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WO2024104294A1 - 一种制备人卵巢体细胞样细胞的方法 - Google Patents

一种制备人卵巢体细胞样细胞的方法 Download PDF

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WO2024104294A1
WO2024104294A1 PCT/CN2023/131312 CN2023131312W WO2024104294A1 WO 2024104294 A1 WO2024104294 A1 WO 2024104294A1 CN 2023131312 W CN2023131312 W CN 2023131312W WO 2024104294 A1 WO2024104294 A1 WO 2024104294A1
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cells
culture
human
somatic cell
concentration
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French (fr)
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吴际
葛均邦
李小勇
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上海交通大学
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/24—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the genital tract, from non-germinal gonad cells
    • C12N2506/243—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the genital tract, from non-germinal gonad cells from cells of the female genital tract cells, from non-germinal ovarian cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention belongs to the field of cell biology, and more specifically, relates to a method for differentiating human pluripotent stem cells into human ovarian somatic cell-like cells.
  • Infertility affects the quality of life and marital stability of millions of families, as well as the mental health of patients and their family members, and has become one of the major health and social issues. Many factors lead to infertility, including malnutrition, mental stress, organic problems such as tumors of the pituitary gland, ovaries or hypothalamus, and endocrine disorders.
  • FGSC has a variety of clinical application values, including the treatment of female infertility, preservation of female fertility and delay of female menopause.
  • the inventors discovered for the first time in the world that FGSC exists in the ovaries of postnatal mice. Using C-terminal DDX4 antibody, magnetic beads were used to sort germ cells from the ovaries of postnatal mice, and FGSC cell lines were established after in vitro culture. Transplantation of FGSC into the ovaries of infertile mice can produce functional oocytes and offspring (Zou, K., Z. Yuan, Z. Yang, et al., Production of offspring from a germline stem cell line derived from neonatal ovaries.
  • Stem cells are a general term for a group of cells that have the potential for self-renewal and differentiation. Stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to their differentiation potential. Although stem cells have been widely studied and successfully used to induce differentiation into a variety of tissue cells, such as liver cells, there are still technical difficulties in inducing human ovarian somatic cell-like cells using stem cells as starting cells.
  • the purpose of the present invention is to provide a method for differentiating human pluripotent stem cells into human ovarian somatic cell-like cells and its application.
  • a method for preparing human ovarian somatic cell-like cells comprising:
  • 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 The basal medium is supplemented with 20 ⁇ 10% KSR, 1 ⁇ 0.5% NEAA, 1 ⁇ 0.5% Glutamax, 0.7 ⁇ 0.2 ⁇ -mercaptoethanol, and 4-10ng/ml bFGF; or
  • 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 8 TM medium from GIBCO
  • mTeSR1 is a medium from Stemcell, Inc. 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 8uM (such as 4, 4.5, 5, 5.5, 6 or 7uM), more preferably 4 to 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.
  • a human ovarian somatic cell-like cell obtained based on human stem cell induction is provided, which is prepared by any of the aforementioned methods; preferably, it has characteristics selected from the following group: the human ovarian somatic cell-like cell is mainly epithelial-like in morphology, with fibroblastic cells appearing in short spindle or triangular shapes; the human ovarian somatic cell-like cell is positive for Foxl2, cyp19a1, and Ptch1 markers (including gene/transcript levels and protein levels); and/or the human ovarian somatic cell-like cell can promote the differentiation of human female germ stem cells.
  • a use of the human ovarian somatic cell-like cells for promoting the differentiation of human female reproductive stem cells into human oocytes.
  • a method for promoting the differentiation of human female reproductive stem cells comprising: using the human ovarian somatic cell-like cells as supporting cells, culturing the human female reproductive stem cells on a cell layer of the human ovarian somatic cell-like cells or mixing the human female reproductive stem cells with the human ovarian somatic cell-like cells.
  • kits for preparing human ovarian somatic cell-like cells in vitro comprising:
  • Culture medium 1 comprising a basal culture medium and a GSK3 inhibitor; preferably, the GSK3 inhibitor is selected from: CHIR99021, LiCl, BIO or Ly2090314, more preferably CHIR99021; Preferably, the amount of CHIR99021 is: when used to prepare functional human ovarian somatic cell-like cells in vitro, it can form a concentration of 3 to 8 uM, more preferably 4 to 6 uM in the culture system;
  • Culture medium 2 comprising a basal culture medium and B/A/W, wherein the B/A/W is BMP4, ActivinA and Wnt3a or a mixture thereof; preferably, when used to prepare functional human ovarian somatic cell-like cells in vitro, the BMP4 can form a concentration of 4-15 ng/ml, preferably 9-12 ng/ml in the culture system, the ActivinA can form a concentration of 5-15 ng/ml, preferably 9-12 ng/ml in the culture system, and the Wnt3a can form a concentration of 5-15 ng/ml, preferably 9-12 ng/ml in the culture system;
  • Culture medium 3 comprising a basal culture medium, Follistain, BMP4 and serum; preferably, when used to prepare functional human ovarian somatic cell-like cells in vitro, the Follistain can form a concentration of 15-40 ng/ml, preferably 20-30 ng/ml, more preferably 22-28 ng/ml in the culture system, the BMP4 can form a concentration of 4-15 ng/ml, preferably 9-12 ng/ml in the culture system, and the concentration of the serum in the culture system is 3-10% (v/v) of the serum concentration (preferably 4-8%, such as 5%, 6%, 7% (v/v));
  • Culture medium 4 includes basal culture medium, Follistain and BMP4; preferably, when used to prepare functional human ovarian somatic cell-like cells in vitro, the Follistain can form a concentration of 15-40 ng/ml, preferably 20-30 ng/ml, more preferably 22-28 ng/ml in the culture system, and the BMP4 can form a concentration of 4-15 ng/ml, preferably 9-12 ng/ml in the culture system.
  • kits for preparing human ovarian somatic cell-like cells based on staged culture of human stem cells are provided.
  • Figure 1 Bright field image of human pluripotent stem cells differentiating into human ovarian somatic cell-like cells.
  • Figure 2 RT-PCR detection of cell markers at various stages of cell differentiation, with GAPDH as an internal reference.
  • FIG. 4 Quantitative analysis results of PCR products (Group 5C) showed that the human ovarian somatic cell-like cells (OSLCs) obtained by this differentiation protocol expressed Foxl2, Cyp19a1, and Ptch1 at high levels.
  • OSLCs human ovarian somatic cell-like cells
  • Figure 6 Determination of hormone secretion of hPSC-derived human ovarian somatic cells. After 48 hours of treatment with 50ug/L testosterone, the supernatant of hPSC-derived human ovarian somatic cells was collected for ELISA to measure estrogen. D14.5 is the culture time of inducing human ovarian somatic cells to the fifth stage, which is the 14.5th day of the entire induction differentiation process; D0 is the cells before the start of induction.
  • Human ovarian somatic cell-like cells induce differentiation of human female germ stem cells; immunofluorescence detection of the expression of GDF9 and ZP3 in cells (oocytes) formed by female germ stem cells after induction by human ovarian somatic cell-like cells.
  • Fig. 8 RT-qPCR detection of the expression of oocyte marker genes c-KIT, FIGLA, GJA4, GDF9 and ZP3 in cells (oocytes) formed by female germ stem cells induced by human ovarian somatic cell-like cells; +OSLC, female germ cells induced by human ovarian somatic cell-like cells; Control, female germ cells induced without adding human ovarian somatic cell-like cells; *, P ⁇ 0.05, **, P ⁇ 0.01, ***, P ⁇ 0.001.
  • the inventors Based on in-depth research, the inventors have established a method for preparing human ovarian somatic cell-like cells in vitro, including cells cultured in stages based on human stem cells or human totipotent stem cells.
  • the method of the present invention can efficiently obtain functional human ovarian somatic cell-like cells, efficiently promote the differentiation ability of female reproductive stem cells cultured in vitro, and provide a new approach for assisted reproduction and infertility treatment.
  • cell culture medium in the present invention is a "basal culture medium”, which is a culture medium generally suitable for cell (stem cell) culture and can provide cells (stem cells) with sufficient nutrients to meet the requirements of cell growth.
  • female reproductive stem cells or “ovarian reproductive stem cells” are used interchangeably, and may be cells of natural origin or cells expanded/passaged based on natural cells. In addition, they may also be cells that have been genetically engineered or modified.
  • ovarian somatic cell-like cells and “ovarian somatic cells” are used interchangeably, and they can be cells of natural origin or cells expanded/passaged based on natural cells, or they can also be cells induced In addition, it can also be a cell that has been genetically engineered or modified.
  • human ovarian somatic cell-like cells refer to "ovarian somatic cell-like cells” that positively express Fox12, Cyp19a1, and Ptch1, also known as “ovarian somatic cells.”
  • the present invention is devoted to the study of in vitro large-scale preparation of functional human ovarian somatic cell-like cells, and discloses a method for differentiating human ovarian somatic cell-like cells from stem cells (including human pluripotent stem cells or human embryonic stem cells, preferably human pluripotent stem cells).
  • the method utilizes the culture system of human pluripotent stem cells, adds key factors for inducing differentiation, and induces differentiation of pluripotent stem cells in vitro, thereby obtaining functional human ovarian somatic cell-like cells.
  • the present invention provides a method for preparing human ovarian somatic cell-like cells in vitro, comprising: (1) cell pre-culture; (2) treating the cells of (1) with a GSK3 inhibitor; (3) treating the cell product of (2) with B/A/W, wherein the B/A/W is BMP4, Activin A 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.
  • fresh culture medium can also be replaced according to the culture conditions.
  • the human embryonic stem cells or human 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.
  • Takahashi et al. selected four effective factors (Oct4, Sox2, c-Myc and K1f4) from 24 transcription factors and transferred them into mouse fetal or adult MEF cells using retrovirus, thereby reversing the "life clock” and successfully transforming them into induced pluripotent stem cells (iPSCs).
  • the induced pluripotent stem cells can be cells from the body itself or pluripotent stem cells that have been expanded, cultured, passaged, or established in the field.
  • the female reproductive stem cells can be female reproductive stem cells from the body, or they can be female reproductive stem cells that have been expanded, cultured, passaged, or established in the art.
  • the inventors have established a female reproductive stem cell line in previous studies.
  • a method for culturing (expanding) female reproductive stem cells includes: placing female reproductive stem cells in an embryonic fibroblast (STO cell) feeder layer, and culturing them in a cell culture medium containing sodium pyruvate, L-glutamine, mercaptoethanol, non-essential amino acids, epidermal growth factor, human basic fibroblast growth factor, glial cell growth factor, and leukemia inhibitory factor. This method can culture female reproductive stem cells, but will not induce them to further differentiate into oocytes.
  • STO cell embryonic fibroblast
  • suspension culture is used for cell induction in the early stage of induction, while adherent culture is used in the later stage.
  • the culture medium used for culture or induction is a liquid culture medium (culture fluid).
  • a specific scheme for inducing differentiation of human stem cells which comprises the following steps:
  • human pluripotent stem cells were cultured in suspension in a low-adhesion culture dish for 2 days to obtain embryoid bodies.
  • mesendoderm differentiation is performed, including treatment with an agonist of the WNT signaling pathway (GSK3 inhibitor, such as CHIR99021) for 30 to 48 hours;
  • GSK3 inhibitor such as CHIR99021
  • the cells were attached to a culture dish treated with 0.2% gelatin;
  • the concentration of CHIR99021 is 5uM
  • the concentration of BMP is 5-10ng/ml
  • the concentration of Activin A is 6-10 ng/ml
  • the concentration of Follistain is 25 ng/ml
  • the concentration of WNT3A is 6-10 ng/ml.
  • the test results of the inventors show that the obtained mesendoderm cells can express brachyury, mixl1, and pax2; and the obtained human ovarian somatic cell-like cells can express foxl2, cyp19a1, and ptch1.
  • the induced human ovarian somatic cell-like cells were identified by reverse transcription polymerase chain reaction and immunocytochemistry, and they can efficiently promote the differentiation of human female germ stem cells into oocytes.
  • the method of the present invention utilizes human stem cells to simulate the developmental laws of human ovarian somatic cells, first differentiating them into early mesoderm cells in vitro, and then continuing to induce differentiation into human ovarian somatic cell-like cells.
  • Several main features and advantages of the method of the present invention are as follows: First, the entire culture system uses cell growth factors and chemical molecules, and does not introduce exogenous genes involving reprogramming or transdifferentiation functions (without changing the genome structure), thereby avoiding the interference of exogenous genes on the genome stability of the original stem cells and the potential safety hazards of transplantation related to exogenous cells.
  • human ovarian somatic cell-like cells can be efficiently obtained, and have the function of secreting estrogen and progesterone.
  • the efficiently obtained human ovarian somatic cell-like cells have an ideal cell state and can well support the differentiation of female reproductive stem cells into oocytes.
  • the human ovarian somatic cell-like cells obtained by the present invention can preferably be used as supporting cells for female reproductive stem cells to promote the efficient differentiation of female reproductive stem cells into human oocytes.
  • the human ovarian somatic cell-like cells obtained by the present invention can be introduced into the ovarian tissue of the body for necessary treatment or adjuvant therapy.
  • administration in the body includes, but is not limited to, local injection (e.g., catheter administration or direct intraovarian injection), systemic injection, intravenous injection, intrauterine injection, and parenteral administration.
  • the recipients are, for example (but not limited to), subjects who have difficulty conceiving, are undergoing infertility treatment, have been treated for cancer, and have undergone cytotoxic therapy (e.g., chemotherapy or radiotherapy) or a combination thereof.
  • the method and product of the present invention can not only provide an excellent model for studying the molecular mechanism of human ovarian somatic cell occurrence, but also provide a new approach for the clinical application of human ovarian somatic cells, and can be applied to assisted reproduction and infertility treatment.
  • the present invention also provides culture media for induction culture at each stage, including the aforementioned culture media 1 to 4.
  • 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 effects.
  • 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 further be added with ingredients for preventing bacterial contamination of cell culture, such as contamination by Gram-positive and Gram-negative bacteria, such as some antibiotics.
  • the cell culture medium may be, for example, but not limited to: DMEM/F12, MEM, DMEM, RPMI1640, Neuronal basal, 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 culture medium 1 to culture medium 4 described in the present invention; preferably, it also contains pluripotent stem cells.
  • the kit also contains a culture medium/reagent for isolating and maintaining cells.
  • the kit also contains instructions for use, so as to facilitate the use of the kit by those skilled in the art in research or clinical application.
  • Example 1 Differentiation of human pluripotent stem cells into human ovarian somatic cell-like cells
  • human ovarian somatic cell-like cells were prepared using human pluripotent stem cells as the basic cells.
  • iPS Human induced pluripotent stem cells
  • 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; cultured for 2 days to form embryoid bodies.
  • human induced pluripotent stem cells can also be replaced by human embryonic stem cells (H9-ES, purchased from Shanghai Chuanqiu Biotechnology Co., Ltd.).
  • BMP4 final concentration 5 ng/mL
  • Activin A final concentration 6 ng/mL
  • Wnt3a final concentration 6 ng/mL
  • GSK3 inhibitor CHIR99021 was added to the basal culture medium DMEM/F12 (containing 20% KSR, 1% NEAA, 1% Glutamax, 0.7% ⁇ -mercaptoethanol) at an amount of 5 uM and cultured for 36 hours.
  • the cell spheres were allowed to settle naturally after standing, and these cell spheres can be used for subsequent culture.
  • Wnt3a final concentration 6 ng/ml
  • Activin A final concentration 6 ng/ml
  • BMP4 final concentration 10 ng/ml
  • the cell spheres were allowed to settle naturally after standing, and these cell spheres can be used for subsequent culture.
  • the cells were attached to a culture dish treated with 0.2% gelatin.
  • Follistain-like protein 1 Follistain or Fstl1
  • BMP4 final concentration 10 ng/ml
  • FBS final concentration 5%
  • hPSCs are human pluripotent stem cells
  • EBs are embryoid bodies
  • MEs are mesendoderm cells
  • OSLCs are human ovarian somatic cell-like cells. It can be seen from the figure that the present invention obtains human ovarian somatic cell-like cells in an ideal state and in large quantities.
  • Example 2 Detection of cell markers at various stages of cell differentiation
  • the cell markers at each stage of differentiation of cells were detected by reverse transcription polymerase chain reaction (RT-PCR).
  • the RT-PCR detection method is as follows:
  • RNA concentration determined above and the requirements of the Hifair III 1st strand Synthesis SuperMix for qPCR (Cat. No.: 11141ES10) kit, 500 ng of total RNA was taken for reverse transcription.
  • the electrophoresis results of the PCR products are shown in Figure 2.
  • the mesendoderm cells (ME) obtained by the differentiation protocol can express Brachyury (Bra), Mixl1, and Pax2 ( Figure 2).
  • the human ovarian somatic cell-like cells (OSLC) obtained by the differentiation protocol can express Foxl2 and Cyp19a1 ( Figure 2), which are typical markers of human ovarian somatic cell-like cells.
  • Example 3 Identification of hPSC-derived human ovarian somatic cell-like cells
  • human ovarian somatic cell-like cells derived from hPSC (induced by group 5C) were identified by immunocytochemistry.
  • Immunocytochemistry detection method is as follows:
  • Antibodies (Fox12, diluted 1:100; Cyp19A1, diluted 1:100; Ptch1, diluted 1:100) were added to antibody diluent (PBS solution containing 5% BSA + 0.2% Triton-X 100);
  • hPSC-derived human ovarian somatic cell-like cells express Cyp19a1, Foxl2, and Ptch1 proteins.
  • the present inventors have found that it is preferable to adopt suspension culture in the first to third stages and then adherent culture.
  • the inventors determined the hormone secretion of hPSC-derived human ovarian somatic cells (induced by group 5C). After 48 hours of treatment with 50ug/L testosterone (added starting from day 6 of the fifth stage), the hPSC-derived human ovarian somatic cells were collected for ELISA to measure estrogen. The results showed that the human ovarian somatic cells of the present invention had high levels of progesterone (prog) and estradiol (E2) ( Figure 6).
  • Example 4 Human ovarian somatic cell-like cells promote the differentiation ability of female germ stem cells cultured in vitro
  • the inventors detected the effect of the human ovarian somatic cell-like cells induced above on the differentiation ability of female germ stem cells cultured in vitro.
  • the inventors inoculated human female germ stem cells onto induced human ovarian somatic cell-like cells (induced by group 5C) for culture, and used a differentiation system without adding human ovarian somatic cell-like cells as a control.
  • the inventors inoculated human female reproductive stem cells into a human ovarian somatic cell layer and cultured for 5 days, and the culture medium contained bFGF, EGF, insulin, transferrin and RA. Subsequently, the inventors used immunofluorescence chemical analysis to detect the expression of marker proteins in oocytes. The results showed that the human oocytes produced by the above method expressed oocyte-specific proteins GDF9 and ZP3, as shown in Figure 7.
  • the inventors detected the expression of oocyte marker genes c-KIT, FIGLA, GJA4, GDF9 and ZP3 in human oocytes produced by the above method by RT-qPCR. The results showed that these marker genes were significantly upregulated, as shown in Figure 8.
  • the hormones secreted by the human ovarian somatic cell-like cells produced by the above method were identified and it was found that they can secrete estrogen and progesterone, and can effectively promote the differentiation of human female reproductive stem cells.

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Abstract

一种制备人卵巢体细胞样细胞的方法，包括基于人干细胞进行分阶段培养，人干细胞包括人多潜能干细胞和人全能干细胞。可以高效地获得功能性人卵巢体细胞样细胞，促进体外培养的雌性生殖干细胞FGSC的分化，为辅助生殖及不孕症治疗提供新的途径。

Description

一种制备人卵巢体细胞样细胞的方法 技术领域
本发明属于细胞生物学领域，更具体地，本发明涉及一种人多能干细胞向人卵巢体细胞样细胞分化的方法。
背景技术
不孕症影响着千千万万家庭的生活质量和婚姻稳定性，也影响患者及其家庭成员的心理健康，已经成为主要的健康和社会问题之一。多方面的因素导致不孕不育的发生，包括营养不良，精神紧张，器质性问题如腺垂体、卵巢或下丘脑的肿瘤以及内分泌紊乱等等。
FGSC具有多种临床应用价值，包括治疗女性不孕症，保存女性生育力和推迟女性更年期等。在以往的工作中，发明人在国际上首次发现出生后小鼠卵巢中存在FGSC。使用C端DDX4抗体从出生后小鼠卵巢中磁珠分选生殖细胞，经体外培养后能建立FGSC细胞系。将FGSC移植到不孕小鼠卵巢中能够产生有功能的卵母细胞和后代(Zou，K.，Z.Yuan，Z.Yang，et al.，Production of offspring from a germline stem cell line derived from neonatal ovaries.Nat Cell Biol，2009.11(5)：p.631-636)。随后，White等人使用相同的抗体从育龄妇女卵巢中流式分选出人FGSC(White，Y.A.R.，D.C.Woods，Y.Takai，et al.，Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.Nat Med，2012.18(3)：p.413-421)。越来越多的研究发现小鼠、大鼠和人卵巢中存在FGSC。临床研究已发现将人FGSC的线粒体注射到患者自身的卵子中能提高卵子质量，增加体外受精(IVF)的成功率(Fakih，M.H.，M.E.Shmoury，J.Szeptycki，et al.，The AUGMENTSM Treatment：Physician Reported Outcomes of the Initial Global Patient Experience.JFIV Reprod Med Genet 2015.3(3)：p.154)。
人FGSC为推迟女性绝经期、治疗女性不孕症以及生育力保持开辟了新的思路。
人FGSC的体外分化需要人卵巢体细胞样细胞的支持和诱导。而可用于人FGSC体外分化的人卵巢体细胞样细胞来源极其有限或缺乏。
干细胞是一类具有自我更新和分化潜能的细胞群体的总称。按干细胞的分 化潜能的大小可以分为全能干细胞，多能(多潜能)干细胞和单能干细胞。尽管干细胞已经被广泛研究，被成功地用于诱导分化为多种组织细胞，如肝脏细胞。然而以干细胞为起始细胞诱导人卵巢体细胞样细胞仍然存在技术难题。
发明内容
本发明的目的在于提供一种人多能干细胞向人卵巢体细胞样细胞分化的方法及其应用。
在本发明的第一方面，提供一种制备人卵巢体细胞样细胞的方法，所述方法是基于人干细胞进行分阶段培养，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；或
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。
在本发明的另一方面，提供前面任一所述的方法的应用，用于基于人干细胞进行分阶段培养，制备人卵巢体细胞样细胞。
在本发明的另一方面，提供一种基于人干细胞诱导获得的人卵巢体细胞样细胞(包括细胞培养物)，其由前面任一所述的方法制备获得；较佳地，其具有选自下组的特征：所述的人卵巢体细胞样细胞为以上皮样形态为主，出现成纤维状的细胞，呈短梭形或三角形；所述的人卵巢体细胞样细胞Foxl2、cyp19a1、Ptch1标志物(包括基因/转录本水平及蛋白水平)呈现阳性；和/或，所述的人卵巢体细胞样细胞能够促进人雌性生殖干细胞分化。
在本发明的另一方面，提供所述的人卵巢体细胞样细胞(包括细胞培养物)的应用，用于促进人雌性生殖干细胞的分化，使其分化为人卵母细胞。
在本发明的另一方面，提供一种促进人雌性生殖干细胞的分化的方法，包括：以所述的人卵巢体细胞样细胞作为支持细胞，将人雌性生殖干细胞培养于所述人卵巢体细胞样细胞的细胞层上或将人雌性生殖干细胞与所述人卵巢体细胞样细胞混合培养。
在本发明的另一方面，提供一种用于体外制备人卵巢体细胞样细胞的试剂盒，其中包括：
培养基1：包括基础培养基以及GSK3抑制剂；较佳地，所述GSK3抑制剂包括选自：CHIR99021、LiCl、BIO或Ly2090314，更佳地为CHIR99021； 较佳地该CHIR99021的量为：当用于体外制备功能性人卵巢体细胞样细胞时，能在培养体系中形成3～8uM，更佳地4～6uM的浓度；
培养基2：包括基础培养基以及B/A/W，其中该B/A/W是BMP4、ActivinA和Wnt3a或它们的混合物；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该BMP4能在培养体系中形成4～15ng/ml、更佳地9～12ng/ml的浓度，该ActivinA能在培养体系中形成5～15ng/ml、较佳地9～12ng/ml的浓度，该Wnt3a能在培养体系中形成5～15ng/ml、较佳地9～12ng/ml的浓度；
培养基3：包括基础培养基以及Follistain、BMP4和血清；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该Follistain能在培养体系中形成15～40ng/ml、较佳地20～30ng/ml、更佳地22～28ng/ml的浓度，该BMP4能在培养体系中形成4～15ng/ml、较佳地9～12ng/ml的浓度，该血清在培养体系中的浓度为血清浓度为3～10％(v/v)(较佳地4～8％，如5％、6％、7％(v/v))；
培养基4：包括基础培养基以及Follistain和BMP4；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该Follistain能在培养体系中形成15～40ng/m、较佳地20～30ng/ml、更佳地22～28ng/ml的浓度，该BMP4能在培养体系中形成4～15ng/ml、较佳地9～12ng/ml的浓度。
在本发明的另一方面，提供所述的试剂盒的应用，用于基于人干细胞分阶段培养，制备人卵巢体细胞样细胞。
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1、人多能干细胞分化为人卵巢体细胞样细胞的明场图。
图2、细胞分化的各阶段细胞标志物的RT-PCR检测，其中，以GAPDH作为内参。
图3、不同方案中的Brachyury基因表达的比较(5C：5uM Chir99021；B：BMP4；A：Activin A；W：Wnt3a)。
图4、PCR产物(5C组)的定量分析结果显示，该分化方案所得到的人卵巢体细胞样细胞(OSLC)高水平表达Foxl2、Cyp19a1，Ptch1。
图5、利用免疫细胞化学方法来对hPSC来源的人卵巢体细胞样细胞进行鉴定。
图6、hPSC来源的人卵巢体细胞样细胞激素分泌的测定。50ug/L的睾酮进行处理hPSC来源的人卵巢体细胞样细胞48h后，收集上清进行ELISA测定雌激素。D14.5为诱导人卵巢体细胞样细胞至第五阶段的培养时间，也即整个诱导分化过程的第14.5d；D0为起始诱导前的细胞。
图7、人卵巢体细胞样细胞诱导人雌性生殖干细胞分化；免疫荧光检测雌性生殖干细胞经人卵巢体细胞样细胞诱导后形成的细胞(卵母细胞)GDF9和ZP3的表达情况。
图8、RT-qPCR检测雌性生殖干细胞经人卵巢体细胞样细胞诱导后形成的细胞(卵母细胞)中卵母细胞标志基因c-KIT、FIGLA、GJA4、GDF9和ZP3表达情况；+OSLC，经人卵巢体细胞样细胞诱导的雌性生殖细胞；Control，不添加人卵巢体细胞样细胞诱导的雌性生殖细胞；*，P＜0.05，**，P＜0.01，***，P＜0.001。
具体实施方式
本发明人在深入研究的基础上，建立了一种在体外制备人卵巢体细胞样细胞的方法，包括基于人干细胞或人全能干细胞进行分阶段培养的细胞。本发明的方法可以高效地获得功能性人卵巢体细胞样细胞，高效促进体外培养的雌性生殖干细胞的分化能力，为辅助生殖及不孕症治疗提供新的途径。
术语
如本文所用，术语“细胞培养基”在本发明中为“基础培养基”，其为普遍适合于细胞(干细胞)培养的，能为细胞(干细胞)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“雌性生殖干细胞”或“卵巢生殖干细胞”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。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，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，人卵巢体细胞样细胞是指Foxl2、Cyp19a1、Ptch1阳性表达的“卵巢体细胞样细胞”，也称为“卵巢体细胞”。
如本文所用，“功能性”是指根据所描述的方法获得的人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(lineage commitment)的发育过程。“谱系”指细胞发育的途径。
培养及诱导方法
本发明致力于功能性人卵巢体细胞样细胞的体外规模化制备的研究，揭示了从干细胞(包括人多能干细胞或人胚胎干细胞，优选地为人多能干细胞)向人卵巢体细胞样细胞分化的方法。该方法利用人多能干细胞的培养体系，添加诱导分化的关键因子，对多能干细胞进行体外诱导分化，从而获得功能性人卵巢体细胞样细胞。
因此，本发明提供了一种体外制备人卵巢体细胞样细胞的方法，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)的细胞产物，其中该B/A/W是BMP4、Activin A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。在培养期间，还可以根据培养情况，更换新鲜的培养基。
本发明中涉及的人胚胎干细胞或人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个人胚胎干细胞系：H1、H13、H14、H7和H9；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。
2006年，Takahashi等通过从24个转录因子中筛选出4个有效的因子(Oct4、Sox2、c-Myc和K1f4)，并利用逆转录病毒将其转入小鼠胎儿或成年MEF细胞，进而实现了“生命时钟”的逆转，成功将其转变为诱导多能干细胞(induced Pluripotent Stem Cells，iPSCs)。所述的诱导多能干细胞可以是来自机体自身的细胞，也可以是来自本领域已经扩增培养/传代/建系的多潜能干细胞。
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，也可以来自本领域已经扩增培养/传代/建系的雌性生殖干细胞。例如，本发明人在前期研究中已经建立了雌性生殖干细胞系。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。
本发明的方法中，诱导前期采用悬浮培养的方式进行细胞诱导，而后期则进行贴壁培养。除非另外说明，用于培养或用于诱导的培养基为液体培养基(培养液)。
在本发明的具体实施例中，提供了一种由人干细胞诱导分化的具体方案，该方案包括如下的步骤：
首先，将人多能干细胞悬浮培养在低黏附的培养皿中2天，获得拟胚体。
在上一步的基础上，进行中内胚层分化，包括以WNT信号通路的激动剂(GSK3抑制剂，如CHIR99021)处理，处理时间30～48小时；
在上一步的基础上，添加Wnt3a、Activin A以及BMP4处理3～6天；
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；
在上一步的基础上，添加Follistain、BMP4、血清处理5-8小时。
在上一步的基础上，添加Follistain、BMP4处理6-8天。
以上的具体实施方案中，CHIR99021的浓度为5uM，BMP浓度为5-10ng/ml， Activin A浓度为6-10ng/ml，Follistain浓度为25ng/ml，WNT3A浓度为6-10ng/ml。
本发明人的检测结果显示，所得到的中内胚层细胞可以表达brachyury、mixl1、pax2；所得到的人卵巢体细胞样细胞可以表达foxl2、cyp19a1、ptch1。
通过逆转录聚合酶链式反应以及免疫细胞化学法鉴定诱导获得的人卵巢体细胞样细胞，其能够高效促进人雌性生殖干细胞分化为卵母细胞。
本发明的方法利用人干细胞模拟人卵巢体细胞的发育规律，将其在体外首先分化为早期中胚层细胞，进而继续诱导分化为为人卵巢体细胞样细胞。本发明的方法的几个主要特点和优势如下：第一，整个培养体系采用细胞生长因子、化学分子，不引入涉及重编程或转分化功能的外源性基因(不改变基因组结构)，因此可以避免外源基因对原干细胞的基因组稳定性的干扰和外源细胞相关的移植安全隐患。第二，可以高效获得人卵巢体细胞样细胞，并且具有能够分泌雌激素与孕激素的功能。第三，高效获得的人卵巢体细胞样细胞，细胞状态理想且能够很好地支持雌性生殖干细胞向卵母细胞的分化。
本发明获得的人卵巢体细胞样细胞，优选地可作为雌性生殖干细胞的支持细胞，促进雌性生殖干细胞向人卵母细胞的高效分化。
本发明获得的人卵巢体细胞样细胞，可被引入到机体的卵巢组织中，用于必要的治疗或辅助治疗。例如，机体内的给予包括但不限于，局部注射(例如，导管给予或直接卵巢内注射)、全身性注射、静脉内注射、子宫内注射和胃肠外给予。接受对象例如(但不限于)受孕困难、处于不孕症治疗中、治疗过癌症和经受过细胞毒治疗(例如，化学治疗或放射治疗)或其组合的受试者。
本发明的方法以及产物不仅能为研究人卵巢体细胞发生的分子机理提供优良的模型，也为为人卵巢体细胞的临床应用提供新的途径，可应用于辅助生殖和不孕症治疗。
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，包括前述的培养基1～培养基4。
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：DMEM/F12、MEM、DMEM、RPMI1640、Neuronal basal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培养基1～培养基4；较佳地，其中还含有多潜能干细胞。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。
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人卵巢体细胞样细胞
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(iPS，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5 x 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。
此外，该阶段中，也可以以人胚胎干细胞(H9-ES，购自上海传秋生物技术有限公司)替换人诱导多潜能干细胞。
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。
(1)方案1(B/A/W)
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B/A/W，也即添加：
BMP4：终浓度5ng/mL；
Activin A：终浓度6ng/mL；和
Wnt3a：终浓度6ng/mL；
培养24小时。
(2)方案2(5C)
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。
以上方案1和方案2的其它培养条件：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人卵巢体细胞样细胞的明场图如图1所示。其中hPSCs为人多能干细胞，EB为拟胚体。ME为中内胚层细胞，OSLC为人卵巢体细胞样细胞。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、细胞分化的各阶段细胞标志物的检测
本实施例中，通过逆转录聚合酶链式反应(RT-PCR)的方法，检测细胞(由5C组诱导)分化的各阶段细胞标志物。
RT-PCR检测方法如下：
(1)总RNA抽提
(a)将收集的细胞(细胞分化的各阶段细胞)移入1ml的Trizol中，反复吹打 3-5次后，室温静置5min，静置后。加入200ul的氯仿，剧烈上下左右混匀20s，室温静置5min；
(b)4℃条件下，12000rpm离心15min；
(c)收集上清液至另一新的无酶离心管中，加入与上清液等量的异丙醇，上下左右混匀后，室温静置10min；
(d)4℃条件下，12000rpm离心10min；
(e)弃上清，加入1ml 70％的无水乙醇，振荡混匀后，4℃条件下，7600rpm离心5min；
(f)弃上清，室温晾干2-5min后，加入适量的无酶水，混匀后，用核酸定量仪测RNA浓度和纯度。
(2)反转录制备cDNA
根据上述测定的RNA浓度以及Hifair III 1st strand Synthesis SuperMix for qPCR(货号：11141ES10)试剂盒的要求，取500ng总RNA进行反转录。
(3)PCR
根据表1的引物序列和2×PCR Master Mix(With Dye)(货号：10102ES03)试剂盒要求进行PCR。
表1
PCR产物的电泳结果如图2，该分化方案所得到的中内胚层细胞(ME)可以表达Brachyury(Bra)、Mixl1、Pax2(图2)。该分化方案所得到的人卵巢体细胞样细胞(OSLC)可以表达Foxl2、Cyp19a1(图2)，它们是典型的人卵巢体细胞样细胞的标志物。
PCR产物的定量分析结果显示(图4)，该分化方案所得到的人卵巢体细胞样细胞(OSLC)高水平表达Foxl2、Cyp19a1，Ptch1。
对于实施例1的第2阶段中的“方案1”(B/A/W)和“方案2”(5C)，本发明人利用PCR法对两方案培养后的细胞培养物分别进行了Brachyury标志物的分析，结果如图3。根据图3可见，5C的诱导更高效地获得中内胚层细胞，能够得到显著更多的中内胚层细胞，细胞状态理想。
实施例3、hPSC来源的人卵巢体细胞样细胞的鉴定
本实施例中，利用免疫细胞化学方法来对hPSC来源的人卵巢体细胞样细胞(由5C组诱导)进行鉴定。
免疫细胞化学检测方法如下：
(a)4％多聚甲醛固定液(PFA)固定细胞，室温放置30min；
(b)弃固定液，用无钙镁的PBS洗3次，每次5min；
(c)用封闭液(含1％BSA+0.2％Triton-X 100的PBS溶液)封闭细胞1h；
(d)将抗体(Foxl2，稀释1∶100；Cyp19A1，稀释1∶100；Ptch1，稀释1∶100)在抗体稀释液(含5％BSA+0.2％Triton-X 100的PBS溶液)中；
(e)吸弃封闭液；将稀释后的一抗加到样品中，4℃孵育过夜；
(f)次日，吸弃一抗，用PBS洗涤细胞3次，每次5min；
(g)将二抗(抗山羊IgG)稀释在抗体稀释液(含5％BSA的PBS溶液)中；
(h)吸弃PBS，将稀释后的二抗(1∶200)加到样品上，室温孵育1h；
(i)吸弃二抗，用PBS洗涤3次，每次5min；
(j)滴加DAPI溶液(10ug/ml)，室温放置2-3min；
(k)吸弃DAPI溶液，再用PBS洗3次，每次5min；
(l)吸弃PBS，抗荧光淬灭剂封片，倒置荧光显微镜下观察拍照。
根据图5可见，hPSC来源的人卵巢体细胞样细胞表达Cyp19a1、Foxl2与Ptch1蛋白。
对于悬浮培养以及贴壁培养的方案，本发明人发现，在第1～3阶段采取悬浮培养，之后是贴壁培养优选的。
同时，本发明人测定了hPSC来源的人卵巢体细胞样细胞(由5C组诱导)的激素分泌。50ug/L的睾酮(第五阶段的第6天开始添加)处理hPSC来源的人卵巢体细胞样细胞48h后，收集上清进行ELISA测定雌激素，结果显示，本发明的人卵巢体细胞样细胞具有高的孕酮(prog)以及雌二醇(E2)水平(图6)。
实施例4、人卵巢体细胞样细胞促进体外培养的雌性生殖干细胞的分化能力
本实施例中，本发明人检测前述诱导获得的人卵巢体细胞样细胞对于体外培养的雌性生殖干细胞的分化能力的影响作用。
首先，本发明人将人雌性生殖干细胞接种到诱导的人卵巢体细胞样细胞(由5C组诱导)上培养。以不添加人卵巢体细胞样细胞的分化体系为对照(Control)。
接着，本发明人将人雌性生殖干细胞接种到人卵巢体细胞样细胞层上培养5天，培养液中含有bFGF、EGF、胰岛素、转铁蛋白和RA。随后，本发明人利用免疫荧光化学分析检测卵母细胞表达标志物蛋白的情况。结果显示，上述方法产生的人卵母细胞表达卵母细胞特异蛋白GDF9和ZP3，如图7。
同时，本发明人通过RT-qPCR检测上述方法产生的人卵母细胞中卵母细胞标志基因c-KIT、FIGLA、GJA4、GDF9和ZP3的表达。结果显示，这些标志基因均显著性上调，如图8。
这些结果表明，诱导的人卵巢体细胞样细胞具有促进雌性生殖干细胞分化的能力，且效果优异、诱导产物状态理想。
对于上述方法产生的人卵巢体细胞样细胞所分泌的激素进行鉴定发现，可以分泌雌激素与孕酮，并且能够高效的促进人雌性生殖干细胞的分化。
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 (14)

  1. 一种制备人卵巢体细胞样细胞的方法，其特征在于，所述方法是基于人干细胞进行分阶段培养，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。
  2. 如权利要求1所述的方法，其特征在于，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。
  3. 如权利要求1所述的方法，其特征在于，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。
  4. 如权利要求1所述的方法，其特征在于，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。
  5. 如权利要求1所述的方法，其特征在于，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。
  6. 如权利要求1所述的方法，其特征在于，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，更佳地4～6uM；或
    培养时间30～48小时。
  7. 如权利要求1所述的方法，其特征在于，步骤(3)中，
    BMP4浓度为4～15ng/ml、较佳地9～12ng/ml；或
    Activin A浓度5～15ng/ml、较佳地9～12ng/ml；或
    Wnt3a浓度为5～15ng/ml、较佳地9～12ng/ml；或
    培养时间2～9天，较佳地2.5～7天；更佳地3～6天。
  8. 如权利要求1所述的方法，其特征在于，步骤(4)中，
    Follistain浓度为15～40ng/ml，较佳地20～30ng/ml，更佳地22～28ng/ml；或
    BMP4浓度为4～15ng/ml、较佳地9～12ng/ml；或
    血清浓度为3～10％(v/v)；或
    培养时间3～12小时，较佳地4～10小时；更佳地5～8小时。
  9. 如权利要求1所述的方法，其特征在于，步骤(5)中，
    Follistain浓度为15～40ng/ml，较佳地20～30ng/ml，更佳地22～28ng/ml；或
    BMP4浓度为4～15ng/ml、较佳地9～12ng/ml；或
    培养时间4～12天，较佳地5～10天；更佳地6～8天。
  10. 一种基于人干细胞诱导获得的人卵巢体细胞样细胞，其由权利要求1～9任一所述的方法制备获得。
  11. 如权利要求10所述的基于人干细胞诱导获得的人卵巢体细胞样细胞，其特征在于，其具有选自下组的特征：
    所述的人卵巢体细胞样细胞为以上皮样形态为主，出现成纤维状的细胞，呈短梭形或三角形；
    所述的人卵巢体细胞样细胞Foxl2、cyp19a1、Ptch1标志物呈现阳性；和/或
    所述的人卵巢体细胞样细胞能够促进人雌性生殖干细胞分化。
  12. 权利要求11所述的人卵巢体细胞样细胞的应用，用于促进人雌性生殖干细胞的分化，使其分化为人卵母细胞。
  13. 一种促进人雌性生殖干细胞的分化的方法，其特征在于，所述方法包括：以权利要求10所述的人卵巢体细胞样细胞作为支持细胞，将人雌性生殖干细胞培养于所述人卵巢体细胞样细胞的细胞层上或将人雌性生殖干细胞与所述人卵巢体细胞样细胞混合培养。
  14. 一种用于体外制备人卵巢体细胞样细胞的试剂盒，其特征在于，其中包括：
    培养基1：包括基础培养基以及GSK3抑制剂；较佳地，所述GSK3抑制剂包括选自：CHIR99021、LiCl、BIO或Ly2090314，更佳地为CHIR99021；较佳地该CHIR99021的量为：当用于体外制备功能性人卵巢体细胞样细胞时，能在培养体系中形成3～8uM，更佳地4～6uM的浓度；
    培养基2：包括基础培养基以及B/A/W，其中该B/A/W是BMP4、ActivinA和Wnt3a或它们的混合物；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该BMP4能在培养体系中形成4～15ng/ml、更佳地9～12ng/ml的浓度，该ActivinA能在培养体系中形成5～15ng/ml、较佳地9～12ng/ml的浓度，该Wnt3a能在培养体系中形成5～15ng/ml、较佳地9～12ng/ml的浓度；
    培养基3：包括基础培养基以及Follistain、BMP4和血清；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该Follistain能在培养体系中形成15～40ng/ml、较佳地20～30ng/ml、更佳地22～28ng/ml的浓度，该BMP4能在培养体系中形成4～15ng/ml、较佳地9～12ng/ml的浓度，该血清在培养体系中的浓度为血清浓度为3～10％(v/v)；
    培养基4：包括基础培养基以及Follistain和BMP4；较佳地，当用于体外制备功能性人卵巢体细胞样细胞时，该Follistain能在培养体系中形成15～40ng/m、较佳地20～30ng/ml、更佳地22～28ng/ml的浓度，该BMP4能在培养体系中形成4～15ng/ml、较佳地9～12ng/ml的浓度。
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