WO2024193062A1 - 制备成熟卵母细胞的方法及应用 - Google Patents

制备成熟卵母细胞的方法及应用 Download PDF

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WO2024193062A1
WO2024193062A1 PCT/CN2023/131356 CN2023131356W WO2024193062A1 WO 2024193062 A1 WO2024193062 A1 WO 2024193062A1 CN 2023131356 W CN2023131356 W CN 2023131356W WO 2024193062 A1 WO2024193062 A1 WO 2024193062A1
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culture medium
cells
human
stem cells
culture
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French (fr)
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吴际
李小勇
葛均邦
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上海交通大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • 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

Definitions

  • the present invention belongs to the field of infertility and fertility preservation; more specifically, the present invention relates to a method for preparing mature oocytes, comprising differentiating human female reproductive stem cells into human mature oocytes.
  • FGSCs are a special type of adult stem cells that not only have the characteristics of stem cells, but also have the ability to transmit genetic information. After a complex development process, female mammalian FGSCs eventually produce functional mature eggs to provide female gametes for the development of offspring.
  • iPSCs human induced pluripotent stem cells
  • PGCLCs PGC-like cells
  • the object of the present invention is to provide a method for preparing mature oocytes, comprising differentiating human female germ stem cells into mature oocytes.
  • an in vitro method for preparing mature human oocytes comprising: (1) co-culturing human female reproductive stem cells with ovarian somatic cell-like cells to establish a human ovarian organoid system, in which follicles are generated; and, (2) isolating follicles from the human ovarian organoids, culturing them in vitro, and obtaining mature human oocytes.
  • the method further comprises: (3) The mother cells are fertilized in vitro and develop in vitro (eg, develop into a blastocyst).
  • the ovarian somatic cell-like cells include ovarian somatic cell-like cells obtained by in vitro differentiation of human pluripotent stem cells.
  • the culturing of ovarian somatic cell-like cells comprises:
  • the human pluripotent stem cells include human induced pluripotent stem cells (iPSCs) and human embryonic stem cells.
  • the human female germ stem cells and ovarian somatic cell-like cells are mixed at a ratio of 1: (100-10000); preferably, at a ratio of 1: (200-8000); and more preferably, at a ratio of 1: (300-6000).
  • the mixed cells are first cultured to form cell clusters and then to form organoids.
  • the human female germ stem cells are mixed with ovarian somatic cell-like cells and then cultured in three dimensions (for example, solidified with Matrigel).
  • the human female reproductive stem cells are mixed with ovarian somatic cell-like cells and then cultured with a first type of organ culture medium to form cell clusters, the culture medium comprising: retinoic acid, L-glutamine, spermidine, sodium pyruvate, ⁇ -mercaptoethanol, EGF, FGF, glial cell-derived neurotrophic factor, leukemia inhibitory factor, and non-essential amino acids; then cultured with a second type of organ culture medium, the culture medium comprising: spermidine, transferrin, sodium selenite, insulin, FSH, L-glutamine, ascorbic acid, EGF, bFGF, glial cell-derived neurotrophic factor, leukemia inhibitory factor, SCF, BMP4, and ⁇ -mercaptoethanol; and then cultured with a third type of organ culture medium, the culture medium comprising: spermidine, transferrin, sodium selenite, insulin, FSH, L-glutamine, ascorbic
  • the culture time in the first type of organ culture medium is 5 to 10 days, preferably 6 to 9 days (such as 7 or 8 days); the culture time in the second type of organ culture medium is 10 to 20 days, preferably 12 to 18 days (such as 13, 14, 15, 16, or 17 days); or the culture time in the third type of organ culture medium is 18 to 45 days, preferably 20 to 40 days (such as 22, 24, 28, 30, 32, 35, or 38 days).
  • the first or second type of organ culture medium is based on MEM ⁇ or a similar culture medium (such as other MEM series culture medium), wherein the first type of organ culture medium contains serum or serum protein substitutes (such as 5-15%, preferably 7-13%, more preferably 8-12%), and the second type of organ culture medium contains serum or serum protein substitutes (preferably 1-5%, such as 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%); the third type of organ culture medium is based on StemPro-34 SFM, which contains serum or serum protein. White substitute (preferably containing 5-15%, such as 7%, 8%, 9%, 10%, 11%, 12% or 13%).
  • the human female germ stem cells are mixed with ovarian somatic cell-like cells at a ratio of 1:400, 500, 600, 800, 1000, 1500, 2000, 3000, 4000, 5000, 7000 or 9000.
  • the medium is replaced (partially replaced) every 12-36 hours.
  • the first organoid culture medium is cultured in a U-shaped plate.
  • the culturing of the second organoid culture medium is carried out in a Transwell.
  • the culturing in the third organoid culture medium is carried out in a Transwell.
  • ⁇ -mercaptoethanol is used to neutralize oxygen free radicals accumulated in the cell culture medium.
  • the content of ⁇ -mercaptoethanol in the first organ culture fluid is 1.5 to 3 times (such as 2 times) that in the second or third organ culture fluid.
  • the first, second and third organ culture media further include antibiotics.
  • the antibiotics include: penicillin and/or streptomycin.
  • the in vitro culture comprises: culturing with a first follicle culture medium, the culture medium comprising: L-glutamine, transferrin, sodium selenite, ascorbic acid, EGF, Activin-A, FSH, ⁇ -mercaptoethanol; subsequently culturing with a second follicle culture medium, the culture medium comprising: L-glutamine, sodium pyruvate, sodium selenite, bFGF, EGF, transferrin, Insulin, PVP, bmp15, gdf9, cAMP, FSH, LH, Estrogen, Progestrone, ⁇ -mercaptoethanol; and then culturing with a third follicle culture medium, the culture medium comprising: the second follicle culture medium and 5-20% (v/v) inactivated human follicular fluid.
  • the first follicle culture fluid is based on MEM ⁇ or a similar culture medium (such as other MEM series culture medium) as the basal culture medium, which contains serum or a serum protein substitute (preferably 3-8%, such as 4%, 5%, 6%, 7%);
  • the second follicle culture fluid is based on MEM ⁇ or a similar culture medium (such as other MEM series culture medium) as the basal culture medium, which contains serum or a serum protein substitute (preferably 5-15%, such as 7%, 8%, 9%, 10%, 11%, 12% or 13%).
  • the culture is a three-dimensional culture (eg, solidified with Matrigel).
  • the medium is replaced (partially replaced) every 12-36 hours.
  • the first, second and third follicle culture fluids are cultured in a Transwell.
  • the first, second and third follicle culture fluids further include ⁇ -Mercaptoethanol.
  • the antibiotics include: penicillin and/or streptomycin.
  • a human mature oocyte is provided, which is prepared by any of the above methods.
  • the mature human oocyte is not able to independently develop into an organism without in vitro fertilization.
  • the human mature oocyte can further develop into a blastocyst in vitro via in vitro fertilization.
  • kits for preparing human mature oocytes in vitro comprising:
  • the ratio of human female germ stem cells to ovarian somatic cell-like cells is 1:(100-10000), preferably 1:(200-8000); more preferably 1:(300-6000).
  • (b) comprises: a first type of organ culture medium, which comprises: retinoic acid, L-glutamine, spermidine, sodium pyruvate, ⁇ -mercaptoethanol, EGF, FGF, glial cell line derived neurotrophic factor, leukemia inhibitory factor, and non-essential amino acids; a second type of organ culture medium, which comprises: spermidine, transferrin, sodium selenite, insulin, FSH, L-glutamine, ascorbic acid, EGF, bFGF, glial cell line derived neurotrophic factor, leukemia inhibitory factor, SCF, BMP4, and ⁇ -mercaptoethanol; a third type of organ culture medium, which comprises: spermidine, transferrin, sodium selenite, insulin, FSH, L-glutamine, ascorbic acid, EGF, bFGF, glial cell line derived neurotrophic factor, leukemia inhibitory factor, SCF, BMP4, and ⁇ -mercaptoethanol; a
  • the first or second type of organ culture medium is based on MEM ⁇ or a similar culture medium, wherein the first type of organ culture medium contains serum or serum protein substitutes (such as 5-15%, preferably 7-13%, more preferably 8-12%), and the second type of organ culture medium contains serum or serum protein substitutes (preferably 1-5%, such as 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%); the third type of organ culture medium is based on StemPro-34SFM, which contains serum or serum protein substitutes (preferably 5-15%, such as 7%, 8%, 9%, 10%, 11%, 12% or 13%).
  • (c) comprises: a first follicle culture medium, which comprises: L-glutamine, transferrin, sodium selenite, ascorbic acid, EGF, Activin-A, FSH, ⁇ -mercaptoethanol; a second follicle culture medium, which comprises: L-glutamine, sodium pyruvate, sodium selenite, bFGF, EGF, transferrin, Insulin, PVP, bmp15, gdf9, cAMP, FSH, LH, Estrogen, Progestrone, ⁇ -mercaptoethanol; a third
  • the follicle culture fluid comprises: a second follicle culture fluid and 5-20% (v/v) inactivated human follicle fluid.
  • FIG. 1A is a bright field image of human pluripotent stem cells obtained in Example 1 differentiated into ovarian somatic cell-like cells.
  • FIG. 1A is a bright field image of human pluripotent stem cells obtained in Example 1 differentiated into ovarian somatic cell-like cells.
  • Figure 1B Representative images of ovarian organoids at week 1 (W1), week 3 (W3), and week 5 (W5) in culture.
  • FIG. 1 Flow cytometric analysis results of ovarian organoid culture at week 1 (W1), week 3 (W3), and week 5 (W5).
  • Fig. 4 Expression of MVH in germ cells of ovarian organoids at the 5th week of culture.
  • Fig. 6 Morphological images of follicles derived from in vitro differentiation of human female germ stem cells at different culture stages.
  • Fig. 7 Morphology of GV-stage oocytes differentiated from human female germline stem cells in vitro.
  • Fig. 8 Morphology of MII oocytes differentiated from human female germline stem cells in vitro.
  • Figure 9 The embryonic development process of mature oocytes differentiated from human female germ stem cells in vitro after in vitro fertilization.
  • FIG. 10 The immature follicles cultured in 3D were placed in in vitro maturation medium and cultured in a 37°C 5% CO 2 incubator for 24-72 hours. The morphology of the oocytes and the looseness of the cumulus cells were observed under a microscope, and the discharge of the first polar body was used as a sign of maturity.
  • basic medium or “basic medium” refers to a medium suitable for cell culture that can provide cells with sufficient nutrients to meet the growth requirements of cells.
  • granulosa cells and “ovarian granulosa cells” are used interchangeably, which may be cells of natural origin or cells expanded/passaged based on natural cells, or may be cells that have been genetically engineered or modified.
  • female reproductive stem cells or “ovarian female reproductive stem cells” are used interchangeably, and may be cells of natural origin or cells expanded/passaged based on natural cells, or may be cells genetically engineered or modified.
  • ovarian somatic cell-like cells and “ovarian somatic cells” are used interchangeably, and may be cells of natural origin or cells expanded/passaged based on natural cells, or may be cells induced; in addition, they may also be cells that have been genetically engineered or modified.
  • Lineage refers to the pathway of cell development in which a precursor or “ancestor” cell (in this case, a stem cell) undergoes progressive physiological changes to become a specific cell type (in this case, an ovarian somatic cell-like cell) with characteristic functions.
  • the female reproductive stem cells may be derived from follicle aspirates, which are often discarded products in assisted reproduction.
  • a method for obtaining female reproductive stem cells comprises: (1) isolating ovarian tissue or cell aggregates from follicle extracts, and performing cell dispersion to obtain single cells; (2) inoculating the cells obtained in step (1) onto inactivated feeder cells for culture, and obtaining single cells from the cultured cells.
  • the ovarian somatic cell-like cells include ovarian somatic cell-like cells obtained by in vitro differentiation of human pluripotent stem cells.
  • the human pluripotent stem cells include human induced pluripotent stem cells (human induced pluripotent stem cells (iPSCs) and human embryonic stem cells.
  • the embryonic stem cells or pluripotent stem cells involved in the present invention can be purchased through commercial channels, and do not necessarily require embryonic stem cells or pluripotent stem cells obtained by destroying embryos.
  • human embryonic stem cells and embryonic germ cells had been successfully established.
  • WiCell Wisconsin Alumni Research Foundation
  • the ovarian somatic cell-like cells include ovarian somatic cell-like cells obtained by in vitro differentiation of human pluripotent stem cells; preferably, the culture of the ovarian somatic cell-like cells includes: (1) cell pre-culture; (2) treating the cells of (1) with a GSK3 inhibitor; (3) treating the cells of (2) with B/A/W, wherein the B/A/W is BMP4, ActivinA and Wnt3a; (4) treating the cells of (3) with Follistain, BMP4 and serum; (5) treating the cells of (4) with Follistain and BMP4, to obtain a culture containing human ovarian somatic cell-like cells.
  • the "treatment” includes “induction” or “induction treatment”.
  • the method is an in vitro/ex vivo culture method.
  • the obtained cells express mesendoderm markers such as brachyury, mixl1, and pax2.
  • the human stem cells include human induced pluripotent stem cells (such as human induced pluripotent stem cells) and human totipotent stem cells (such as human embryonic stem cells).
  • the “ovarian somatic cell-like cells” refer to cells expressing Foxl2, Cyp19a1 and Ptch1.
  • steps (1) to (3) suspension culture is performed, and in steps (4) to (5), adherent culture is performed.
  • step (1) cells are cultured in a basal culture medium; in steps (2) to (5), GSK3 inhibitor, B/A/W, Follistain, BMP4, and serum are added to the basal culture medium; preferably, the basal culture medium is a stem cell culture medium (a culture medium suitable for culturing stem cells); more preferably, the basal culture medium is E8, mTesr or DMEM/F12 basal culture medium; more preferably, the basal culture medium is a basal culture medium supplemented with 20 ⁇ 10% KSR, 1 ⁇ 0.5% NEAA, 1 ⁇ 0.5% Glutamax, 0.7 ⁇ 0.2 ⁇ -mercaptoethanol, and 4-10ng/ml bFGF.
  • the basal culture medium is a stem cell culture medium (a culture medium suitable for culturing stem cells); more preferably, the basal culture medium is E8, mTesr or DMEM/F12 basal culture medium; more preferably, the basal culture medium is a basal culture medium supplemented with 20 ⁇
  • the cell pre-culture time is 1 to 4 days; preferably 1.5 to 3 days; more preferably 1.5 to 2.5 days (such as 2 days).
  • the culture medium for suspension culture is a liquid culture medium (culture solution).
  • the culture medium for adherent culture is a corresponding liquid culture medium (culture solution).
  • low-adhesion culture dishes are used for culture.
  • the cells are attached to a culture dish treated with gelatin; preferably, the dish is treated with 0.2% gelatin.
  • E8 is Essential 8TM medium from GIBCO
  • mTeSR1 is a medium from Stemcell. Both culture media can be used to culture pluripotent stem cells.
  • the GSK3 inhibitor is selected from: CHIR99021, LiCl, BIO or Ly2090314; preferably CHIR99021; preferably, the concentration of CHIR99021 is 3 to 8 uM (such as 4, 4.5, 5, 5.5, 6 or 7uM), more preferably 4-6uM.
  • the culturing time is 30 to 48 hours (eg, 32, 34, 36, 38, 40, 42 or 44 hours).
  • the concentration of BMP4 is 4-15 ng/ml (such as 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng/ml), preferably 9-12 ng/ml.
  • the concentration of Activin A is 5 to 15 ng/ml (such as 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng/ml), preferably 9 to 12 ng/ml.
  • the concentration of Wnt3a is 5-15 ng/ml (eg, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng/ml), preferably 9-12 ng/ml.
  • the culture time is 2 to 9 days (such as 2.5, 3, 4, 5, 6, 7 or 8 days), preferably 2.5 to 7 days; more preferably 3 to 6 days.
  • the concentration of Follistain is 15-40 ng/ml (such as 18, 20, 22, 25, 28, 30, 32 or 35 ng/ml), preferably 20-30 ng/ml, and more preferably 22-28 ng/ml.
  • the concentration of BMP4 is 4-15 ng/ml (such as 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng/ml), preferably 9-12 ng/ml.
  • the serum concentration is 3-10% (v/v) (preferably 4-8%, such as 5%, 6%, 7% (v/v)).
  • the culturing time is 3 to 12 hours (such as 4, 5, 6, 7, 8, 9, 10 or 11 hours), preferably 4 to 10 hours; more preferably 5 to 8 hours.
  • the concentration of Follistain is 15-40 ng/ml (such as 18, 20, 22, 25, 28, 30, 32 or 35 ng/ml), preferably 20-30 ng/ml, and more preferably 22-28 ng/ml.
  • the concentration of BMP4 is 4-15 ng/ml (such as 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ng/ml), preferably 9-12 ng/ml; or
  • the culture time is 4 to 12 days (such as 4.5, 5, 6, 7, 8, 9, 10 or 11 days), preferably 5 to 10 days; more preferably 6 to 8 days.
  • the present invention provides a method for differentiating human female reproductive stem cells into mature oocytes in vitro.
  • the method first co-cultures human female reproductive stem cells with ovarian somatic cells to establish a human ovarian organoid system, in which follicles are generated; then, follicles are separated from human ovarian organoids, cultured in vitro, and mature oocytes are obtained.
  • the method of the present invention comprises: (i) 3D co-culture of ovarian somatic cells differentiated in vitro from human female reproductive stem cells and human induced pluripotent stem cells (iPSCs) to establish a human ovarian organoid system; (ii) follicles are separated from ovarian organoids and cultured in 3D, and mature oocytes are obtained after in vitro maturation; further optionally, (iii) the obtained mature oocytes are fertilized in vitro and finally developed into blastocysts.
  • iPSCs human induced pluripotent stem cells
  • the human female reproductive stem cells and ovarian somatic cell-like cells are separated to obtain female reproductive stem cells.
  • the human female reproductive stem cells are preferably mixed with ovarian somatic cell-like cells at a ratio of about 1: (100-10000); preferably mixed with 1: (200-8000); more preferably mixed with 1: (300-6000).
  • human female reproductive stem cells can obtain good support from ovarian somatic cell-like cells and differentiate better.
  • the human female reproductive stem cells are mixed with ovarian somatic cell-like cells at a ratio of about 1: 1000.
  • the first type of organ culture medium is used, and some active components of the culture medium and their recommended or preferred dosages are listed in Table 1.
  • a second type of organ culture medium is applied.
  • Some active components of the culture medium and their recommended or preferred dosages are listed in Table 2, among which bFGF, insulin, spermidine and BMP4 play a major role.
  • the second organoid culture medium uses MEM ⁇ or a similar culture medium (such as other MEM series culture medium) as a basal culture medium, which is conducive to providing nutrients suitable for cell growth, and compared with the first organoid culture medium, it further promotes the development of follicles in the organoids.
  • a small amount of serum is added to the culture medium, which is more conducive to promoting the development of follicles in the organoids.
  • the third organoid culture medium is based on StemPro-34, which is different from the previous MEM culture medium.
  • StemPro-34 can provide more suitable and sufficient nutrition for organoid growth.
  • Preferably, about 10% serum is added to the culture medium, so as to better utilize the multi-layer follicle development in the organoid.
  • a 3D culture method is used in combination with the organoid culture medium to provide good conditions for the generation of follicles.
  • follicles are generated in the human ovarian organoid system; thereafter, follicles are isolated from the human ovarian organoids and cultured in vitro to obtain mature oocytes.
  • the first follicle culture medium is used, and some active components of the culture medium and their recommended or preferred dosages are listed in Table 4, among which FSH and PVP play a major role.
  • the first follicle culture medium uses MEM ⁇ or similar culture medium (such as other MEM series culture medium) as the basic culture medium.
  • a second follicle culture medium After culturing in the above culture medium, a second follicle culture medium is applied. Some active components of the culture medium and their recommended or preferred dosages are listed in Table 5.
  • the second follicle culture medium is MEM ⁇ or a similar culture medium (such as other MEM series culture medium) is used as the basic culture medium.
  • a third follicle culture medium is applied, which is based on the second follicle culture medium and adds inactivated human follicular fluid.
  • the inactivated human follicular fluid can be added at 5-15% (v/v); more preferably, 8-12% (v/v); for example, 6, 7, 9, 10, 11, 13, 14% (v/v).
  • the addition of human follicular fluid is conducive to the development of late follicles and the formation of antral follicles.
  • antibiotics are added to the follicle culture medium to avoid contamination.
  • the antibiotics include penicillin and/or streptomycin.
  • a 3D culture method is used in combination with the organoid culture medium to provide good conditions for the generation of follicles.
  • the present invention also provides a human oocyte product obtained by the method of the present invention, including a human mature oocyte.
  • the present invention provides a human mature oocyte, which is obtained by using human female reproductive stem cells as starting cells, mixing them with ovarian somatic cell-like cells, establishing a human ovarian organoid system, and having a characteristic selected from the following group: the first polar body is expelled.
  • ICSI fertilization is performed and the appearance of pronuclei is observed after 16-24 hours.
  • the appearance of pronuclei and bipolar bodies is used as a sign of fertilization.
  • embryo culture medium is used to continue culturing until the blastocyst develops.
  • the method and product of the present invention can not only provide an excellent model for studying the molecular mechanism of production and maturation of human oocytes, but also provide a new approach for the clinical application of human oocytes, and can be applied to assisted reproduction.
  • the present invention also provides culture medium for induction culture at each stage, including the aforementioned first type organ culture medium, second type organ culture medium, third type organ culture medium; first follicle culture medium, second follicle culture medium, third follicle culture medium.
  • cytokines or chemical components known in the art that have the same or similar functions as them can also be applied to the present invention.
  • the analogs, proteins with the same functions (such as proteins with the same functions of growth factors) or compounds of the specifically listed components, equivalent compounds, analogs, derivatives and/or their salts, hydrates or precursors that induce the same target can also be used to replace the above-mentioned specific components to achieve the same technical effect.
  • These analogs, proteins with the same functions or compounds should also be included in the present invention.
  • Analogs of compounds include, but are not limited to: isomers and racemates of compounds. Compounds have one or more asymmetric centers.
  • the "precursor of a compound” refers to a compound that can be converted into a compound of any of the above compounds, or a salt or solution composed of a compound of any of the above compounds in the culture medium after being applied or treated by an appropriate method.
  • the culture medium may also be added with a bacterial contaminant for preventing cell culture.
  • contamination such as Gram-positive and Gram-negative bacteria, and components such as some antibiotics.
  • the cell culture medium may be, for example, but not limited to: MEM, StemPro-34, DMEM, DMEM/F12, RPMI1640, Neuronal basal or Fischers, etc. It should be understood that those skilled in the art are familiar with the preparation or purchase of the basal cell culture medium.
  • the preferred cell culture medium is provided in the embodiments of the present invention.
  • the present invention also provides a kit, which contains the first type of organ culture medium, the second type of organ culture medium, and the third type of organ culture medium described in the present invention; the first follicle culture medium, the second follicle culture medium, and the third follicle culture medium.
  • the kit also contains starting cells, human female reproductive stem cells, and ovarian somatic cell-like cells. Alternatively, it also contains pluripotent stem cells for generating ovarian somatic cell-like cells.
  • the kit also contains culture medium/reagents for isolating and maintaining cells.
  • the kit also contains instructions for use, so as to facilitate the use of those skilled in the art in research or clinical application.
  • Matrigel is used to treat Transwell, 3 kinds of follicle culture fluids are used, and some factors/reagents such as Transferrin, Insulin, cAMP, LH, Estrogen, Progestrone, inactivated human follicular fluid, etc. are selected. These factors are all related to human ovarian or follicular development; (3) the follicle culture time is greatly extended. The mouse culture is about 3-4 weeks, while the human culture time is about 6 weeks.
  • human 3D ovarian organoids were constructed using ovarian somatic cells differentiated from human female germ stem cells and iPSCs.
  • Example 1 Differentiation of human pluripotent stem cells into ovarian somatic cell-like cells
  • human ovarian somatic cell-like cells were prepared using human pluripotent stem cells as the basic cells.
  • iPSCs Human induced pluripotent stem cells (iPSCs, obtained from Shanghai Oriental Hospital (Oriental Hospital affiliated to Tongji University) National Stem Cell Transformation Resource Bank) were suspended and cultured in low-adhesion culture bottles, with an inoculation volume of 5x 10 5 cells/bottle.
  • DMEM 20% KSR, 1% NEAA, 1% Glutamax, 0.7% ⁇ -mercaptoethanol (Sigma) were used as the basic culture medium, and the culture conditions were: suspension culture at 37°C and 5% CO 2 constant temperature; culture for 2 days to form embryoid bodies. Let it stand for 5 minutes, wait for the embryoid bodies to precipitate naturally, and this part of the cell spheres can be used for subsequent culture.
  • Change to fresh medium and carry out the third stage of culture add Wnt3a (final concentration 6ng/ml), Activin A (final concentration 6ng/ml) and BMP4 (final concentration 10ng/ml) to the basic medium DMEM/F12 (containing 20% KSR, 1% NEAA, 1% Glutamax, 0.7% ⁇ -mercaptoethanol) for 3 days. Let the cell spheres settle naturally, and these cell spheres can be used for the subsequent culture.
  • Follistain or Fstl1 Final concentration 25 ng/ml
  • BMP4 final concentration 10 ng/ml
  • FBS final concentration 5%
  • FIG. 1A The bright field image of human pluripotent stem cells differentiated into ovarian somatic cell-like cells obtained by the above method is shown in Figure 1A. According to the figure, it can be seen that the present invention obtains human ovarian somatic cell-like cells in an ideal state and in large quantities.
  • Example 2 Establishment of ovarian organoids based on human FGSCs and acquisition of follicles
  • Human FGSCs were obtained from follicular aspirates and used to establish a co-culture system. FGSCs were passaged in vitro for about 10 generations.
  • Ovarian somatic cell-like cells are obtained by inducing differentiation of iPSCs in vitro, as described in Example 1.
  • Human FGSCs and ovarian somatic cell-like cells were mixed in a 15 ml centrifuge tube at a ratio of 1:1000 and centrifuged at 1000 rpm/min for 5 min.
  • organoid culture medium I (Table 6).
  • 10% FBS in MEM ⁇ can be replaced by 10% SPS (serum protein substitute).
  • the suspension was mixed with Matrigel at a ratio of 1:1, and the cell suspension and Matrigel mixture was quickly added to a 96-well U-shaped plate, 5 ⁇ l per well.
  • the culture plate was placed in a 37°C 5% CO 2 incubator for 30 min to allow Matrigel to solidify.
  • the 96-well U-shaped plate was taken out, 100 ⁇ l of organoid culture medium I was added thereto, and the U-shaped plate was placed in a 37°C 5% CO 2 incubator for 7 days, during which the medium was changed every 24 h.
  • the cell clusters formed by the aggregation of FGSCs and ovarian somatic cells were transferred to the upper chamber of the Transwell using a 1 ml pipette, and 6-10 cell clusters were cultured in each Transwell.
  • 1 ml of organoid culture medium II (Table 7) was added to the upper chamber of the Transwell, and 1.5 ml of organoid culture medium II was added to the lower chamber of the Transwell.
  • the culture plate was placed in a 37°C 5% CO 2 incubator, during which 1 ml of culture medium was aspirated from the lower chamber of the Transwell and 1 ml of fresh culture medium was added every 24 hours.
  • Organoids were cultured in organoid culture medium II for about 2 weeks and then replaced with organoid culture medium III (Table 8). Organoids were cultured in organoid culture medium III for about 4 weeks or longer, during which 1 ml of culture medium was aspirated from the lower chamber of the Transwell and 1 ml of fresh culture medium was added every 24 hours.
  • Tissue section results also showed that the organoids contained follicles at different developmental stages (Figure 2).
  • tissue permeabilization of ovarian organoids combined with immunohistochemistry confirmed that oocytes in the follicles of ovarian organoids were positive for the germ cell marker DDX4 ( Figure 4).
  • Granulosa cells in ovarian organoid follicles also expressed the granulosa cell marker PTCH1 ( Figure 5).
  • the inventors In order to detect the hormone secretion ability of ovarian organoids, the inventors detected the concentrations of progesterone and estradiol in the culture medium during the ovarian organoid culture process. The results showed that during the ovarian organoid culture period, the concentrations of progesterone (prog) and estradiol (E2) continued to increase, indicating that ovarian organoids have the ability to secrete hormones (Figure 6).
  • Ovarian organoids were permeabilized and combined with immunohistochemistry to confirm that granulosa cells in the ovarian organoid follicles expressed the hormone secretion-related marker CYP19A1, further confirming the hormone secretion ability of ovarian organoids (Figure 7).
  • the culture medium was changed to follicular culture medium II (Table 10). During the period of 24 hours, 1 ml of culture medium was aspirated and discarded from the lower chamber of Transwell and 1 ml of fresh culture medium was added.
  • follicle culture medium III (10% (v/v) inactivated human follicle fluid was added to follicle culture medium II) and cultured for about 3 weeks, during which 1 ml of culture medium was discarded from the lower chamber of the Transwell every 24 hours and 1 ml of fresh culture medium was added.
  • the inactivated human follicle fluid was obtained from the Reproductive Medicine Center (its function is to promote follicle growth).
  • the oocytes and granulosa cells in the follicles are close to maturity.
  • the immature follicles after 3D culture were placed in an in vitro maturation culture medium (basal maturation culture medium with 15% human follicular fluid) and cultured in a 37°C 5% CO 2 incubator for 24-72 hours.
  • the morphology of the oocytes and the looseness of the cumulus cells were observed under a microscope, and the discharge of the first polar body was used as a sign of maturation. As shown in Figure 10, the first polar body was discharged, and the discharge of the first polar body was a sign of oocyte maturation.

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Abstract

一种制备成熟卵母细胞的方法及应用。由雌性生殖干细胞(FGSCs)衍生的卵巢类器官系统，利用该系统将雌性生殖干细胞体外高效分化至功能性卵母细胞。所述卵母细胞经体外受精能进一步发育。所述方法可以高效地获得功能性卵母细胞，为辅助生殖提供了新的途径，以及为女性不孕不育治疗提供了新方法。

Description

制备成熟卵母细胞的方法及应用 技术领域
本发明属于不孕以及生育力保存领域；更具体地，本发明涉及一种制备成熟卵母细胞的方法，包括从人雌性生殖干细胞分化至人成熟卵母细胞。
背景技术
近年来，全球不孕不育症逐年增加，平均发病率越来越高。不孕不育已经成为继心脑血管、肿瘤之后威胁人类健康的第三大疾病。此外，严峻的数据背后不孕不育现象还朝着年轻化趋势发展，其中25-30岁人群所占比例最高。卵母细胞匮乏和质量下降是导致女性不孕的关键因素。雌性生殖干细胞(FGSCs)的发现为解决卵母细胞匮乏提供了新的视角，其作为补充卵母细胞的生殖系干细胞，可维持卵巢的生殖功能，间接的维持卵巢的内分泌，延缓卵巢早衰，对雌性哺乳动物提高卵泡质量与受孕率具有重要意义，正成为医疗界关注的焦点。FGSCs是一类特殊的成体干细胞，它不但具有干细胞的特征，而且还具有传递遗传信息的能力。雌性哺乳动物FGSCs经过复杂的发育过程，最终产生有功能的成熟卵子，为发育后代提供雌性配子。
早在2012年，日本科学家Mitinori Saitou和Katsuhiko Hayashi就已经研发出将小鼠胚胎干细胞、诱导性多能干细胞诱导出原始生殖细胞的技术。但局限是，体外培养后，研究人员需要将这些原始生殖细胞移植入成年小鼠体内，才能获得卵母细胞；并且，基于物种的不同，其技术不易于套用于人。
2016年10月，Katsuhiko Hayashi研究团队将小鼠的皮肤细胞诱导出人工卵子，并成功受精发育成健康小鼠，这是世界上首次完全体外诱导出人工卵子。但是，其诱导自皮肤细胞，且基于物种的不同、生殖细胞的性能及培养条件差异大，其技术不易于套用于人。
2018年Mitinori Saitou研究团队通过将人诱导多能干细胞(iPSCs)诱导成PGC样细胞(PGCLCs)，然后将其与其他细胞共培养。最终将iPSCs诱导为具有卵母细胞特性的细胞。然而，经鉴定，它们属于卵原细胞，不能在体外成熟并受精。
综上，本领域目前还没有体外得到人成熟卵母细胞的相关报道。
发明内容
本发明的目的在于提供一种制备成熟卵母细胞的方法，包括从人雌性生殖干细胞分化至成熟卵母细胞。
在本发明的第一方面，提供一种制备人成熟卵母细胞的体外方法，所述方法包括：(1)将人雌性生殖干细胞与卵巢体细胞样细胞进行共培养，建立人卵巢类器官系统，所述人卵巢类器官系统中生成卵泡；和，(2)从人卵巢类器官中分离卵泡，体外培养，获得人成熟卵母细胞。
在一种或多种优选实施方式中，获得人成熟卵母细胞后，还包括：(3)将人成熟卵 母细胞进行体外受精，体外发育(如发育至囊胚)。
在一种或多种优选实施方式中，(1)中，所述卵巢体细胞样细胞包括人多潜能干细胞体外分化获得的卵巢体细胞样细胞。
在一种或多种优选实施方式中，所述卵巢体细胞样细胞的培养包括：
(1)细胞预培养；
(2)以GSK3抑制剂处理(1)的细胞；
(3)以B/A/W处理(2)的细胞，其中该B/A/W是BMP4、ActivinA和Wnt3a；
(4)以Follistain、BMP4以及血清处理(3)的细胞；
(5)以Follistain和BMP4处理(4)的细胞，获得含有人卵巢体细胞样细胞的培养物。
在一种或多种优选实施方式中，所述的人多潜能干细胞包括人诱导多潜能干细胞(人诱导多能干细胞(iPSCs)和人胚胎干细胞。
在一种或多种优选实施方式中，(1)中，所述人雌性生殖干细胞与卵巢体细胞样细胞以1∶(100～10000)混合；较佳地以1∶(200～8000)混合；更佳地以1∶(300～6000)混合。
在一种或多种优选实施方式中，所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，先将混合细胞培养以形成细胞团，之后形成类器官。
在一种或多种优选实施方式中，所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，进行三维培养(例如以Matrigel凝固)。
在一种或多种优选实施方式中，所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，以第一类器官培养液进行培养、形成细胞团，该培养液包括：视黄酸、L-谷氨酰胺、亚精胺、丙酮酸钠、β-巯基乙醇、EGF、FGF、胶质细胞源性神经营养因子、白血病抑制因子、非必需氨基酸；随后以第二类器官培养液进行培养，该培养液包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；之后以第三类器官培养液进行培养，该培养液包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇。
在一种或多种优选实施方式中，以第一类器官培养液进行培养的时间为5～10天，较佳地6～9天(如7、8天)；以第二类器官培养液进行培养的时间为10～20天，较佳地12～18天(如13、14、15、16、17天)；或以第三类器官培养液进行培养的时间为18～45天，较佳地20～40天(如22、24、28、30、32、35、38天)。
在一种或多种优选实施方式中，所述第一或第二类器官培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基，其中第一类器官培养液含血清或血清蛋白替代物(如5～15％，较佳地7～13％，更佳地8～12％)，第二类器官培养液含血清或血清蛋白替代物(较佳地含1～5％，如含1.5％、2％、2.5％、3％、3.5％、4％或4.5％)；所述第三类器官培养液以StemPro-34 SFM为基础培养基，其含血清或血清蛋 白替代物(较佳地含5～15％，如含7％、8％、9％、10％、11％、12％或13％)。
在一种或多种优选实施方式中，所述人雌性生殖干细胞与卵巢体细胞样细胞例如以1∶400、500、600、800、1000、1500、2000、3000、4000、5000、7000或9000混合。
在一种或多种优选实施方式中，所述培养在进行时，每隔12-36小时进行换液(部分换液)。
在一种或多种优选实施方式中，第一类器官培养液进行培养时，在U型板中进行。
在一种或多种优选实施方式中，第二类器官培养液进行培养时，在Transwell中进行。
在一种或多种优选实施方式中，第三类器官培养液进行培养时，在Transwell中进行。
在一种或多种优选实施方式中，所述第一、第二、第三类器官培养液中，β-巯基乙醇用于中和掉细胞培养基中积累的氧自由基。
在一种或多种优选实施方式中，所述第一、第二、第三类器官培养液中，第一类器官培养液的β-巯基乙醇的含量为所述第二或第三类器官培养液中含量的1.5～3倍(如2倍)。
在一种或多种优选实施方式中，所述第一、第二、第三类器官培养液中，还包括抗生素。
在一种或多种优选实施方式中，所述抗生素包括：青霉素和/或链霉素。
在一种或多种优选实施方式中，(2)中，所述体外培养包括：以第一卵泡培养液进行培养，该培养液包括：L-谷氨酰胺、转铁蛋白、亚硒酸钠、抗坏血酸、EGF、Activin-A、FSH、β-巯基乙醇；随后以第二卵泡培养液进行培养，该培养液包括：L-谷氨酰胺、丙酮酸钠、亚硒酸钠、bFGF、EGF、转铁蛋白、Insulin、PVP、bmp15、gdf9、cAMP、FSH、LH、Estrogen、Progestrone、β-巯基乙醇；之后以第三卵泡培养液进行培养，该培养液包括：第二卵泡培养液以及5～20％(v/v)灭活人卵泡液。
在一种或多种优选实施方式中，所述第一卵泡培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基，其含血清或血清蛋白替代物(较佳地含3～8％，如含4％、5％、6％、7％)；所述第二卵泡培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基，其含血清或血清蛋白替代物(较佳地含5～15％，如含7％、8％、9％、10％、11％、12％或13％)。
在一种或多种优选实施方式中，所述的培养为三维培养(例如以Matrigel凝固)。
在一种或多种优选实施方式中，所述培养在进行时，每隔12-36小时进行换液(部分换液)。
在一种或多种优选实施方式中，第一、第二和第三卵泡培养液进行培养时，在Transwell中进行。
在一种或多种优选实施方式中，所述第一、第二、第三卵泡培养液中，还包括β -巯基乙醇。
在一种或多种优选实施方式中，所述第一、第二、第三卵泡培养液中，还包括抗生素。
在一种或多种优选实施方式中，所述抗生素包括：青霉素和/或链霉素。
在本发明的另一方面，提供一种人成熟卵母细胞，其由前面任一所述的方法制备获得。
在一种或多种优选实施方式中，所述人成熟卵母细胞未经体外受精，不能独立地发育为生物体。
在本发明的另一方面，提供人雌性生殖干细胞与卵巢体细胞样细胞的应用，用于体外制备人成熟卵母细胞。
在一种或多种优选实施方式中，所述人成熟卵母细胞经由体外受精，能进一步体外发育至囊胚。
在本发明的另一方面，提供一种用于体外制备人成熟卵母细胞的试剂盒，其中包括：
(a)人雌性生殖干细胞和卵巢体细胞样细胞，或其混合细胞；
(b)类器官培养液；以及
(c)卵泡培养液。
在一种或多种优选实施方式中，(a)中，所述人雌性生殖干细胞与卵巢体细胞样细胞的比例为1∶(100～10000)，较佳地1∶(200～8000)；更佳地1∶(300～6000)。
在一种或多种优选实施方式中，(b)包括：第一类器官培养液，其包括：视黄酸、L-谷氨酰胺、亚精胺、丙酮酸钠、β-巯基乙醇、EGF、FGF、胶质细胞源性神经营养因子、白血病抑制因子、非必需氨基酸；第二类器官培养液，其包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；第三类器官培养液，其包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；较佳地，所述第一或第二类器官培养液以MEMα或类似培养基为基础培养基，其中第一类器官培养液含血清或血清蛋白替代物(如5～15％，较佳地7～13％，更佳地8～12％)，第二类器官培养液含血清或血清蛋白替代物(较佳地含1～5％，如含1.5％、2％、2.5％、3％、3.5％、4％或4.5％)；所述第三类器官培养液以StemPro-34SFM为基础培养基，其含血清或血清蛋白替代物(较佳地含5～15％，如含7％、8％、9％、10％、11％、12％或13％)。
在一种或多种优选实施方式中，(c)包括：第一卵泡培养液，其包括：L-谷氨酰胺、转铁蛋白、亚硒酸钠、抗坏血酸、EGF、Activin-A、FSH、β-巯基乙醇；第二卵泡培养液，其包括：L-谷氨酰胺、丙酮酸钠、亚硒酸钠、bFGF、EGF、转铁蛋白、Insulin、PVP、bmp15、gdf9、cAMP、FSH、LH、Estrogen、Progestrone、β-巯基乙醇；第三 卵泡培养液，其包括：第二卵泡培养液以及5～20％(v/v)灭活人卵泡液。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1A、实施例1获得的人多能干细胞分化为卵巢体细胞样细胞的明场图。
图1B、卵巢类器官培养第1周(W1)、第3周(W3)和第5周(W5)的代表性图像。
图2、卵巢类器官培养的第1周(W1)、第3周(W3)和第5周(W5)流式细胞分析结果。
图3、培养第5周卵巢类器官中有明显的RFP信号。
图4、培养第5周卵巢类器官生殖细胞中MVH的表达情况。
图5、卵巢类器官培养过程激素浓度变化。
图6、人雌性生殖干细胞体外分化衍生的卵泡不同培养时期的形态图。
图7、人雌性生殖干细胞体外分化的GV期卵母细胞形态。
图8、人雌性生殖干细胞体外分化的MII卵母细胞形态。
图9、人雌性生殖干细胞体外分化的成熟卵卵母细胞经体外受精后的胚胎发育过程。
图10、将经过3D培养的未成熟卵泡放入体外成熟培养液中在37℃5％CO2培养箱中培养24-72h，显微镜观察卵母细胞的形态、卵丘细胞的松散程度，以第一极体排出作为成熟的标志。
图11、一旦卵母细胞成熟，进行ICSI受精，16-24小时后观察有无原核出现，以出现原核及双极体作为受精的标志。
具体实施方式
本发明人经过深入的研究，建立了由雌性生殖干细胞(FGSCs)衍生的卵巢类器官系统(较佳地利用三维(3D)培养系统建立)，并利用该系统将雌性生殖干细胞体外高效分化至功能性卵母细胞，经体外受精能进一步发育(最终可形成健康后代)。本发明的方法可以高效地获得功能性卵母细胞，为辅助生殖提供了新的途径，以及为女性不孕不育治疗提供了新方法。
术语
如本文所用，术语“基础培养基”或“基本培养基”，其为适合于细胞培养的，能为细胞提供足够营养成分的、满足细胞生长所需的培养基。
如本文所用，所述的“颗粒细胞”与“卵巢颗粒细胞”可互换使用，其可以是天然来源的细胞或基于天然细胞扩增/传代的细胞，或者也可以是经由遗传改造或遗传修饰的细胞。
如本文所用，所述的“雌性生殖干细胞”或“卵巢雌性生殖干细胞”可互换使用，其可以是天然来源的细胞或基于天然细胞扩增/传代的细胞，或者也可以是经由遗传改造或遗传修饰的细胞。
如本文所用，所述的“卵巢体细胞样细胞”与“卵巢体细胞”可互换使用，其可以是天然来源的细胞或基于天然细胞扩增/传代的细胞，或者也可以是经诱导得到的细胞；此外，也可以是经由遗传改造或遗传修饰的细胞。
如本文所用，“功能性”是指根据所描述的方法获得的卵巢体细胞样细胞具有与所预期的相同或相似的功能。
如本文所用，“分化”指谱系定型(1ineage commitment)的发育过程。“谱系”指细胞发育的途径，其中前体或“祖先”细胞(本发明中为干细胞)经历渐进的生理变化以成为具有特征性功能的特定细胞类型(本发明中为卵巢体细胞样细胞)。
雌性生殖干细胞
本发明中，所述的雌性生殖干细胞可以是来自机体的雌性生殖干细胞，也可以来自本领域在先技术已经扩增培养/传代/建系的雌性生殖干细胞。例如，本发明人的在先研究中已经扩繁了雌性生殖干细胞，可以应用这些体外扩增获得的雌性生殖干细胞。例如，一种雌性生殖干细胞的培养(扩增)方法包括：将雌性生殖干细胞置于胚胎成纤维细胞(STO细胞)饲养层，在含有丙酮酸钠、L-谷氨酰胺、巯基乙醇、非必需氨基酸、表皮生长因子、人碱性成纤维细胞生长因子、神经胶质细胞生长因子、白血病抑制因子的细胞培养基中培养。这一方法可培养雌性生殖干细胞，但不会诱导其进一步分化为卵母细胞。
所述雌性生殖干细胞可来自于卵泡抽吸物，其为辅助生殖中常被废弃的产品。例如，一种获得雌性生殖干细胞的方法包括：(1)从卵泡抽提物中分离出卵巢组织或细胞团块，进行细胞分散以获得单细胞；(2)将步骤(1)获得的细胞接种到灭活的饲养层细胞上培养，从经过培养的细胞
卵巢体细胞样细胞
本发明中，所述卵巢体细胞样细胞包括人多潜能干细胞体外分化获得的卵巢体细胞样细胞。所述的人多潜能干细胞包括人诱导多潜能干细胞(人诱导多能干细胞(iPSCs)和人胚胎干细胞。
本发明中涉及的胚胎干细胞或多潜能干细胞是可通过商业途径购买的，并非必需通过破坏胚胎的方式获得的胚胎干细胞或多潜能干细胞。早在1998年人胚胎干细胞和胚胎生殖细胞就已经建系成功，例如1998年Thomson领导的小组从14个囊胚中最终建立起5个人类ES细胞系：H1、H13、H14、H7和H19；Gearhart领导的小组从5-9周龄的流产胎儿的性腺嵴及肠系膜中分离原始的干细胞，以期避免因直接利用胚胎所造成的伦理学上的麻烦。见晁岚等，“人胚胎干细胞的研究进展”，《现代 妇产科进展》，2003年7月第12卷第4期。基于上述工作，在2000年的2月份，Wisconsin Alumni Research Foundation(WARF)建立了WiCell，WiCell是一家非官方的、非盈利性的附属机构，它以低费用向合格的科学家分配人胚胎干细胞。提供这种现成的人胚胎干细胞的机构还有很多。
所述卵巢体细胞样细胞包括人多潜能干细胞体外分化获得的卵巢体细胞样细胞；较佳地，所述卵巢体细胞样细胞的培养包括：(1)细胞预培养；(2)以GSK3抑制剂处理(1)的细胞；(3)以B/A/W处理(2)的细胞，其中该B/A/W是BMP4、ActivinA和Wnt3a；(4)以Follistain、BMP4以及血清处理(3)的细胞；(5)以Follistain和BMP4处理(4)的细胞，获得含有人卵巢体细胞样细胞的培养物。
在一个或多个实施方式中，所述“处理”包括“诱导”或“诱导处理”。
在一个或多个实施方式中，所述方法为体外/离体培养方法。
在一个或多个实施方式中，步骤(3)之后，获得的细胞表达brachyury、mixl1、pax2的中内胚层标志物。
在一个或多个实施方式中，所述的人干细胞包括人潜能干细胞(如人诱导多潜能干细胞)和人全能干细胞(如人胚胎干细胞)。
在一个或多个实施方式中，所述“卵巢体细胞样细胞”中，该“体细胞样细胞”是指表达Foxl2、Cyp19a1与Ptch1的细胞。
在一个或多个实施方式中，步骤(1)～步骤(3)中，进行悬浮培养，步骤(4)～步骤(5)中，进行贴壁培养。
在一个或多个实施方式中，步骤(1)中，以基础培养基培养细胞；步骤(2)～步骤(5)中，GSK3抑制剂、B/A/W、Follistain、BMP4、血清被添加于基础培养基中；较佳地，所述基础培养基为干细胞培养基(培养干细胞适用的培养基)；更佳地所述基础培养基为E8，mTesr或DMEM/F12基础培养液；更佳地，所述基础培养基为添加20±10％KSR、1±0.5％NEAA、1±0.5％Glutamax、0.7±0.2β-巯基乙醇、4～10ng/ml bFGF的基础培养基。
在一个或多个实施方式中，步骤(1)中，细胞预培养的时间为1～4天；较佳地1.5～3天；更佳地1.5～2.5天(如2天)。
在一个或多个实施方式中，用于悬浮培养的培养基为液体培养基(培养液)。用于贴壁培养的培养基为相应的液体培养基(培养液)。
在一个或多个实施方式中，悬浮培养时，利用低黏附的培养皿进行培养。
在一个或多个实施方式中，贴壁培养时，将细胞贴于经明胶处理的培养皿中；较佳地，经0.2％明胶处理。
在一个或多个实施方式中，E8为GIBCO公司的Essential 8TM培养基，mTeSR1为Stemcell公司的培养基。这两种培养基均可用于多能干细胞的培养。
在一个或多个实施方式中，步骤(2)中，所述GSK3抑制剂包括选自：CHIR99021、LiCl、BIO或Ly2090314；较佳地为CHIR99021；较佳地该CHIR99021浓度为3～8uM (如4、4.5、5、5.5、6或7uM)，更佳地4～6uM。
在一个或多个实施方式中，步骤(2)中，培养时间30～48小时(如32、34、36、38、40、42或44小时)。
在一个或多个实施方式中，步骤(3)中，BMP4浓度为4～15ng/ml(如5、6、7、8、9、10、11、12、13或14ng/ml)、较佳地9～12ng/ml。
在一个或多个实施方式中，Activin A浓度5～15ng/ml(如6、7、8、9、10、11、12、13或14ng/ml)、较佳地9～12ng/ml。
在一个或多个实施方式中，Wnt3a浓度为5～15ng/ml(如6、7、8、9、10、11、12、13或14ng/ml)、较佳地9～12ng/ml。
在一个或多个实施方式中，步骤(3)中，培养时间2～9天(如2.5、3、4、5、6、7或8天)，较佳地2.5～7天；更佳地3～6天。
在一个或多个实施方式中，步骤(4)中，Follistain浓度为15～40ng/ml(如18、20、22、25、28、30、32或35ng/ml)，较佳地20～30ng/ml，更佳地22～28ng/ml。
在一个或多个实施方式中，步骤(4)中，BMP4浓度为4～15ng/ml(如5、6、7、8、9、10、11、12、13或14ng/ml)、较佳地9～12ng/ml。
在一个或多个实施方式中，步骤(4)中，血清浓度为3～10％(v/v)(较佳地4～8％，如5％、6％、7％(v/v))。
在一个或多个实施方式中，步骤(4)中，培养时间3～12小时(如4、5、6、7、8、9、10或11小时)，较佳地4～10小时；更佳地5～8小时。
在一个或多个实施方式中，步骤(5)中，Follistain浓度为15～40ng/ml(如18、20、22、25、28、30、32或35ng/ml)，较佳地20～30ng/ml，更佳地22～28ng/ml。
在一个或多个实施方式中，步骤(5)中，BMP4浓度为4～15ng/ml(如5、6、7、8、9、10、11、12、13或14ng/ml)、较佳地9～12ng/ml；或
在一个或多个实施方式中，步骤(5)中，培养时间4～12天(如4.5、5、6、7、8、9、10或11天)，较佳地5～10天；更佳地6～8天。
卵巢类器官的建立
本发明提供了一种由人雌性生殖干细胞体外分化至成熟卵母细胞的方法。所述方法首先将人雌性生殖干细胞与卵巢体细胞样细胞进行共培养，建立人卵巢类器官系统，所述人卵巢类器官系统中生成卵泡；之后，从人卵巢类器官中分离卵泡，体外培养，获得成熟卵母细胞。在一种优选的实施方式中，本发明的方法包括：(i)利用人雌性生殖干细胞与人诱导多能干细胞(iPSCs)体外分化的到的卵巢体细胞样细胞进行3D共培养，建立人卵巢类器官系统；(ii)从卵巢类器官中分离卵泡并进行3D培养，体外成熟后获得成熟卵母细胞；进一步可选地，进行(iii)将所获得的成熟卵母细胞经体外受精最终发育至囊胚。
本发明中，所述人雌性生殖干细胞与卵巢体细胞样细胞以中分选出雌性生殖干细 胞标志物阳性细胞(参见PCT/CN2016/073429)。1∶(100～10000)混合；较佳地以1∶(200～8000)混合；更佳地以1∶(300～6000)混合。从而，人雌性生殖干细胞能够获得卵巢体细胞样细胞的良好支持，较好地发生分化。在最为优选的方式中，所述人雌性生殖干细胞与卵巢体细胞样细胞以约1∶1000的比例进行混合。
本发明中，应用了第一类器官培养液，该培养液的一些活性组分及其建议的或优选的用量列举如表1。
表1
在优选的实施方式中，所述的第一类器官培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基，有利于提供适合于细胞生长的营养成分。优选的，该培养液中不添加血清，而是采用SPS血清替代物，以更有利于形成细胞团。
经上述培养液的培养后，应用第二类器官培养液，该培养液的一些活性组分及其建议的或优选的用量列举如表2，其中，尤其bFGF、胰岛素、亚精胺、BMP4发挥了主要的作用。
表2

在优选的实施方式中，所述的第二类器官培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基，有利于提供适合于细胞生长的营养成分，与第一类器官培养液相比，进一步有利用类器官中的卵泡发育。优选的，该培养液中添加少量的血清，从而更有利于促进类器官中的卵泡发育。
经上述培养液的培养后，应用第三类器官培养液，该培养液的一些活性组分及其建议的或优选的用量列举如表3。
表3
在优选的实施方式中，所述的第三类器官培养液以StemPro-34为基础培养基，不同于前面的MEM培养液，StemPro-34能为类器官生长提供更适合且充足的营养。优选的，该培养液中添加10％左右的血清，从而更有利用类器官中多层卵泡发育。
本发明的优选方式中，所述的类器官培养液中添加抗生素，从而避免污染的产生。较佳地，所述抗生素包括：青霉素和/或链霉素。
本发明的优选方式中，在进行类器官的建立时，采用3D培养的方法，配合所述的类器官培养液，为卵泡的生成提供良好的条件。
成熟卵母细胞的获得
如前述建立人卵巢类器官系统后，所述人卵巢类器官系统中生成卵泡；之后，从人卵巢类器官中分离卵泡，体外培养获得成熟卵母细胞。
本发明中，应用了第一卵泡培养液，该培养液的一些活性组分及其建议的或优选的用量列举如表4，其中，尤其FSH、PVP发挥了主要的作用。
表4
在优选的实施方式中，所述的第一卵泡培养液以MEMα或类似培养基(如其它MEM系列培养基)为基础培养基。
经上述培养液的培养后，应用第二卵泡培养液，该培养液的一些活性组分及其建议的或优选的用量列举如表5。
表5
在优选的实施方式中，所述的第二卵泡培养液以MEMα或类似培养基(如其它 MEM系列培养基)为基础培养基。
经上述培养液的培养后，应用第三卵泡培养液，其在第二卵泡培养液基础上，加入灭活人卵泡液。较佳地，灭活人卵泡液可以添加5～15％(v/v)；更佳地添加8～12％(v/v)；例如添加6、7、9、10、11、13、14％(v/v)。此时，人卵泡液的添加有利于后期卵泡发育，形成窦卵泡。
本发明的优选方式中，所述的卵泡培养液中添加抗生素，从而避免污染的产生。较佳地，所述抗生素包括：青霉素和/或链霉素。
本发明的优选方式中，在进行类器官的建立时，采用3D培养的方法，配合所述的类器官培养液，为卵泡的生成提供良好的条件。
卵母细胞
基于本发明的新发现，本发明还提供了一种由本发明所述的方法获得的人卵母细胞产物，包括人成熟卵母细胞。
本发明提供了一种人成熟卵母细胞，其是以人雌性生殖干细胞为出发细胞、将其与卵巢体细胞样细胞混合，建立人卵巢类器官系统，从该系统中获得，其具有选自下组的特征：第一极体被排出。
一旦人卵母细胞成熟，进行ICSI受精，16-24小时后观察有无原核出现，以出现原核及双极体作为受精的标志。受精后使用胚胎培养液继续培养，直至发育至囊胚
本发明的方法以及产物不仅能为研究人卵母细胞的产生以及成熟的分子机理提供优良的模型，也为人卵母细胞的临床应用提供了新的途径，可应用于辅助生殖。
培养基/试剂盒
本发明也提供了用于各阶段诱导培养的培养基，包括前述的第一类器官培养液、第二类器官培养液、第三类器官培养液；第一卵泡培养液、第二卵泡培养液、第三卵泡培养液。
除了本发明实施例中所列举的具体细胞因子或化学组分以外，本领域公知的与它们具有相同或相近功能的细胞因子或化学组分也可被应用于本发明中。所述具体列举的成分的类似物、同功能蛋白(如生长因子的同功能蛋白)或化合物、诱导相同靶点的等效化合物、类似物、衍生物和/或它们的盐、水合物或前体，也可用于替换上述具体列举的成分，以实现同样的技术效果。这些类似物、同功能蛋白或化合物也应被包含在本发明中。化合物的类似物包括但不限于：化合物的异构体、外消旋体。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。所述的“化合物的前体”指当用适当的方法施用或处理后，该化合物的前体在培养基中可转变成上述任一化合物的一种化合物，或上述任一化合物的一种化合物所组成的盐或溶液。
作为本发明的优选方式，所述的培养基中还可加入用于预防细胞培养的细菌污 染，例如革兰氏阳性和阴性细菌污染的成分，例如一些抗生素。
所述的细胞培养基(基础培养基)例如可以是但不限于：MEM、StemPro-34、DMEM、DMEM/F12、RPMI1640、Neuronal basal或Fischers等。应理解，本领域技术人员熟悉所述的基础细胞培养基的配制或购买途径。而本发明的实施例中则提供了的优选的细胞培养基。
本发明还提供了一种试剂盒，其中含有本发明所述的包括前述的第一类器官培养液、第二类器官培养液、第三类器官培养液；第一卵泡培养液、第二卵泡培养液、第三卵泡培养液。较佳地，所述试剂盒中还含有出发细胞人雌性生殖干细胞、卵巢体细胞样细胞。或者，其中还含有用于生成卵巢体细胞样细胞的多潜能干细胞。较佳地，在需要的情况下，所述的试剂盒中还含有用于分离和维持细胞的培养基/试剂。较佳地，所述试剂盒中还包含使用说明书，从而便于本领域人员在研究中或在临床上应用。
本发明的技术方案，适用于从人雌性生殖干细胞出发，将之与卵巢体细胞样细胞进行共培养，最终形成成熟卵母细胞。出于物种的差异，针对人雌性生殖干细胞的诱导与针对鼠的生殖干细胞的诱导相比，区别是极其显著的，主要在于：(1)人3D类器官培养时间与鼠相比，时间大大延长，鼠的类器官培养只需要3周左右，而人类器官培养需要7天+2周+4周＝7周；(2)3D培养时培养条件差异，例如本发明中利用Matrigel处理Transwell，利用了3种卵泡培养液，优选了一些发挥作用的因子/试剂如Transferrin，Insulin，cAMP，LH，Estrogen，Progestrone，灭活人卵泡液等，这些因素均为人卵巢或卵泡发育相关；(3)卵泡培养时间大大延长，鼠大约培养3-4周，人的培养时间约为6周左右。
本领域中，至今没有成功建立由人雌性生殖干细胞衍生的卵巢类器官系统并利用该系统将雌性生殖干细胞体外高效分化至功能卵母细胞的方案。而本发明则成功获得成熟卵母细胞，其经体外受精能产生健康后代。
申请专利时，建议强调物种来源以及出发细胞的特定性，以及对所获得的功能性卵母细胞提供完善的活性验证数据。在此基础上强调：首次利用人雌性生殖干细胞和iPSCs诱导分化的卵巢体细胞样细胞构建了人3D卵巢类器官；以及首次将人雌性生殖干细胞体外分化至功能卵母细胞。
本发明的优异效果在于：
(1)首次利用人雌性生殖干细胞和iPSCs诱导分化的卵巢体细胞样细胞构建了人3D卵巢类器官。
(2)首次将人雌性生殖干细胞体外分化至功能卵母细胞。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社中所述的 条件，或按照制造厂商所建议的条件。
实施例1、人多能干细胞分化为卵巢体细胞样细胞
本实施例中，以人多能干细胞为基础细胞，制备人卵巢体细胞样细胞。
第一阶段
将人诱导多潜能干细胞(iPSCs，获自上海市东方医院(同济大学附属东方医院)国家干细胞转化资源库)悬浮培养在低黏附的培养瓶中，接种量为5x 105细胞/瓶。以DMEM、20％KSR、1％NEAA、1％Glutamax、0.7％β-巯基乙醇(Sigma公司)为基础培养基，培养条件：37℃、5％CO2恒温条件下悬浮培养；培养2天，形成拟胚体。静置5min，待拟胚体自然沉淀，该部分细胞球可用于后面的培养。
第二阶段
换新鲜培养基，进行第二阶段培养。该阶段中，本发明人先后采取了两种培养方案。在基础培养基DMEM/F12(含20％KSR、1％NEAA、1％Glutamax、0.7％β-巯基乙醇)中添加WNT信号通路的激动剂：GSK3抑制剂CHIR99021，添加量为5uM。培养36小时。培养条件：37℃、5％CO2恒温条件下悬浮培养。静置，细胞球自然沉淀，该部分细胞球可用于后面的培养。
第三阶段
换新鲜培养基，进行第三阶段培养：在基础培养基DMEM/F12(含有20％KSR、1％NEAA、1％Glutamax、0.7％β-巯基乙醇)中添加Wnt3a(终浓度6ng/ml)、Activin A(终浓度6ng/ml)以及BMP4(终浓度10ng/ml)处理3天。静置，细胞球自然沉淀，该部分细胞球可用于后面的培养。
第四阶段
换新鲜培养基，进行第四阶段培养。在上一步的基础上，将细胞贴于经0.2％明胶处理过的培养皿中。在基础培养基DMEM/F12(含有20％KSR、1％NEAA、1％Glutamax、0.7％β-巯基乙醇)中添加卵泡抑素样蛋白1(Follistain-like protein 1，简称Follistain或Fstl1)(终浓度25ng/ml)、BMP4(终浓度10ng/ml)、FBS(终浓度5％)处理5-8h。
第五阶段
换新鲜培养基，进行第五阶段培养。在基础培养基DMEM/F12(含有20％KSR、1％NEAA、1％Glutamax、0.7％β-巯基乙醇)中，添加Follistain(25ng/ml)、BMP4(10ng/ml)处理8天；不添加血清。
经由上述方法获得的人多能干细胞分化为卵巢体细胞样细胞的明场图如图1A所示。根据该图可见，本发明获得了状态理想的人卵巢体细胞样细胞，且数量多。
实施例2、基于人FGSCs的卵巢类器官的建立以及卵泡的获得
从卵泡抽吸物中获得人FGSCs，用于建立共培养体系。FGSCs在体外传代10代左右。
卵巢体细胞样细胞由iPSCs体外诱导分化获得，如实施例1。
将人FGSCs与卵巢体细胞样细胞按照1∶1000的比例混合至15ml离心管中，1000rpm/min离心5min。
离心结束后，用类器官培养液I(Organoid culture medium I；表6)重悬。
表6
MEMα中10％FBS可以由10％SPS(serum protein substitute)替代。
然后将悬液按照1∶1的比例与Matrigel混合，迅速将细胞悬液与Matrigel混合物滴加至96孔U型板中，每孔5μl。将培养板放入37℃ 5％CO2培养箱中培养30min，使Matrigel凝固。取出96孔U型板，向其中加入100μl类器官培养液I，将U型板放置于37℃ 5％CO2培养箱中培养7天，期间每24h换液一次。
上述96孔U型板中培养7天后，将FGSCs与卵巢体细胞样细胞聚合形成的细胞团用1ml移液器转移到Transwell上室，每个Transwell培养6-10个细胞团。向Transwell上室添加类器官培养液II(Organoid culture medium II；表7)1ml，向Transwell下室添加类器官培养液II 1.5ml。
将培养板放入37℃ 5％CO2培养箱中，期间每24h从Transwell下室吸弃1ml培养液并补加1ml新鲜培养液。
表7

*MEMα中2％FBS可以由2％SPS替代。
类器官在类器官培养液II中培养约2周，更换为类器官培养液III(表8)。类器官在类器官培养液III(Organoid culture medium III)培养约4周或更久，期间每24h从Transwell下室吸弃1ml培养液并补加1ml新鲜培养液。
表8
*StemPro-34中10％FBS可以由10％SPS替代。
本发明人研究了人卵巢类器官形成的发育特征。在培养的第三周(W3)，发现类器官中开始出现较小的卵泡，随着连续培养，卵泡长大，在培养的第五周(W5)可以清晰的看到由卵母细胞组成的卵泡(图1B)。
组织切片结果也显示类器官中含有不同发育时期的卵泡(图2)。
荧光显微镜可检测到卵巢类器官中明显的RFP信号(图3)。这表明卵巢类器官中形成卵泡且来源于FGSCs。
此外，卵巢类器官进行组织透化并结合免疫组化证实卵巢类器官卵泡中的卵母细胞呈现生殖细胞标记物DDX4阳性(图4)。
卵巢类器官卵泡中的颗粒细胞也表达颗粒细胞标志物PTCH1(图5)。
为了检测卵巢类器官的激素分泌能力，本发明人检测了卵巢类器官培养过程中培养基中的孕酮和雌二醇浓度。结果表明，在卵巢类器官培养期间，孕酮(prog)和雌二醇(E2)浓度持续增加，表明卵巢类器官具有激素分泌能力(图6)。
卵巢类器官进行组织透化并结合免疫组化证实，卵巢类器官卵泡中颗粒细胞表达激素分泌相关的标志物CYP19A1，进一步证实卵巢类器官的激素分泌能力(图7)。
上述结果表明，来源于人FGSCs的卵巢类器官含有卵泡，并具有激素分泌功能。
实施例3、获得成熟卵母细胞
准备一个新的Transwell，在上室加入1ml Matrigel，将培养板放入37℃ 5％CO2培养箱中培养30min，使Matrigel凝固。使用胰岛素注射针头慢慢将单个卵泡(实施例2中培养至5-7周后的卵泡)剥离，用口吸管将单个卵泡转移至Matrigel预处理的Transwell上室，用卵泡培养液I(Follicle culture medium I；表9)培养，期间24h从Transwell下室吸弃1ml培养液并补加1ml新鲜培养液。
表9
*MEMα中5％FBS可以由5％SPS替代。
1周后，更换为卵泡培养液II(Follicular culture medium II；表10)培养，期间24h从Transwell下室吸弃1ml培养液并补加1ml新鲜培养液。
表10

*MEMα中10％FBS可以由10％SPS替代。
2周后更换为卵泡培养液III(卵泡培养液II中加入和10％(v/v)灭活人卵泡液)培养3周左右，期间24h从Transwell下室吸弃1ml培养液并补加1ml新鲜培养液。灭活人卵泡液获自：生殖医学中心(其作用是促进卵泡生长)。
卵泡培养过程中，可以看到卵泡外周颗粒细胞增殖明显，卵泡直径变大(图8)。
在培养5周(此处5周，从分离出卵泡开始培养算起)后，卵泡中的卵母细胞已经发育至GV期(图9)。
随着继续培养，到第6周时，卵泡中卵母细胞及颗粒细胞都接近成熟。
将经过3D培养的未成熟卵泡放入体外成熟培养液(基础成熟培养液中加入15％人卵泡液)中在37℃5％CO2培养箱中培养24-72h，显微镜观察卵母细胞的形态、卵丘细胞的松散程度，以第一极体排出作为成熟的标志。根据图10可见：第一极体被排出，排出第一极体为卵母细胞成熟的标志。
一旦卵母细胞成熟，进行ICSI受精，16-24小时后观察有无原核出现，以出现原核及双极体作为受精的标志。受精后使用胚胎培养液继续培养，直至发育至囊胚，如图11。
上述结果说明，本发明的方法可分化获得成熟卵母细胞，且其后续可实现良好的生长发育。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。同时，在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。

Claims (15)

  1. 一种制备人成熟卵母细胞的体外方法，其特征在于，所述方法包括：
    (1)将人雌性生殖干细胞与卵巢体细胞样细胞进行共培养，建立人卵巢类器官系统，所述人卵巢类器官系统中生成卵泡；和
    (2)从人卵巢类器官中分离卵泡，体外培养，获得人成熟卵母细胞。
  2. 如权利要求1所述的方法，其特征在于，获得人成熟卵母细胞后，还包括：
    (3)将人成熟卵母细胞进行体外受精，体外发育。
  3. 如权利要求1所述的方法，其特征在于，(1)中，所述卵巢体细胞样细胞包括人多潜能干细胞体外分化获得的卵巢体细胞样细胞。
  4. 如权利要求3所述的方法，其特征在于，所述卵巢体细胞样细胞的培养包括：
    (1)细胞预培养；
    (2)以GSK3抑制剂处理(1)的细胞；
    (3)以B/A/W处理(2)的细胞，其中该B/A/W是BMP4、ActivinA和Wnt3a；
    (4)以Follistain、BMP4以及血清处理(3)的细胞；
    (5)以Follistain和BMP4处理(4)的细胞，获得含有人卵巢体细胞样细胞的培养物。
  5. 如权利要求1所述的方法，其特征在于，(1)中，所述人雌性生殖干细胞与卵巢体细胞样细胞以1∶(100～10000)混合；较佳地以1∶(200～8000)混合；更佳地以1∶(300～6000)混合。
  6. 如权利要求1所述的方法，其特征在于，所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，先将混合细胞培养以形成细胞团，之后形成类器官；或
    所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，进行三维培养。
  7. 如权利要求5所述的方法，其特征在于，所述人雌性生殖干细胞与卵巢体细胞样细胞混合后，以第一类器官培养液进行培养、形成细胞团，该培养液包括：视黄酸、L-谷氨酰胺、亚精胺、丙酮酸钠、β-巯基乙醇、EGF、FGF、胶质细胞源性神经营养因子、白血病抑制因子、非必需氨基酸；随后以第二类器官培养液进行培养，该培养液包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；之后以第三类器官培养液进行培养，该培养液包括：亚精胺、转铁蛋白、亚硒 酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；或
  8. 如权利要求7所述的方法，其特征在于，以第一类器官培养液进行培养的时间为5～10天，较佳地6～9天；以第二类器官培养液进行培养的时间为10～20天，较佳地12～18天；或以第三类器官培养液进行培养的时间为18～45天，较佳地20～40天；
    较佳地，所述第一或第二类器官培养液以MEMα或类似培养基为基础培养基，其中第一类器官培养液含血清或血清蛋白替代物，第二类器官培养液含血清或血清蛋白替代物；所述第三类器官培养液以StemPro-34SFM为基础培养基，其含血清或血清蛋白替代物。
  9. 如权利要求1所述的方法，其特征在于，(2)中，所述体外培养包括：以第一卵泡培养液进行培养，该培养液包括：L-谷氨酰胺、转铁蛋白、亚硒酸钠、抗坏血酸、EGF、Activin-A、FSH、β-巯基乙醇；随后以第二卵泡培养液进行培养，该培养液包括：L-谷氨酰胺、丙酮酸钠、亚硒酸钠、bFGF、EGF、转铁蛋白、Insulin、PVP、bmp15、gdf9、cAMP、FSH、LH、Estrogen、Progestrone、β-巯基乙醇；之后以第三卵泡培养液进行培养，该培养液包括：第二卵泡培养液以及5～20％(v/v)灭活人卵泡液；
  10. 如权利要求1所述的方法，其特征在于，所述第一卵泡培养液以MEMα或类似培养基为基础培养基，其含血清或血清蛋白替代物；所述第二卵泡培养液以MEMα或类似培养基为基础培养基，其含血清或血清蛋白替代物；较佳地，所述的培养为三维培养。
  11. 一种人成熟卵母细胞，其由权利要求1～10任一所述的方法制备获得。
  12. 人雌性生殖干细胞与卵巢体细胞样细胞的应用，用于体外制备人成熟卵母细胞。
  13. 如权利要求12所述的应用，其特征在于，所述人成熟卵母细胞经由体外受精，能进一步体外发育至囊胚。
  14. 一种用于体外制备人成熟卵母细胞的试剂盒，其特征在于，其中包括：
    (a)人雌性生殖干细胞和卵巢体细胞样细胞，或其混合细胞；
    (b)类器官培养液；以及
    (c)卵泡培养液。
  15. 如权利要求14所述的试剂盒，其特征在于，(a)中，所述人雌性生殖干细胞与卵巢体细胞样细胞的比例为1∶(100～10000)，较佳地1∶(200～8000)；更佳地1∶(300～6000)；或
    (b)包括：第一类器官培养液，其包括：视黄酸、L-谷氨酰胺、亚精胺、丙酮酸钠、β-巯基乙醇、EGF、FGF、胶质细胞源性神经营养因子、白血病抑制因子、非必需氨基酸；第二类器官培养液，其包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；第三类器官培养液，其包括：亚精胺、转铁蛋白、亚硒酸钠、胰岛素、FSH、L-谷氨酰胺、抗坏血酸、EGF、bFGF、胶质细胞源性神经营养因子、白血病抑制因子、SCF、BMP4、β-巯基乙醇；较佳地，所述第一或第二类器官培养液以MEMα或类似培养基为基础培养基，其中第一类器官培养液含血清或血清蛋白替代物，第二类器官培养液含血清或血清蛋白替代物；所述第三类器官培养液以StemPro-34 SFM为基础培养基，其含血清或血清蛋白替代物；或
    (c)包括：第一卵泡培养液，其包括：L-谷氨酰胺、转铁蛋白、亚硒酸钠、抗坏血酸、EGF、Activin-A、FSH、β-巯基乙醇；第二卵泡培养液，其包括：L-谷氨酰胺、丙酮酸钠、亚硒酸钠、bFGF、EGF、转铁蛋白、Insulin、PVP、bmp15、gdf9、cAMP、FSH、LH、Estrogen、Progestrone、β-巯基乙醇；第三卵泡培养液，其包括：第二卵泡培养液以及5～20％(v/v)灭活人卵泡液。
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