Method for purifying ovarian progenitor cells and application of ovarian progenitor cells in generating steroid secreting cells in vitro

Abstract

The invention provides a method for purifying ovarian progenitor cells and application of the ovarian progenitor cells to in-vitro generation of steroid secretion cells, and belongs to the technical field of biology. The method for purifying the ovarian progenitor cells comprises the following steps: digesting, filtering and washing an ovary, purifying through a magnetic bead or flow cytometry sorting procedure, and screening to obtain the ovarian progenitor cells, a marker used in the purification process is LY6A, LGR5 or PDGFRA (Platelet Derived Growth Factor Receptor A). Experimental results show that the PDGFRA < + > progenitor cells and the LY6A < + > progenitor cells can form androgen synthetic cells for expressing CYP11A1 and CYP17A1; and the LGR5 < + > progenitor cells can only form progestational hormone synthetic cells which express CYP11A1 but do not express CYP17A1.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology, and particularly relates to a method for purifying ovarian progenitor cells and their application in inducing development into steroid-secreting cells in vitro. Background Art

[0002] The ovary has two main functions: producing germ cells (eggs) and producing sex hormones, which are two important components of successful reproduction. Ovarian hormone disorders are often accompanied by symptoms such as irregular menstruation, amenorrhea, infertility, etc., which not only affect reproduction but also the overall health of the individual. The three major endocrine cells of the ovary are theca cells, granulosa cells, and luteal cells. Granulosa cells produce estrogen, theca cells provide androgen precursors for granulosa cells to synthesize estrogen, and luteal cells produce progesterone, which supports pregnancy and fetal growth. These three types of cells are all necessary for sex steroid hormones that maintain ovarian and overall health. Different from steroid cells in the testis, the ovary has active follicular development and regular ovulation cycles, which involve the regeneration and transformation of steroid cells. The dynamics of theca cells, especially their progenitor cells, have not been well studied.

[0003] The ovarian theca layer surrounds the developing follicles, and this layer contains various cell types, including steroid theca cells, vascular endothelial cells, immune cells, and fibroblast-like cells. Since theca cells are not found around early follicles and are only observed when the follicles develop into secondary follicles containing two or more layers of granulosa cells, this indicates that even in adults, theca cells must be recruited or newly developed from progenitor cells during follicle formation. Researchers have isolated and cultured potential steroidogenic theca progenitor cells in various species including mice, pigs, sheep, and humans. These studies have all consistently demonstrated that adult mammalian ovaries contain theca cell progenitor cells. However, these in vitro studies have all used procedures similar to those for culturing ordinary mesenchymal stem cells without pre-purifying the cells. Under appropriate induction conditions, although these cells can express some theca cell-specific markers and produce steroids, they are also often found to express other ovarian cell markers such as granulosa cells and / or oocytes, indicating that the cultured cells are heterogeneous. In addition, their location in vivo is largely unknown.

[0004] In addition to these theca progenitor cells that study steroidogenesis, researchers have also identified a common population of progenitor cells in the ovarian surface epithelium (OSE) and stromal compartment for ovulation-related tissue repair and maintenance of ovarian cellular homeostasis in vivo. During fetal development, progenitor cells from both OSE and perivascular stromal locations contribute to the formation of granulosa and theca cells. OSE is a single-layered, poorly differentiated epithelium that contains progenitor cells for repairing ovarian ruptures during ovulation. Marker proteins used to identify these populations include stem cell antigen-1 (Sca1, encoded by the gene Ly6a), LGR5, and endothelial protein C receptor (EPCR, encoded by the gene Procr). However, the relationship between these well-defined tissue-repair progenitor cells and those steroid progenitor cells identified by in vitro methods remains unclear. In addition, whether these ovulation-related tissue-repair progenitor cells can form steroidogenic cells is still unknown. Summary of the Invention

[0005] In view of this, an object of the present invention is to provide a method for purifying ovarian progenitor cells and its application in generating steroid-secreting cells in vitro.

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for purifying ovarian progenitor cells, comprising: digesting, filtering, and washing the ovary, and then purifying by magnetic beads or flow cytometry sorting procedures to screen out ovarian progenitor cells; the markers used in the purification process are LY6A, LGR5, or PDGFRA.

[0008] Preferably, the cells positive after purification can differentiate into steroid hormone-secreting cells in vitro.

[0009] Preferably, the steroids include androgens and progesterones.

[0010] Preferably, the ovarian progenitor cells include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

[0011] The present invention also provides the application of ovarian progenitor cells in generating steroid-secreting cells in vitro. Preferably, the ovarian progenitor cells are the ovarian progenitor cells screened by the above method.

[0012] Preferably, the ovarian progenitor cells include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

[0013] Preferably, the method for generating steroid-secreting cells in vitro includes: after the ovarian progenitor cells screened by the above method are proliferated in vitro, they are induced to differentiate in vitro for 10-15 days in a DMEM / F12 medium containing insulin-transferrin-selenium additive, Smoothened agonist and luteinizing hormone.

[0014] Preferably, the concentration of the insulin-transferrin-selenium additive is 10 μg / ml - 5.5 μg / ml - 5 ng / ml. The concentration of the Smoothened agonist is 0.5 μM, and the concentration of the luteinizing hormone is 2 ng / ml.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a method for purifying ovarian progenitor cells, including: after digesting, filtering and washing the ovaries, purifying them by magnetic beads or flow cytometry sorting procedures to screen out ovarian progenitor cells; the markers used in the purification process are LY6A, LGR5 or PDGFRA. The experimental results show that both PDGFRA+ and LY6A+ progenitor cells can form androgen-synthesizing cells expressing CYP11A1 and CYP17A1; while LGR5+ progenitor cells can only form progesterone-synthesizing cells expressing CYP11A1 but not CYP17A1. The ovarian progenitor cells screened by the method of the present invention and the formula of the differentiation medium used can provide a reference for the later generation of human androgen and progesterone cells. And the in vitro preparation of human ovarian hormone-synthesizing cells is one of the main directions of future cell therapy for female infertility. In addition, this technology also provides a new basis and foundation for optimizing the development theory of ovarian hormone-synthesizing cells. Description of the Drawings

[0017] Figure 1 : Immunofluorescence staining of 5 potential stem cell markers; LY6A and PDGFRA antibodies label ovarian stromal cells, and granulosa cells are not stained; EPCR and LGR5 antibodies label ovarian surface epithelial (OSE) cells and a small number of stromal cells; LY6A also labels OSE cells; however, CD51 stains almost all stromal cells and some follicular granulosa cells; red arrows: OSE positive cells; red short arrows: OSE negative cells; white arrows: theca cell positive; white short arrows: theca cell negative; yellow arrows: stromal cell positive; green arrows: granulosa cell positive; The scale bar represents a length of 50 μm;

[0018] Figure 2: Purification of LY6A- and CD51-positive cells by flow cytometry or magnetic bead (FACS / MACS) method; (A) Flow cytometry analysis of ovarian cells stained with LY6A-PE antibody separated by MACS; The purification rate of LY6A+ cells was over 99%, and the purification rate of CD51+ cells was over 95%; Positive cells were located within the blue or red frame; LY6A untagged: Cells before staining with LY6A-PE antibody; LY6A tagged: Unseparated cells (Impurified) after staining with LY6A-PE antibody; LY6A+: LY6A+ cells separated by MACS method (Purified); CD51+: CD51+ cells separated by MACS method; (B): PE-labeled LY6A+ and CD51+ cells before and after purification; (C): QPCR analysis of two steroid genes before and after purification of LY6A- or CD51-labeled cells; Data were from 3-4 repeated experiments; Data were expressed as Mean±SE; *, **, ***, **** showed significant differences from positive cells at P<0.05, 0.01, 0.001 or 0.0001 respectively;

[0019] Figure 3 : Cells separated by LY6A- and CD51-labeling expressed steroid markers (HSD3B and CYP11A1); LY6A- and CD51+ cells only expressed these two proteins, while LY6A+ and CD51- cells did not express these two proteins; The scale bar length was 50μm;

[0020] Figure 4 : Proliferation and differentiation of LY6A- / + cells in vitro; (A) EdU labeling and quantitative analysis of LY6A- / + cells; (B) Androgens (androstenedione and testosterone) were produced by LY6A+ cells after 12 days of steroid cell differentiation induction culture; (C) Expression of two steroid genes before (d0) and during (d6) 12 days of in vitro differentiation of LY6A+ cells; Data were from 4 repeated experiments; Data were expressed as mean±SEM; ***, **** showed significant differences compared with LY6A- cells (A) or d0 cells (B, C) (P<0.01, 0.001 or 0.0001); The scale bar indicated a length of 50μm;

[0021] Figure 5 : Ovarian LY6A- / + cells were induced to differentiate in vitro by steroid cells for 10-12 days; The changes in the expression of CYP11A1 and HSD3B before (d0) and after (d10 or d12) differentiation of the two types of cells were compared; The two enzymes were not expressed before differentiation and began to be expressed after differentiation;

[0022] Figure 6: Ovarian LGR5+ and PDGFRA+ cells were induced to differentiate in vitro by steroid cells for 10 days; the changes in the expression of CYP11A1 and CYP17A1 before (d0) and after (d10) differentiation of the two types of cells were compared; neither of the two enzymes was expressed in the two types of cells before differentiation, but the LGR5+ cells expressed CYP11A1 after 10 days of differentiation and did not express CYP17A1; while the PDGFRA+ cells expressed both enzymes after differentiation; the scale bar represents a length of 10 μm. Detailed implementation mode

[0023] The present invention provides a method for purifying ovarian progenitor cells, including: digesting, filtering, and washing the ovary, and then purifying it through a magnetic bead or flow cytometry sorting program to screen out ovarian progenitor cells; the markers used in the purification process are LY6A, LGR5, or PDGFRA.

[0024] The cells that are positive after purification according to the present invention can be differentiated into steroid hormone-secreting cells in vitro; the steroids include androgens and progesterones.

[0025] The ovarian progenitor cells according to the present invention include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

[0026] The present invention also provides the application of ovarian progenitor cells in generating steroid-secreting cells in vitro. Preferably, the ovarian progenitor cells are the ovarian progenitor cells screened by the above method.

[0027] The ovarian progenitor cells according to the present invention include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

[0028] The method for generating steroid-secreting cells in vitro according to the present invention preferably includes: after the ovarian progenitor cells screened by the above method are proliferated in vitro, they are induced to differentiate in vitro in a DMEM / F12 medium containing insulin-transferrin-selenium additive, Smoothened agonist, and luteinizing hormone for 10-15 days; the concentration of the insulin-transferrin-selenium additive is 10 μg / ml - 5.5 μg / ml - 5 ng / ml. The concentration of the Smoothened agonist is 0.5 μM, and the concentration of the luteinizing hormone is 2 ng / ml.

[0029] Functions of the components required for the differentiation medium of the present invention: The insulin-transferrin-selenium additive can reduce the concentration of fetal bovine serum required for cell culture differentiation, ensure sufficient nutrient supply for cells, and enable cells to reach the best growth state during the culture differentiation process; under in vivo physiological conditions, luteinizing hormone (LH) binds to the receptor of this protein on ovarian theca cells, stimulating the development of theca cells and the synthesis of testosterone and androstenedione. Therefore, this component is added to the differentiation medium to simulate the in vivo physiological situation; Desert hedgehog signaling molecule (DHH) is a necessary regulatory molecule during the development of Leydig cells in the testis. SAG is an agonist of the DHH signaling pathway. Although the mechanism of action of DHH in promoting steroid cell differentiation is unknown, experiments have found that it is indispensable in the development of testicular Leydig cells. It is speculated that it may be related to the expression of SF1 and the expression of LH receptor. For the first time, this technical solution uses a simple formula of these three components to very effectively differentiate ovarian progenitor cells into steroid-secreting cells.

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0031] In in vitro experiments, ovarian epithelial LGR5+ progenitor cells formed steroidogenic cells that did not express CYP17A1 but expressed CYP11A1, while PDGFRA+ stromal progenitor cells generated steroidogenic cells that expressed both CYP11A1 and CYP17A1, indicating that progenitor cells in both locations have lineage preferences in forming steroidogenic cells (progesterone-producing cells or androgen-producing cells).

[0032] The statistical analysis of the data in the embodiments is as follows: The student t-test is used to detect the significant differences between the two groups of experiments. One-way analysis of variance (ANOVA) is used for multiple group comparisons. If P < 0.05, the differences between groups are considered significant. The significant differences between individual groups are tested using the SNK test, and the SPSS (IBM, US) statistical software package is used. The significance at different confidence levels is defined as P < 0.05, 0.01, 0.001, or 0.0001.

[0033] Example 1

[0034] 1. Chemicals and reagents

[0035] DMEM / F12 medium, fetal bovine serum (FBS), insulin-transferrin-selenium (ITS), dexamethasone, and bovine serum albumin (BSA) were all from Sigma-Aldrich (St. Louis, MO). β-mercaptoethanol, N2 and B27 medium additives, and fibroblast growth factor 2 (FGF2) were purchased from Thermo Fisher (Waltham, MA, USA). Epidermal growth factor (EGF) was from PeproTech (Rocky Hill, NJ, USA). Chicken embryo extract was from USBiological (Salem, MA, USA). Leukemia inhibitory factor (LIF) was from Millipore (Burlington, MA, USA). Smoothened agonist (SAG) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Oncostatin-M and platelet-derived growth factor bb (PDGFBB) were from ProSpec (East Brunswick, NJ, USA). Human LH was from MyBioSource (San Diego, CA, USA). Anti-r-Phycoerythrin (PE) magnetic particles, IMag TM Cell isolation magnet holder and IMag TM Buffer (10x) was from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase-conjugated secondary antibody (MultiSciences Biotech, China).

[0036] Abbreviation definitions: BSA, bovine serum albumin; CD51, cluster of differentiation 51 (integrin αV); CYP11A1, cytochrome P450 family 11a1; CYP17A1, cytochrome P450 family 17a1; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; DHH, desert hedgehog; DIM, differentiation induction medium; EDTA, ethylenediaminetetraacetic acid; EdU, ethynyl-2-deoxyuridine; EM, proliferation medium; EPCR, endothelial protein C receptor; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HBSS, Hank’s balanced salt solution; HSD3B, hydroxysteroid dehydrogenase 3b; IS, internal standard; ITS, insulin / transferrin / selenium tissue culture supplement; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; LH, luteinizing hormone; LHCGR, luteinizing hormone / choriogonadotropin receptor; LY6A, lymphocyte antigen 6 family member a; PBS, phosphate-buffered saline; PDGFRA, platelet-derived growth factor receptor a; PE, phycoerythrin; Rps16, ribosomal protein S16; SAG, smoothened agonist (DHH agonist); SLC, testicular Leydig cell stem cell.

[0037] 2. Experimental animals

[0038] Female 9-week-old C57BL / 6 mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). The mice were housed in the Experimental Animal Center of Wenzhou Medical University under controlled lighting (14 h light: 10 h dark) and temperature (22 °C), and had free access to water and food. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council and were approved by the Animal Care and Use Committee of Wenzhou Medical University.

[0039] 3. Techniques for studying the distribution of progenitor cells in the ovaries of adult mice

[0040] Before exploring the differentiation potential of different ovarian progenitor cells, their specific locations in the ovaries of adult mice were first determined. Ovarian sections were stained with antibodies against four ovarian progenitor cell markers, EPCR, LGR5, LY6A, PDGFRA, and an antibody against testicular Leydig cell stem cell CD51.

[0041] Among them, the method of immunohistochemical staining of ovarian sections is as follows: The tissue is fixed with 4% paraformaldehyde for 24 h, dehydrated with sucrose (20% for 12 h, 30% for 12 h), and embedded in OCT (optimal cutting temperature compound) at -20 °C. Frozen sections (8 μm thick) are incubated overnight at 4 °C with a primary antibody mixture (1:100 - 1:500, see Table 1), and then incubated for 45 min at room temperature in the dark with a fluorescently labeled secondary antibody mixture (1:100, see Table 1). Before microscopic examination, the cell nuclei are visualized with a drop of mounting medium containing DAPI.

[0042] For immunohistochemical staining of ovarian tissue, the labeled dextran polymer (LDP) method is used: Ovarian sections are treated with 3% hydrogen peroxide to quench endogenous peroxidase. After antigen exposure by microwave heating with citrate buffer (pH 6), they are incubated overnight at 4 °C with a PDGFRA primary antibody (1:100, see Table 2), and then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (1:3000). Then, the bound antibody is visualized with diaminobenzidine substrate, and the cell nuclei are counterstained with hematoxylin.

[0043] Table 1 Antibodies used for immunofluorescence and immunohistochemistry

[0044]

[0045] The technical effects are as Figure 1 shown: LY6A, EPCR, and LGR5 are all expressed by cells associated with the OSE (red arrows), and very few (EPCR) or no (LGR5) positive cells are detected in the stromal compartment. However, for the LY6A marker protein, a large number of positive cells are also found in the stromal compartment (yellow arrows), including some in the theca layer (white arrows). PDGFRA+ cells are only detected in the stromal compartment (yellow arrows), and no cells are found in the OSE (red arrowheads) or theca layer (white arrowheads). On the other hand, the CD51 label covers almost all cells in the entire parenchymal region, including some cells distributed inside the follicles (green arrows).

[0046] It can be seen that EPCR+ and LGR5+ cells are mainly distributed along the ovarian surface epithelium (OSE), and LY6A+ cells are distributed in both the OSE and ovarian parenchyma. However, PDGFRA+ cells are only located in the stromal region.

[0047] 4. In vitro isolation and expansion of progenitor cells

[0048] (1) Twenty adult female mouse ovaries were taken, washed with PBS, chopped with sterile scissors for 20 s, digested in DMEM / F12 medium containing type IV collagenase (1 mg / ml) at 37 °C for 30 min, and gently shaken (90 times / min). The digested cells were filtered through a 70-μm nylon mesh, washed, and then purified by magnetic bead / fluorescence-activated cell sorting (MACS / FACS) procedure.

[0049] Progenitor cell marker proteins used for cell isolation include LY6A, EPCR, LGR5, PDGFRA, and CD51.

[0050] Isolation of progenitor cells using the MACS procedure: Cell pellets were suspended in cold BD IMag 7 (BI) buffer at a density of 1×10 TM cells / ml, stained with a PE-conjugated primary antibody (1:100, see Table 2) in the dark at 4 °C for 40 min. After washing and labeling with anti-r-PE magnetic particles (1:100) at 4 °C for 30 min, the cells were transferred to a collection tube and immediately placed on a BD IMag TM Cell Separation Magnet Stand for 8 min. The negative cell fraction (supernatant) was collected. The positive cell fraction (adsorbed on the tube wall) was resuspended in BI buffer. Depending on the antibody used (see Tables 1 and 2), the sorting process was repeated 2 - 6 times. The final positive and negative cell fractions were combined, and the percentage of positive cells was determined by flow cytometry or used for in vitro culture and differentiation.

[0051] Table 2 Antibodies used in flow cytometry

[0052]

[0053] (2) Some positive and negative fractions were collected, and the expression of steroid genes / proteins (Cyp11a1 and Cyp17a1) was detected by QPCR and / or immunofluorescence staining.

[0054] RNA extraction and real-time qPCR analysis: Total RNA was extracted immediately after cell sorting or culturing using the RNeasy Mini Kit (QIAGEN, USA) according to the manufacturer's instructions. The extracted RNA was quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher, Roskilde, Denmark) and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, USA). The Green PCR Master Mix Kit was used for qPCR amplification. The amplification conditions were 95°C for 5 min, followed by 40 cycles of 95°C (10 s) and 60°C (30 s). The total reaction volume was 15 μl, which contained 7.5 μl SYBR Green Mix, 1.5 μl forward and reverse primer mixtures, and 0.02 μg of diluted cDNA. The ubiquitously expressed gene ribosomal protein S16 (Rps16) was used as an internal reference. The expression levels were calculated using the Delta-Ct method and normalized to Rps16. The primer sequences are shown in Table 3.

[0055] Table 3 PCR primer sequences

[0056]

[0057] The technical effects are as Figure 2 shown. Taking LY6A as an example, the original cell suspension before antibody labeling was distributed in clusters below the diagonal ( Figure 2 A; LY6A untagged and impurified). For cells labeled with the PE-LY6A antibody, approximately 7.1% were labeled and moved above the diagonal ( Figure 2 A and 2B, LY6A tagged and impurified). After the FACS procedure or 4 rounds of MACS purification, the cell purity reached over 99% ( Figure 2 A, 2B; LY6Apurified). Similarly, CD51+, PDGFRA+, EPCR+, and LGR5+ cells were all significantly enriched to approximately 70% - 96% ( Figure 2 A, 2B).

[0058] The Cyp11a1 and Cyp17a1 mRNA levels were significantly enriched in LY6A- cells and undetectable in the LY6A+ fraction, indicating that LY6A labeling successfully excluded all steroidogenic cells ( Figure 2 C). Similar enrichments were also achieved for LGR5+ and PDGFRA+ cells, and the expression of these two marker genes was also enriched by CD51, but the technical effects were exactly the opposite of those of LY6A ( Figure 2 C).

[0059] The CD51-positive fraction, rather than the negative fraction, enriched two steroidogenic genes (Cyp11a1 and Cyp17a1). EPCR also failed to exclude or enrich any of the two steroidogenic genes tested, indicating that CD51 and EPCR are not suitable as markers for isolating steroid cell progenitors.

[0060] (3) Since LY6A and CD51 showed opposite patterns, the expression differences of steroidogenic enzymes HSD3B and CYP11A1 were further compared between the cells sorted by these two markers. The specific results are as Figure 3 shown.

[0061] Immunofluorescence and enzyme staining of the marker proteins: The isolated cells / cultured cells were washed three times with PBS, then incubated overnight at 4°C with a mixture of primary antibodies (1:100 - 1:500, see Table 1), and then incubated in the dark for 45 min at room temperature with a mixture of fluorescently labeled secondary antibodies (1:100, see Table 1). Before microscopic examination, the cell nuclei were visualized with a drop of mounting medium containing DAPI. For HSD3B enzyme staining, freshly isolated cells or cultured cells were dried on a slide for approximately 10 min. Then the cells were stained for 40 min with a solution containing 5β-androstane-3β-ol-17-one steroid substrate (0.4 mM), NAD (1 mg / ml), and nitroblue tetrazolium (0.2 mg / ml). After staining, the cells were washed with HBSS and fixed with 10% formalin / HBSS for 5 min.

[0062] The technical effects are shown in Figure 3 : Consistent with the qPCR results, LY6A - cells co-expressed HSD3B and CYP11A1, while LY6A+ cells did not. Compared with LY6A cells, CD51 cells showed the opposite pattern in the expression of the two enzymes.

[0063] 5. Progenitor cells differentiate into steroid cells in vitro

[0064] To further characterize the differentiation potential of LY6A+ cells, LY6A+ and LY6A - cells were cultured in vitro, and their abilities to proliferate and differentiate into steroidogenic cells were evaluated.

[0065] (1) The cells were amplified in DMEM / F12 medium containing FBS (5%), ITS (1X), penicillin-streptomycin (100 IU / ml - 100 μg / ml), dexamethasone (0.5 nM), LIF (0.5 ng / ml), chicken embryo extract (2.5%), β-mercaptoethanol (50 μM), non-essential amino acids (0.5%), FGF2 (10 ng / ml), EGF (10 ng / ml), PDGFBB (10 ng / ml), Oncostatin-M (10 ng / ml), N2 (0.5%), and B27 (1%). The medium was changed every two days until the cells reached approximately 60% confluence. To detect the proliferation activity of the cells, after 3 days of culture, the cells were then labeled with EdU (ethyl-2'-deoxyuridine, 2 μM) for 24 h to detect the cell division activity.

[0066] The specific results are asFigure 4 As shown in A, 87% of the LY6A+ cells could be labeled by EdU, while only 14% of the LY6A- cells were EdU-positive.

[0067] (2) To determine the potential of LY6A+ cells to form theca steroid cells, they were cultured in vitro for 12 days using a differentiation induction medium containing LH (2 ng / ml), SAG (DHH agonist, 0.25 μM) and 1X ITS. The specific steps are as follows:

[0068] After the cells reached a confluence of about 60%, they were transferred into an induction differentiation medium composed of DMEM / F12, 1-fold ITS, 0.5 μM SAG and 2 ng / ml LH for differentiation for 12 days. The medium was replaced every other day, and the used medium collected was used to measure androgens (androstenedione and testosterone) by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC: Acquity XEVO TQD triple quadrupole mass spectrometer, Waters, USA). RNA was isolated from the cells, and QPCR was used to detect the expression of Cyp11a1 and Cyp17a1. Steroid synthesis pathway proteins (CYP11A1, CYP17A1 and HSD3B1) were detected by immunofluorescence or enzymatic methods. The specific results are as Figure 4 B- Figure 4 C and Figure 5 shown.

[0069] Method for determining androgens by ultra-high performance liquid chromatography-tandem mass spectrometry: The medium collected from the cells separately isolated from 4 markers for in vitro culture was used to measure the concentrations of androstenedione and testosterone by liquid chromatography-mass spectrometry. The internal control used a cell culture medium without androgens. An internal standard (IS) working solution was prepared using the androgen-d3 stock solution. The collected cell culture medium (50 μl) was mixed with 100 μl of acetonitrile and 5 μl of the IS working solution. After vibrating for 3 min, the mixture was centrifuged at 12,000 g for 15 min. The supernatant (10 μl) was injected into the system through a self-sampling protocol. For experimental accuracy, all samples were repeatedly detected, and the coefficient of variation of the internal detection was less than 10%.

[0070] The differentiation effect of LY6A cells is shown in Figure 4 B and Figure 4 C: In the first 4 days of differentiation, no testosterone or androstenedione was detected in the medium ( Figure 4 B). However, these two androgens could be detected on the 6th day, and the concentration increased continuously before the 10th day. To confirm the characteristics of theca cells, we detected the expression of key steroid synthesis genes (Cyp11a1 and Cyp17a1) in the cells before and after in vitro differentiation ( Figure 4C). Before differentiation, neither of these two genes was expressed, but significant expression levels were detected after differentiation.

[0071] The expression effect of steroid synthesis proteins is shown in Figure 5 : Before differentiation (d0), the cells were negative for CYP11A1 and HSD3B protein staining, and after 10 days of differentiation, the cells were positive for these two enzymes (d10 or d12).

[0072] 6. Differences in the offspring generated by progenitor cells in vitro

[0073] Since LY6A+ cells are distributed in the OSE and parenchymal tissues, the LY6A+ cells that generate steroid cells may come from one or two locations. To further confirm the differences between the progenitor cells at these two locations, cells isolated from LGR5 (derived only from the OSE) and PDGFRA (derived only from the parenchymal tissue) were compared to understand their ability to generate steroid cells. The isolation and culture of LY6A, PDGFRA, and LGR5 cells are detailed in Example 2 and Example 3. Both the isolated LGR5+ cells and PDGFRA+ cells did not contain CYP11A1 and CYP17A1 ( Figure 6 , d0). After 10 days of in vitro differentiation, most LGR5+ cells acquired the ability to express CYP11A1 ( Figure 6 , d10), while no cells expressed CYP17A1. However, after 10 days of in vitro differentiation, the cells generated from PDGFRA+ cells were able to express both CYP11A1 and CYP17A1 ( Figure 6 , d10), indicating that PDGFRA+ cells have the characteristics of theca-like steroid synthesis cells.

[0074] In the present invention, immunofluorescence staining and fluorescence / magnetic-activated cell sorting (FACS / MACS) techniques were used to compare the distribution of ovarian tissue repair progenitor cells (expressing Ly6A, LGR5, or EPCR) and ovarian stromal stem cells (PDGFRA and CD51), as well as their ability to generate steroidogenic cells in vitro. The results showed that although progenitor cells from both the OSE and stroma (LY6A+) can generate steroid cells, there are important differences between the cells from the two sources. The steroid cells generated from progenitor cells from the stroma (PDGFRA+) expressed CYP11A1 and CYP17A1, indicating that they are theca-like cells.

[0075] While the steroid cells generated from progenitor cells from the OSE (LGR5+) expressed CYP11A1 but could not express CYP17A1, indicating that they are granulosa-like or lutein-like cells. These results suggest that there may be lineage preferences when ovarian surface and stromal progenitor cells differentiate into steroid cells.

[0076] Example 2

[0077] Method for in vitro identification of steroidogenic theca cells using PDGFRA as a marker

[0078] (1) Take the ovaries of 20 adult female mice, wash them with PBS, chop them with sterile scissors for 20 s, and digest them in DMEM / F12 medium containing type IV collagenase (1 mg / ml) at 37 °C for 30 min with slow shaking (90 times / min).

[0079] (2) Filter the digested cells through a 70 μm nylon mesh, wash them, and then purify them once by flow cytometry sorting (FACS) procedure.

[0080] Suspend the digested ovarian cell pellets at a density of 1×10 7 cells / ml in pre-cooled 1×Binding Buffer, and stain them with PE-conjugated anti-PDGFRA primary antibody (1:100) in the dark at 4 °C for 40 min.

[0081] After labeling with the conjugated antibody, centrifuge at 1300 r / min for 5 min, discard the supernatant, and wash the cells 3 times with pre-cooled 1×Binding Buffer. Subsequently, resuspend the cell pellets at a density of 1×10 5 cells with FACS buffer, transfer them to a flow tube, and then perform cell sorting with a flow cytometer. The positive cells are ovarian stromal PDGFRA+ progenitor cells.

[0082] After in vitro proliferation of the cells, induce differentiation in vitro for 12 days using DMEM / F12 medium containing ITS (the concentration of insulin-transferrin-selenium additive is 10 μg / ml - 5.5 μg / ml - 5 ng / ml), 0.5 μM SAG, and 2 ng / ml LH. Then detect the expression of steroid hormones and their synthetic enzymes.

[0083] From the results of Example 1, it can be seen that ovarian stromal PDGFRA+ progenitor cells can develop into androgen cells expressing CYP11A1 and CYP17A1.

[0084] Example 3

[0085] Method for in vitro identification of steroidogenic theca cells using LGR5 as a marker

[0086] (1) Take the ovaries of 20 adult female mice, wash them with PBS, chop them with sterile scissors for 20 s, and digest them in DMEM / F12 medium containing type IV collagenase (1 mg / ml) at 37 °C for 30 min with slow shaking (90 times / min).

[0087] (2) Filter the digested cells through a 70-μm nylon mesh, wash them, and then purify them 4 times by magnetic-activated cell sorting (MACS) procedure.

[0088] Select the labeled cells using the BD IMag TM protocol according to the manufacturer's instructions. Suspend the cell pellet at a density of 1 × 10 7 cells / ml in cold BD IMag TM (BI) buffer and stain with PE-conjugated anti-LGR5 primary antibody (1:100) for 40 min at 4°C in the dark.

[0089] After washing and labeling with anti-r-PE magnetic particles (1:100) for 30 min at 4°C, transfer the cells to a collection tube and immediately place them on the BD IMag TM Cell Separation Magnet Stand (BD Biosciences, USA) for 8 min. Collect the negative cell fraction (supernatant) and resuspend the positive cell fraction (adhered to the tube wall) in BI buffer. The positive cells are ovarian surface epithelial LGR5+ progenitor cells.

[0090] After proliferating the cells in vitro, induce differentiation in vitro for 12 days using DMEM / F12 medium containing ITS (the concentrations of insulin-transferrin-selenium additive are 10 μg / ml - 5.5 μg / ml - 5 ng / ml), 0.5 g / mSAG, and 2 ng / ml luteinizing hormone (LH). Then detect the expression of steroid hormones and their synthetic enzymes.

[0091] As can be seen from the results of Example 1, ovarian surface epithelial LGR5+ progenitor cells can express CYP11A1 but do not express CYP17A1, which is in line with the characteristics of progesterone-producing cells.

[0092] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying ovarian progenitor cells, characterized in that, Comprising: After digesting, filtering, and washing the ovary, purify it through magnetic beads or flow cytometry sorting procedures to screen out ovarian progenitor cells; The markers used in the purification process are LY6A, LGR5, or PDGFRA.

2. The method according to claim 1, wherein The cells that are positive after purification can differentiate into steroid hormone-secreting cells in vitro.

3. The method according to claim 2, wherein The steroids include androgen and progesterone.

4. The method according to claim 1, wherein The ovarian progenitor cells include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

5. Use of ovarian progenitor cells for generating steroid-secreting cells in vitro, characterized in that, The ovarian progenitor cells are the ovarian progenitor cells screened by the method according to any one of claims 1 to 4.

6. The application according to claim 5, characterized in that, The ovarian progenitor cells include ovarian LY6A+ progenitor cells, ovarian stromal PDGFRA+ progenitor cells, and ovarian surface epithelial LGR5+ progenitor cells.

7. The application according to claim 5 or 6, characterized in that, The method for generating steroid-secreting cells in vitro includes: after proliferating the ovarian progenitor cells screened by the method according to any one of claims 1 to 4 in vitro, inducing differentiation in DMEM / F12 medium containing insulin-transferrin-selenium additive, Smoothened agonist, and luteinizing hormone for 10 to 15 days in vitro.

8. The application according to claim 7, wherein The concentration of the insulin-transferrin-selenium additive is 10 μg / ml - 5.5 μg / ml - 5 ng / ml. The concentration of the Smoothened agonist is 0.5 μM, and the concentration of the luteinizing hormone is 2 ng / ml.

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