic or recombinant protein, mutant protein can be used as long as the acquisition of the pluripotent stem cell can be achieved. Includes insertions, substitutions and deletion mutants, fragments, and chemically modified derivatives thereof.
  • each cytokin And a protein substantially homologous to the wild-type amino acid sequence.
  • substantially homologous means that the degree of homology to the wild-type amino acid sequence is preferably
  • the basal medium of the medium used in Method III of the present invention the same medium as in Method I described above can be used.
  • the medium used in the method III of the present invention may contain an additive known per se. As the additive, those similar to the above-mentioned method I can be used. Each additive is preferably contained within a known concentration range. [0176] Furthermore, the medium used in the method of the present invention may contain serum. As the serum, those similar to the above-mentioned method I can be used. The serum concentration range is the same as in Method I above.
  • a male germline stem cell precursor may be cultured in the presence of a feeder cell.
  • a feeder cell the same one as in the above method I can be used.
  • one feeder cell for example, about 10 5 to 10 6 cells per hole of a 6-well plate are usually used.
  • a male germline stem cell precursor may be cultured in an adherent state to an insoluble carrier via an extracellular matrix such as laminin. Good.
  • the cell passage interval and dilution rate are appropriately set according to the culture conditions. Usually, cells are subcultured at an interval of 2 to 10 days, 1Z2 to: LZ 10-fold dilution. .
  • the cultured cells are about 3 to 6 weeks after the start of culture. Forms two types of colonies.
  • One colony has a morphology characterized by an intercellular bridge and a morula-like structure, which is a male germline stem cell colony.
  • the other colony is more tightly packed and has a morphology very similar to that of ES cells, which is a pluripotent stem cell colony. Therefore, male germline stem cell colonies and pluripotent stem cell colonies can be clearly distinguished visually.
  • the pluripotent stem cells obtained by the method of the present invention can form teratomas, and within the teratomas, various cells (for example, nerve, epidermis, muscle, bronchial epithelium) divided into three germ layers. Cartilage, bone, squamous epithelium, neuroepithelium, etc.).
  • male germline stem cells when injected into the seminiferous tubule, form spermatogenic colonies and do not form teratomas. Therefore, the pluripotent stem cells obtained by the method of the present invention are clearly distinguished from male germline stem cells.
  • the cells obtained by the method of the present invention retain pluripotency by analyzing the expression of cell surface markers and the like of the obtained cells using a flow cytometer or the like. And you can check if you have the power.
  • the ability of the cells to retain pluripotency is also confirmed. Can be confirmed.
  • the pluripotent stem cells obtained by the method III of the present invention are positive for alkaline phosphatase as in the case of normal ES cells.
  • the pluripotent stem cells obtained by the method III of the present invention can be proliferated while maintaining pluripotency over a long period of time (for example, usually 2 months or more).
  • the medium used in the above-mentioned method III may be used! Use a medium (maintenance medium) that is more suitable for long-term culture of stem cells.
  • the maintenance medium may not contain the above-mentioned cyto force-in (FGF, GDNF, FGF2, EGF, etc.), but preferably contains LIF.
  • the concentration of LIF is usually 10 to: L0 6 units / ml, For example, 10 to: L0 5 units / ml, preferably 10 2 to 10 4 units / ml, more preferably 3 ⁇ 10 2 to
  • the maintenance medium may contain serum.
  • the serum concentration in the maintenance medium is usually 2-30 (vZ v)%, preferably 10-20 (v / v)%.
  • the basal medium of the maintenance medium the same medium as used in the above-mentioned method III can be used, but preferably, the basal medium suitably used for ES cell culture (for example, DME)
  • the basal medium suitably used for ES cell culture for example, DME
  • the maintenance medium may contain the same additives as those used in the above-mentioned method III.
  • the pluripotent stem cells obtained by the method III of the present invention can be cryopreserved semipermanently, and can be used after thawing and asleep as necessary.
  • the pluripotent stem cells maintain pluripotency even after freezing and thawing.
  • the cells are suspended in a cell cryopreservation composition known per se, such as a cell banker (manufactured by DIA-IATRON) containing dimethyl sulfoxide and urinary fetal serum albumin, and 80--200 ° C.
  • the cells are preferably stored at 196 ° C (in liquid nitrogen).
  • the pluripotent stem cells obtained by the method III of the present invention are allowed to sleep after cryopreservation, they are thawed in a solvent according to a conventional method, similar to the above-described method for growing male germline stem cells, Suspend to make a cell suspension.
  • Pluripotency is maintained even if pluripotent stem cells once asleep are cultured and then frozen again.
  • the pluripotent stem cells obtained by the method III of the present invention can proliferate while maintaining pluripotency over a long period of time
  • the pluripotent stem cells can be isolated by a method known per se.
  • Genetically modified pluripotent stem cells such as pluripotent stem cells introduced with a specific foreign gene or pluripotent stem cells deficient in a specific gene can be produced by modifying the gene.
  • Examples of the method for gene introduction into pluripotent stem cells obtained by Method III of the present invention include the same methods as those for gene introduction into male germline stem cells described in Method I above. I can do it.
  • Method II a method similar to that described in Method I above may be used to select a stable genetically modified pluripotent stem cell into which a foreign gene has been stably introduced, or a pluripotent lacking a specific gene. It is also possible to obtain sex stem cells.
  • the pluripotent stem cells obtained by the method III of the present invention have the ability to differentiate into all somatic cells constituting the living body, and all test techniques applied to ES cells and EG cells,
  • the method can be applied to the diverse stem cells, and various functional cells, tissues, animals (excluding humans), etc. can be produced using the pluripotent stem cells. If pluripotent stem cells with modified genes are used, various functional cells, tissues, animals (excluding humans), etc. with modified genes can be produced.
  • mesenchymal cells can be produced by culturing the pluripotent stem cells obtained by the method III of the present invention under conditions of mesoderm cell sorting.
  • the mesoderm cell sorting conditions include conditions known per se that allow ES or EG cells to be sorted into mesoderm cells.
  • a plate coated with type IV collagen for example, in a plate coated with type IV collagen.
  • Culture see, for example, Blood, vol. 93, pl253-1263, 1999
  • culture in methylcellulose medium (Development, vol 125, pl747-1757, 1998)
  • feeder cell for inducing differentiation of mesodermal cells for example, Co-culture with stromal cells such as OP9 cells (Proc. Natl. Aca d. Sci.
  • Ectodermal cells can be produced by culturing pluripotent stem cells obtained by the method III of the present invention under conditions known per se for ectodermal cell sorting.
  • differentiation conditions for ectoderm cells include known conditions that allow ES or EG cells to differentiate into ectoderm cells, and are not particularly limited.
  • gelatin using a neuronal differentiation induction medium for example, N2B27 medium
  • Examples include culturing on a coated plate (for example, see Nature Biotechnology, vol. 21, 183-186, 2003, etc.).
  • Endodermal cells can be produced by culturing pluripotent stem cells obtained by the method III of the present invention under endodermal cell differentiation conditions known per se.
  • endodermal cell differentiation conditions include known conditions that allow ES or EG cells to differentiate into endoderm cells, and are not particularly limited. For example, differentiation conditions for insulin-producing cells (Proc Natl Acad Sci USA) , 97, 11307-11312).
  • pluripotent stem cells obtained by the method m of the present invention can be differentiated from, for example, ES cells into vascular cells (such as vascular endothelial cells) (Development, vol. 125, 1747-1757, 1998).
  • ES cells Differentiation of ES cells into neurons (Neuron, vol.28, 31-40, 2000), differentiation of ES cells into pigment cells (Development, vol.125, 2915-2923, 1998), ES cells To differentiate into insulin-producing cells (Proc Natl Acad Sci USA, 97, 11307-11312, 20000), to differentiate ES cells into ectoderm cells (WO01Z088100 pamphlet), ES cell mass (D Endodermal cells, ectodermal cells, mesoderm cells, blood cells, endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, glial cells, nerve cells, epithelium Using methods for producing cells, melanocytes, and keratinocytes (Reprod.Fertil.Dev., 10, 31, 1998) Te, the Rukoto be induced to differentiate into various functional cells, it is possible to produce a variety of functional cells.
  • the pluripotency obtained by the method ⁇ of the present invention by the method described in Proc Natl Acad Sci U SA, vol. 100, pi 1457-11462, 2003 and Nature, vol. 427, pl48-154, 2004 It may be possible to obtain progeny animals of the pluripotent stem cells by producing germ cells such as sperm from sex stem cells and using them for mating.
  • the pluripotent stem cells obtained by the method ⁇ of the present invention are transferred to immunodeficient animals such as nude mice or animals that have induced immune tolerance to form teratomas, and the teratomas have various abilities. Functional cells can also be isolated.
  • the gene-modified functional cell is obtained by modifying the gene of the pluripotent stem cell obtained by the method III of the present invention and applying the differentiation method described above to the gene-modified pluripotent stem cell obtained. It is possible to obtain.
  • the production of an animal using the pluripotent stem cells obtained by the method of the present invention can be carried out according to a method known per se, such as a method using a chimeric embryo.
  • pluripotent stem cells obtained by the method III of the present invention are introduced into a host embryo to obtain a chimeric embryo.
  • the animal species of the “host” is preferably the same as the animal species of the pluripotent stem cell to be introduced.
  • the “embryo” is not particularly limited, and examples thereof include blastocysts and 8-cell stage embryos.
  • the “embryo” is a hormonal agent (eg PMSG with FSH-like action and LH action) It can be obtained by mating a female animal that has undergone superovulation treatment (eg, using hCG) with a male animal.
  • a method for introducing pluripotent stem cells into a host embryo a microinstruction method, an aggregation method, and the like are known. However, a deviation method can also be used.
  • the chimeric embryo is transferred to the pupa or oviduct of the host animal to obtain a chimeric animal.
  • the host animal is preferably a pseudopregnant animal.
  • a pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by ligation or the like. The host animal into which the chimeric embryo has been transferred becomes pregnant and gives birth to the chimeric animal.
  • the chimeric animal is crossed with normal animals or the chimeric animals, and the pluripotent stem cell-derived gene is selected from the next generation (F1) individuals.
  • An animal carrying a stem cell-derived gene an animal derived from the pluripotent stem cell
  • various forms can be used as an index. For example, body color or coat color is used as an index. It is also possible to select by extracting partial force DNA from the body and performing Southern blot analysis or PCR assay.
  • the pluripotent stem cells obtained by the method III of the present invention are introduced into a tetraploid embryo to obtain a tetraploid chimeric embryo, and the tetraploid chimeric embryo is used as a uterus or egg of a host animal.
  • Animals derived from the pluripotent stem cells can also be obtained directly by transfer to a tube (Proc. Natl. Acad. Sci. USA, vol. 90, p8424-8428, 1993).
  • a tetraploid embryo can be obtained by electrofusion of a blastocyst by a method known per se, but a 2-cell blastocyst may be electrofused by applying an electric pulse in a Mantol solution. .
  • an animal (transgenic animal) carrying the introduced foreign gene can be obtained from, for example, a pluripotent stem cell into which a specific foreign gene has been introduced.
  • a heterozygous animal can be obtained from a pluripotent stem cell deficient in a specific gene.
  • gene-deficient homozygous animals can be obtained by breeding the obtained gene-deficient heterozygous animals.
  • the present invention also relates to a composition (agent) for producing pluripotent stem cells derived from male germline stem cells, including FGF (excluding FGF2).
  • FGF excluding FGF2
  • Pluripotent stem cells derived from vesicle precursors eg primordial germ cells
  • the FGF used in the composition of the present invention is preferably a member of the FGF9 subfamily, more preferably FGF9.
  • composition of the present invention may further contain a GDNF receptor ligand.
  • the GDNF receptor ligand is preferably GDNF.
  • composition of the present invention may further contain FGF2.
  • composition of the present invention may further contain EGF.
  • composition of the present invention may further contain a physiologically acceptable carrier (eg, physiological isotonic solution (physiological saline, basal medium as described above, glucose and other adjuvants (eg, D-sorbitol)). , D-mannitol, sodium isotonic solution, etc.), excipients, preservatives, stabilizers (eg, human serum albumin, polyethylene glycol, etc.), binders, solubilizers, non An ionic surfactant, a buffering agent (for example, phosphate buffer or sodium acetate buffer), a preservative, an antioxidant, the above-mentioned additives, and the like can be included.
  • physiological isotonic solution physiological saline, basal medium as described above, glucose and other adjuvants (eg, D-sorbitol)
  • D-mannitol sodium isotonic solution, etc.
  • excipients eg, preservatives, stabilizers (eg, human serum albumin
  • the mixing ratio of each of the above-mentioned constituents contained in the composition of the present invention is such that when the composition of the present invention is used by being added to the medium used in Method III of the present invention, It is preferable that the concentration of the component in the culture medium is configured to fall within the above-mentioned preferable range.
  • composition of the present invention is in the form of an isotonic aqueous solution or powder, and the method of the present invention.
  • composition of the present invention may be a medium used in the above-described method III of the present invention.
  • Testicular cells are SD rats, BN rats, Lewis rats, Donryu rats, F344 rats, F1 rats of BN (female) and SD (male), or GFP transgenic rats that have been backcrossed to SD background (hereinafter GFP rats) (Collected by Dr. Masaru Okabe, Osaka University) (7-21 days old). Since this GFP rat expresses the EGFP gene in virtually all cell types, it is possible to trace the rat-derived cells using the fluorescence of EGFP as an indicator.
  • Testis cells were prepared according to the method described in Biology of Reproduction, vol.69, No.2, p.612-616, 2003, collagenase (type IV, manufactured by Sigma) and trypsin (manufactured by Invitrogen). ) was collected by two-step enzymatic digestion.
  • testis cell suspension was passed through a 20-30 ⁇ m nylon mesh to remove the undigested cell mass, centrifuged at 600 X g for 5 minutes to collect testis cells, and anti-testis cells were treated with anti-testis cells.
  • the spermatogonial stem cells were concentrated as CD9 positive cells by the magnetic bead method using CD9 antibody. The concentration operation was performed according to the description in (Proc Natl Acad Sci USA, 1999, 96 ⁇ , pages 5504-5509).
  • Laminin-coated plates are prepared by treating the culture dish with PBS containing laminin (BD Biosciences) at a concentration of 20 g / ml at 37 ° C, then removing the solution and washing twice with PBS. did.
  • Basal culture medium was StemPro supplement (Invitrogen), 25 gZml insulin, 100 ⁇ g / ml transferrin, 60 putrescine, 30 ⁇ 30 sodium selenate, 30 ⁇ g / ml pyruvic acid, 1 lZml DL lactate (Sigma), 2 mM L-Glutamine, 5 X 10 " 5 M 2-Mercaptoethanol, MEM vitamin solution (Invitrogen), MEM non-essential amino acid solution (Invitrogen), 10" 4 M ascorbic acid, 10 / z gZml d-Piotin, 30 ng StemPro-34 SFM (Invitrogen) supplemented with / par ⁇ estradiol, 60 ng Zml progesterone (Sigma) and 1 ⁇ g / ml heparin. If desired, a cytodynamic force-in at a desired concentration was appropriately added to the medium. 0.625 ml of FCS was added per
  • the cells were maintained at 37 ° C in air containing 5% carbon dioxide and subcultured onto new laminin coated dishes as appropriate.
  • recipient mouse testes were collected 2 months after donor cell transplantation and analyzed by observing fluorescence under UV light (Okabe M, Ikawa M, K ominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of ubiquitous gree n cells. FEBS Lett 1997; 407: 313-319).
  • Donor germ cells were specifically identified because host testis cells do not have intrinsic fluorescence.
  • a germ cell cluster is defined as a colony if it occupies the entire circumference of the tubule and has a length of at least 0.1 mm (Na gano M, Avarbock MR, Bnnster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient tests. Biol Reprod 1999; 60: 1429-1436). All animal experiment protocols were approved by the Animal Protection and Use System Committee of Kyoto University.
  • the recipient testicular seminiferous tubule was carefully dissected and germ cells were mechanically recovered. Microinsemination was performed as previously described (Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709-720). Embryos that reached the 4-cell stage after 24 hours of culture were transferred to the oviducts of day-1 pseudopregnant Wistar female rats. Growing fetuses taken on day 21 were raised by feeding Wistar strain foster rats.
  • Testis cells of 2-week-old GFP rats are cultured on a ultralow attachment plate (Nunc) for 1 week to 10 days in a medium containing 15 ng / ml GDNF, 20 ng / ml EGF and lOngZml FGF2.
  • the spermatogonial stem cells were concentrated by collecting the colony forming cells adhering to the top.
  • the obtained spermatogonial stem cells were cultured in the above medium on a laminin-coated dish to establish rat GS cells. 1. 8 ⁇ 10 5 Zwell rat GS cells were seeded in a laminin-coated 6-well plate, and further cultured in the above medium supplemented with various FGFs for 7 to 13 days. After culturing, the number of GS cells in the well was counted, and the multiplication factor from the start of culturing was calculated. The results of multiple tests are shown in Tables 3-10.
  • the proliferation efficiency of rat GS cells was significantly increased by adding FGF to the medium. This effect is related to any type of FGF tested (FGF4, FGF5, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19 and FGF20) were also confirmed. In particular, FGF9 strongly promoted the proliferation of GS cells even at relatively low concentrations.
  • Testicular cells from 2-week-old SD strain rats were cultured on a ultralow attachment plate (Nunc) for 1 week to 10 days in a medium containing 15 ng / ml GDNF, 20 ng / ml EGF and lOngZml FGF2.
  • the spermatogonial stem cells were concentrated by collecting the colony forming cells.
  • the obtained spermatogonial stem cells were cultured in the above medium on a laminin-coated dish to establish rat GS cells. From 61 days after the start of culture, lOngZml of FGF9 was further added to the medium, and GS cells were subsequently cultured. The number of cells was measured over time, and the multiplication factor at the start of culture was calculated. The results are shown in Figure 1.
  • Testicular cells from 2-week-old SD strain rats were cultured on a ultralow attachment plate (Nunc) for 1 week to 10 days in a medium containing 15 ng / ml GDNF, 20 ng / ml EGF and lOngZml FGF2.
  • the spermatogonial stem cells were concentrated by collecting the colony forming cells.
  • the obtained spermatogonial stem cells were cultured in the above medium on a laminin-coated dish to establish rat GS cells. From 29 days after the start of culture, lOngZml of FGF9 was further added to the medium, and GS cells were subsequently cultured. The number of cells was measured over time, and the multiplication factor from the beginning of the culture was calculated.
  • Figure 2 shows the results of three independent tests.
  • testicular cell power of SD rats was also established in GS cell culture. 1. 8 ⁇ 10 5 / well rat GS cells were seeded in a laminin-coated 6-well plate and further cultured for 7 days in a medium supplemented with the following combination of cytodynamic ins. After culturing, the number of GS cells in the well was counted, and the multiplication factor from the start of culturing was calculated. The results are shown in Table 12 and FIG.
  • the spermatogonial stem cells concentrated by ultralow attachment plate (Nunc) from the testis cells of GFP rat that had been backcrossed to a 2-week-old SD background were transferred to a 15 ngZml GDNF, Rat GS cells were established by culturing in a medium containing 20 ng / ml EGF and 10 ng / ml F GF2. 122 days after the start of culture, lOngZml FGF9 was added to the medium, and the culture was further continued. To confirm whether the cultured cells maintain their activity as male germline stem cells and the stem cells proliferate in vitro, the cultured cells are placed in the seminiferous tubules at two different times. After transplanting, the number of colonies formed was measured.
  • GS cells were collected and transplanted into the seminiferous tubule of busulfan-treated nude mice.
  • FGF9 supplementation day 122
  • GS cells were collected again for transplantation, and the increase in the number of stem cells during this period was measured. Colonies in transplantation experiments were measured under UV irradiation 7-8 weeks after transplantation.
  • GS cells proliferated 27.7 times as the total number of cells and 69.3 times as the number of stem cells within 100 to 172 days after the start of culture.
  • FGF9 was added 122 days after the start of the culture
  • GS cells proliferated 166.2 times as the total number of cells and 4155.9 times as the number of stem cells. That is, the addition rate of FGF9 increased not only the total number of cells but also the increase rate of the number of stem cells.
  • FGF9 was shown to strongly promote the proliferation (ie, amplification) of male germline stem cells while maintaining the ability as male germline stem cells.
  • the cultured cells are transplanted into the seminiferous tubules of KSN strain nude mice treated with busulfan and derived from the cultured cells. Attempts were made to produce progeny animals derived from cultured cells by microinsemination using sperm.
  • GFP rat-derived GS cells were cultured in a medium containing 15 ngZml GDNF, 20 ng / ml EGF, lOngZml FGF2 and lOng / ml FGF9 on a laminin-coated dish for 116 days.
  • a large number of GFP-positive seminiferous tubules filled with apparently normal spermatogenic cells are produced, and a large spermatogenic colony derived from the cultured cells is formed.
  • Figure 5 Since nude mice (recipients) do not have intrinsic fluorescence, GFP-positive spermatogenesis in host mice in transplantation experiments is only from cultured donor stem cells.
  • GFP-positive sperm or GFP-positive sperm cells were collected from testes of nude mice and injected into oocytes.
  • a pCXN-based plasmid vector carrying the neomycin resistance gene and the EGFP structural gene (pCAG-EGFP) operably linked to the CAG promoter was used for gene transfer.
  • GS cells were transfected with Cell Line Nucleofector Kit T (Amaxa) according to the manufacturer's instructions. The isolated 5 ⁇ 10 6 GS cells were suspended in 100 ⁇ 1 of solution T (Nucleofector Kit T, Amaxa), and 5 ⁇ g of plasmid DNA was added thereto, followed by electopore positioning.
  • Cells were suspended in medium containing 4 ml of 15 ⁇ g / ml GDNF, 20 ng / ml EGF, lOng / ml FGF2, and lOng / ml FGF9 and seeded on laminin coated dishes (30 cm 2 ).
  • the G418 selection (40 ⁇ g / ml, dieneticin; manufactured by Invitrogen) also started the transfer force 6 days later. After 14 days of selection with G418, G418 was also removed. Cells were cleaved 34 days after transfusion. Since the growth of GS cells is affected by density, each colony contains 5000 untransfected GS cells And transferred to a 96-well laminin-coated culture plate. Modified cells were passaged every 10-14 days, repeating G418 selection.
  • Primordial germ cells were collected from pregnant ICR strain mice maintained in a controlled environment with a 12:12 day: night cycle from 8 am to 8 pm (SLC, Shizuoka, Japan). When the sexual plug was recognized, the day was set at 0.5 dpc. Embryo gender was determined using dienotyping based on the Ubel PCR method (D ev. Biol., Vol. 229, p. 468-479, 2001), and only male embryos were used in this study. 12. 5- 15.
  • Laminin coated plates were prepared by treating the culture dish with PBS containing laminin (BD Biosciences) at a concentration of 20 g / ml at 37 ° C, then removing the solution and washing twice with PBS. .
  • Example 2 The same basal culture medium as used in Example 1 was used. 15 ngZml GDNF, 20 ng / ml EGF and lOngZml FGF2 were added to the medium, and further lOngZml FGF9 was added to the medium as desired. 0.625 ml of FCS was added per ml of basal culture medium.
  • the site force in used in Example 2 is the same as in Example 1. Cells are maintained at 37 ° C in air containing 5% carbon dioxide, and new laminin coated It was passed on to Itush.
  • Figure 10 shows the state of the cells one month after the start of culture.
  • male germline stem cells can be obtained by culturing primordial germ cells in a medium containing FGF9.
  • Example 2 a tissue enriched in reproductive cells was obtained by dissection of the reproductive ridge of 12.5 dpc and 14.5 dpc embryos, and the tissue contained ImM EDTAdnvitrogen, Carlsbad, CA). Primordial germ cells of ICR strain mice were collected by 10-minute enzymatic digestion with% trypsin.
  • Laminin coated plates are obtained by treating culture dishes with PBS containing laminin (BD Biosciences) at a concentration of 20 g / ml overnight at 37 ° C, then removing the solution and washing twice with PBS. Prepared.
  • Example 3 The same basal culture medium as in Example 1 was used. 15 ngZml GDNF, 20 ng / ml EGF and lOngZml FGF2 were added to the medium, and further lOngZml FGF9 was added to the medium as desired. 0.625 ml of FCS was added per ml of basal culture medium.
  • the site force in used in Example 3 is the same as in Example 1. The cells were maintained in air containing 5% carbon dioxide at 37 ° C and passaged onto new laminin coat dishes as appropriate.
  • ES cell-like colonies were isolated and further grown under similar culture conditions.
  • about 2 X 10 6 pieces of ES-like cells are injected into the skin under the KSN nude mice (Japan SLC), it was analyzed in three weeks after transplantation.
  • the formed tissue was fixed with 10% neutral buffered formalin and processed for norafine sections. Sections were stained with hematoxylin and eosin and observed under a microscope.
  • the cancer included derivatives of three embryonic germ layers, such as muscle, nerve, blood vessel, bronchial epithelium and the like.
  • male germline stem cells proliferated by the method of the present invention can be used to produce genetically modified animals (transgenic animals, gene-deficient animals, etc.), treat male infertility, develop therapeutic agents for male infertility, and perform gene therapy at the germline level. Therefore, the present invention is useful in the fields of biotechnology and medicine.
  • pluripotent stem cells can be stably and efficiently induced from male germline stem cells regardless of the age difference of the animal from which the primitive germline stem cells are derived. It becomes possible to do.
  • pluripotent stem cells obtained by the method of the present invention it is possible to construct various tissues having histocompatibility for autotransplantation, which is useful in the medical fields such as regenerative medicine and gene therapy. It is.
  • the pluripotent stem cells are useful in the biotechnology field because they can be used to create genetically modified animals such as transgenic animals and knockout animals.

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Abstract

La présente invention concerne un procédé qui permet la prolifération de cellules souches germinales mâles et inclut la culture des cellules souches germinales mâles dans un milieu contenant du FGF. L'utilisation de ce procédé permet la prolifération stable et efficace de cellules souches germinales mâles indépendamment de l'espèce animale. De plus, l'utilisation de ce procédé permet la prolifération stable et efficace de cellules souches germinales mâles indépendamment de l'âge de l'animal duquel sont issues les cellules testiculaires. En particulier, il est ainsi possible de produire des cellules souches germinales mâles issues de cellules testiculaires d'un animal avant la naissance avec une grande efficacité.
PCT/JP2007/055213 2006-03-16 2007-03-15 Procédé permettant la prolifération in vitro de cellules souches germinales mâles Ceased WO2007108393A1 (fr)

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Cited By (1)

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CN119192326A (zh) * 2024-10-23 2024-12-27 四川大学 重组cFGF2蛋白和鸟类原始生殖细胞培养基及其应用

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119192326A (zh) * 2024-10-23 2024-12-27 四川大学 重组cFGF2蛋白和鸟类原始生殖细胞培养基及其应用
CN119192326B (zh) * 2024-10-23 2025-06-17 四川大学 重组cFGF2蛋白和鸟类原始生殖细胞培养基及其应用

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