Method for Obtaining Female Germline Stem Cells from Follicular Aspirates

Abstract

Provided is a method for obtaining female germline stem cells from follicular aspirates.

Claims

1. A method for separating female germline stem cells from follicular aspirates, comprising: (1) separating ovarian tissues or cell masses from the follicular aspirates and performing cell dispersion to obtain single cells; and (2) inoculating and culturing the cells obtained in step (1) on inactivated feeder layer cells, and sorting female germline stem cell marker-positive cells from the cultured cells.

2. The method of claim 1, further comprising: (3) proliferation-culturing and subculturing the female germline stem cell marker-positive cells obtained in step (2) on feeder layer cells.

3. The method of claim 1, wherein step (1) comprises: filtering the follicular aspirates by using a cell strainer with a pore size of 30-100 m, with solids retained on the strainer as the ovarian tissues or cell masses.

4. The method of claim 3, wherein the solids retained on the strainer as the ovarian tissues or cell masses are separated from the strainer, and the ovarian tissues or cell masses are separated from the follicular aspirates by the method of centrifugation.

5. The method of claim 1, wherein step (1), before performing the cell dispersion, further comprises: further treating the obtained ovarian tissues or cell masses to remove red blood cells.

6. The method of claim 1, wherein in step (1), the cell dispersion is performed by using one or more enzymes selected from the group consisting of collagenase IV, trypsin, trypsin substitute, or Acutase.

7. The method of claim 1, wherein in step (2), the feeder layer cells include STO cells, MEF cells or SNL cells.

8. The method of claim 1, wherein the inactivated feeder layer cells in step (2) are mitomycin C-inactivated feeder layer cells; or the culture period on the feeder layer cells is 10-18 days in step (2).

9. The method of claim 1, wherein in step (2), an antibody specific to a female germline stem cell marker is conjugated to magnetic beads and incubated with the cells cultured on the feeder layer cells in order to sort the female germline stem cell marker-positive cells.

10. The method of claim 1, wherein the inactivated feeder layer cells are obtained as follows: the feeder layer cells are treated with mitomycin C, digested into single cells after washing, and inoculated on a gelatin-coated cell culture plate.

11. The method of claim 1, wherein the female germline stem cell marker includes DDX4 and/or IFITM3.

12. A method for obtaining ovarian tissues or cell masses, comprising separating the ovarian tissues or cell masses from follicular aspirates.

13. The method of claim 12, further comprising filtering the follicular aspirates by using a cell strainer with a pore size of 30-100 m, with solids retained on the strainer as the ovarian tissues or cell masses.

14. The method of claim 2, further comprising proliferation-culturing and subculturing the female germline stem cell marker-positive cells obtained in step (2) on feeder layer cells in a stem cell culture medium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1. A schema diagram of obtaining oocytes via transvaginal puncture at an IVF center, showing that preantral follicles and FGSCs can be obtained with mature oocytes.

[0030] COC, cumulus-oocyte complexes; and

[0031] OSE, ovarian surface epithelium.

[0032] FIG. 2. Appearance and components of follicular aspirates.

[0033] A. The appearance of the follicular aspirates obtained from an IVF center; and

[0034] B. Tissues or cell masses adhered to the nylon strainer after filtration of the follicular aspirates over a 30-m cell strainer. The right panel is an enlarged view of the red box area.

[0035] FIG. 3. The ovarian tissues and granular cell masses contained in the follicular aspirates.

[0036] A. Ovarian tissues (left and middle panels) and granular cell masses (right panel), which have different shapes and sizes, can be obtained from the follicular aspirates.

[0037] B. The proportion of the ovarian tissues obtained at two IVF centers.

[0038] FIG. 4. Female germline stem cells (FGSCs) separated from the follicular aspirates.

[0039] A. DDX4.sup.+ cells sorted after a small amount of ovarian tissues in the follicular aspirates are digested and cultured in vitro for 2 weeks. White arrows indicate magnetic beads.

[0040] B. Immunocytochemical staining is used to confirm that the sorted cells are DDX4.sup.+ cells.

[0041] FIG. 5. The morphology of FGSCs cultured in vitro.

[0042] A. FGSCs in the division stage.

[0043] B. The cell morphology of the fourth generation (p4) FGSCs.

[0044] C. The cell morphology of the 30th generation (p30) FGSCs.

[0045] FIG. 6. RT-PCR analysis of human ovarian tissues (28 years old), FGSC cell lines (1# and 2#) and FGSCs obtained from the surgically removed ovaries (srFGSCs).

[0046] FIG. 7. Immunofluorescence staining of DDX4, OCT4, IFITM3, BLIMP-1 and DAZL proteins in the FGSC cell lines.

[0047] FIG. 8. Dual immunofluorescence staining of BrdU and DDX4 in the FGSC cell lines.

[0048] FIG. 9. G-banding karyotype analysis of FGSC cell lines, showing a normal karyotype (46, XX) and its proportion (right panel). n=59.

DETAILED DESCRIPTION OF EMBODIMENTS

[0049] Female germline stem cells (FGSCs) are a kind of germline stem cells present in the ovaries and have a variety of clinical application values. The present inventors have discovered for the first time in the study that follicular aspirates comprise ovarian tissues or cell masses from which female germline stem cells can be obtained.

[0050] As used herein, the follicular aspirates are follicular aspirates clinically produced by follicular puncture and aspiration. In general, oocyte-containing aspirates can be obtained after clinical follicular puncture and aspiration (e.g., oocyte retrieval via transvaginal ovarian puncture). The follicular aspirates of the present invention are usually follicular aspirates remained after picking out mature oocytes. The follicular aspirates can also be wastes generated when transvaginal ultrasound-guided follicular puncture and aspiration is used to treat benign ovarian cysts (Katayama, K. P., M. Roesler, C. Gunnarson, et al., Ultrasound-guided transvaginal needle aspiration of follicles for in vitro fertilization. Obstetrics and Gynecology, 1988. 72(2): p. 271-274; and Nikolaou, M., G Adonakis, P. Zyli, et al., Transvaginal ultrasound-guided aspiration of benign ovarian cysts. Journal of Obstetrics and Gynaecology, 2014. 34(4): p. 332-335). The follicular aspirates can also be wastes generated when ultrasound-guided transvaginal ovarian needle drilling (UTND) is used to treat polycystic ovary syndrome (PCOS) (Badawy, A., M. Khiary, A. Ragab, et al., Ultrasound-guided transvaginal ovarian needle drilling (UTND) for treatment of polycystic ovary syndrome: a randomized controlled trial. Fertility and Sterility, 2009. 91(4): p. 1164-1167).

[0051] As used herein, the female germline stem cell marker refers to a specific molecule expressed on the surface of female germline stem cells (including transmembrane structures), and female germline stem cells can be identified or sorted with an antibody that specifically binds to the marker. In the present invention, the female germline stem cell marker includes but is not limited to DDX4 and IFITM3. Most preferably, DDX4 is selected as the marker for female germline stem cell sorting.

[0052] As used herein, the female germline stem cells (FGSCs) are mammalian female germline stem cells; preferably human female germline stem cells or non-human primate female germline stem cells.

[0053] The present invention provides a method for separating female germline stem cells from follicular aspirates, comprising: (1) separating ovarian tissues or cell masses from the follicular aspirates and performing cell dispersion to obtain single cells; and (2) inoculating and culturing the cells obtained in step (1) on inactivated feeder layer cells, and sorting female germline stem cell marker-positive cells from the cultured cells.

[0054] The follicular aspirates may be clinically discarded follicular aspirates, such as discarded follicular aspirates obtained after transvaginal puncture and oocyte retrieval at some IVF centers (FIG. 1). That is, the follicular aspirates may be by-products produced from an IVF center. It has been confirmed in the present invention that follicular aspirates contain a small amount of ovarian tissues, and the tissues can be used to obtain female germline stem cells.

[0055] The follicular aspirates may be derived from one donor, or may be derived from multiple donors, for example, from 1 to 100 donors, including 2, 5, 10, 20, 40, 60 donors, and so on.

[0056] The follicular aspirates can be filtered using a cell strainer with a suitable pore size to obtain valuable ovarian tissues or cell masses from the follicular aspirates. As a preferred embodiment of the present invention, the follicular aspirates are filtered by using a cell strainer with a pore size of 30-100 m, and solids retained on the strainer serve as the ovarian tissues or cell masses. For example, the pore size of the cell strainer may be 30 m, 40 m, 50 m, 60 m, 70 m, 80 m, 90 m, or the like.

[0057] In order to obtain relatively pure cells and avoid the interference of impurity cells, the step of removing red blood cells in the follicular aspirates is also included during or after the filtration with a strainer. The red blood cells in the follicular aspirates can be removed by rinsing with some commonly used rinse solutions (such as buffers), or the red blood cells can be lysed and removed by using a red blood cell lysis solution. If rinsing is selected to remove the red blood cells, rinsing can be performed during the filtration with a strainer.

[0058] The ovarian tissues or cell masses separated from the strainer, preferably also with red blood cells removed, are further centrifuged, the supernatant is discarded, and the ovarian tissues or cell masses at the bottom of the centrifuge tube are collected and ready for subsequent cell dispersion.

[0059] The obtained ovarian tissues or cell masses are subjected to cell dispersion to obtain single cells that can be cultured. The cell dispersion can be performed using various physical or chemical methods of cell dispersion well known in the art. As a preferred embodiment of the present invention, the cell dispersion is performed by using one or more enzymes selected from the group consisting of collagenase, trypsin, trypsin substitute (TrypleE Express) or Acutase. As the most preferred embodiment of the present invention, the ovarian tissues are digested into single cells by two-step enzymatic digestion successively comprising: step one, collagenase IV digestion; and step two, trypsin-EDTA digestion. The full name of collagenase is collagen hydrolytic enzyme (collagenase) and it can specifically hydrolyze the three-dimensional helical structure of natural collagen without damaging other proteins and tissues. Collagenase IV comprises at least 7 protease components with molecular weights ranging from 68 to 130 kD. Trypsin is a protease that degrades a protein at a specific site, thus degrading the protein at the juncture between cells. At this time, the cells become spherical due to the tension of their internal cytoskeleton, thereby separating the cells. EDTA is a non-enzymatic digestant, and its mechanism of action is generally believed to be as follows: the adhering and growth of some cells must require the presence of Ca.sup.2+, while EDTA can form a chelate with Ca.sup.2+, which can promote the separation of cells.

[0060] The obtained single cells are inoculated and cultured on inactivated feeder layer cells. As a preferred embodiment of the present invention, the inactivated feeder layer cells are mitomycin C-inactivated feeder layer cells. As a preferred embodiment of the present invention, the feeder layer cells may include STO cells, MEF cells, SNL cells, or the like.

[0061] The culture period on the feeder layer cells is generally 10-18 days, for example, it may be 12, 14, or 16 days. Afterwards, the female germline stem cell marker-positive cells are sorted from the cultured cells.

[0062] The female germline stem cell marker is a specific molecule expressed on the surface of female germline stem cells (including transmembrane structures), and female germline stem cells can be identified or sorted with an antibody that specifically binds to the marker. The specific antibody may be a monoclonal antibody or a polyclonal antibody. The female germline stem cell marker can be used to immunize an animal such as rabbit, mouse, rat, etc. to produce a polyclonal antibody; and various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. Similarly, cells that express a female germline stem cell marker or an antigenic fragment thereof can be used to immunize an animal to produce an antibody. The monoclonal antibody can be prepared using hybridoma technology.

[0063] In some embodiments of the present invention, the antibody used for sorting the female germline stem cells may be a C-terminal DDX4 (e.g., available from Abeam, ab13840) antibody, or IFITM3 (e.g., available from Abeam; ab15592) antibody may also be used.

[0064] DDX4 or IFITM3 is a female germline stem cell marker, and is also an oocyte marker. Therefore, in fact, the separated cells also contain oocytes when using DDX4 or IFITM3 for cell sorting. However, the present inventors have observed that oocytes cannot survive and proliferate for a long time during the subsequent culture, and gradually disappear. The disappearance of oocytes is also demonstrated in Example 2 of the present invention. The cultured FGSC cell lines do not express the oocyte-related genes C-KIT, FIGLA, GDF9, GJA4, ZP1, ZP2, and ZP3. Therefore, those skilled in the art can understand that DDX4 or IFITM3 can be used as a sorting marker applied in the method of the present invention, and relatively pure FGSC cell lines can also be obtained by the method of the present invention.

[0065] An antibody specific to a female germline stem cell marker is conjugated to magnetic beads and incubated with the cells cultured on the feeder layer cells in order to sort the female germline stem cell marker-positive cells. Magnetic beads used for sorting the female germline stem cells may be magnetic beads with a diameter of 2.8 m (e.g., available from Dynabeads, 112.03 D), or magnetic beads with a diameter of 50 nm (e.g., available from Miltenyi Biotec, 130-048-602) may also be used.

[0066] Culture medium for in vitro proliferation of the female germline stem cells may be a culture medium known in the art. As a preferred embodiment of the present invention, the culture medium may be MEM containing 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM -mercaptoethanol, 10 ng/mL leukemia inhibitory factor, 20 g/ml transferrin, 5 g/ml insulin, 60 M putrescine, 10 ng/mL epidermal growth factor, 40 ng/mL glial cell-derived neurotrophic factor, 1 ng/mL basic fibroblast growth factor and 15 mg/L penicillin. The formulation of the various components mentioned above can properly fluctuate up and down (for example, 30% up and down; preferably 20% up and down; more preferably 10% up and down; and more preferably 5% up and down), which is easily operated by a person skilled in the art.

[0067] As a preferred embodiment of the present invention, the female germline stem cells can also be cultured in MEM containing 10% fetal bovine serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, 1 concentration of penicillin-streptomycin-glutamine solution, 0.1 mM -mercaptoethanol, 1 concentration of N-2 supplement, 10.sup.3 units/ml leukemia inhibitory factor, 10 ng/ml epidermal growth factor, 1 ng/ml basic fibroblast growth factor and 40 ng/ml glial cell-derived neurotrophic factor. The formulation of the various components mentioned above can properly fluctuate up and down (for example, 30% up and down; preferably 20% up and down; more preferably 10% up and down; and more preferably 5% up and down), which is easily operated by a person skilled in the art.

[0068] The present inventors have discovered for the first time in the art that follicular aspirates comprise ovarian tissues or cell masses. Based on this new finding of the present inventors, the present invention also provides a method for obtaining ovarian tissues or cell masses, which comprises: separating the ovarian tissues or cell masses from follicular aspirates. As a preferred embodiment of the present invention, the method comprises: filtering the follicular aspirates by using a cell strainer with a pore size of 30-100 m, with solids retained on the strainer as the ovarian tissues or cell masses.

[0069] The excellent effects of the present invention lie in:

[0070] (1) discovering for the first time that female germline stem cells can be separated from follicular aspirates, and providing a more preferred method for separating and culturing female germline stem cells; and

[0071] (2) obtaining female germline stem cells from follicular aspirates not only reduces the surgical risks, but also makes full use of clinical resources, as well as provides a new approach for obtaining female germline stem cells.

[0072] The present invention is further illustrated below in combination with particular examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not specify the specific conditions in the following examples are generally performed in accordance with conventional conditions, such as those described in J. Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or in accordance with the conditions recommended by the manufacturer.

EXAMPLE 1

Obtaining a Small Amount of Human Ovarian Tissues from the Follicular Aspirates Produced at an IVF Center

[0073] 1. Obtaining Human Follicular Aspirates

[0074] After the informed consent was signed, follicular aspirates were obtained from two IVF centers. Patients were between 30 and 35 years old. After oocytes were picked out, follicular aspirates produced from 2-6 patients were collected in a sterile 50 mL centrifuge tube and delivered to the laboratory at room temperature within two hours. The study was approved by the Ethics Committees of the First Maternal and Child Health Hospital affiliated with Tongji University and the First People's Hospital of Chenzhou City, Hunan Province, China.

[0075] 2. Collecting a Small Amount of Ovarian Tissues from the Follicular Aspirates

[0076] The follicular aspirates were filtered by using a cell strainer with 30 m pore size and rinsed several times with sterile PBS buffer until there were no visible blood cells. The cell strainer was poured on a cell culture dish or centrifuge tube upside down, and the tissue masses adhered to the nylon strainer (the cell strainer with 30 m pore size) were washed with sterile PBS buffer. The strainer was then placed under a dissecting microscope to see if it was clean and if there was any residue, the tissue masses can be collected with a tweezer. After centrifugation at 300 g for 5 minutes, the supernatant was discarded, and the tissue masses collected at the bottom of the centrifuge tube were used for subsequent experiments.

[0077] 3. Results

[0078] Clinically, the shortage of ovarian specimens of patients restricts the basic research and clinical applications of FGSCs. Previous studies have found that follicular aspirates contain preantral follicles and mature oocytes can be obtained after in vitro culture.

[0079] The present inventors obtained discarded follicular aspirates from two IVF centers. Since follicular aspirates contain a variety of somatic cells, in order to remove these cell contaminations, the present inventors used a strainer with 30 m pore size to remove these cells. During the operation, it was found that there were many tissues or cell masses on the strainer after filtration (FIG. 2).

[0080] These tissues or cell masses were observed under a microscope and they were found to contain small pieces of ovarian tissues and granular cell masses (FIG. 3A). Statistics found that ovarian tissues were detected in the samples derived from both IVF centers, and 91.7% and 100% of the total samples had ovarian tissues detected, respectively (FIG. 3B).

[0081] In summary, these results demonstrate that follicular aspirates contain a small amount of ovarian tissue masses.

Example 2

Separation and Identification of FGSCs from a Small Amount of Ovarian Tissues in the Follicular Aspirates

[0082] 1. Preparing STO Feeder Layer

[0083] STO cells were thawed, and then inoculated and cultured into a culture dish with DMEM (Life Technologies) culture medium containing the following components: 1 mM non-essential amino acids (NEAA; Life Technologies), 10% fetal bovine serum (Hyclone), and 6 mg/L penicillin (Sigma). When preparing the feeder layer, STO cells were treated with 10 g/mL mitomycin C (Sigma) for 2-3 hours, washed several times with PBS, and then digested with trypsin into single cells which were inoculated on a 0.1% (w/v) gelatin-coated cell culture plate and incubated with STO culture medium.

[0084] 2. Separating FGSCs

[0085] The ovarian tissue masses collected in the previous example were placed in PBS containing 1 mg/mL collagenase IV and then slowly shaken for 5 minutes in a water bath at 37 C. The solution was centrifuged at 300 g for 5 minutes, and then washed once with PBS buffer, and centrifuged again. PBS containing 0.5 mM EDTA and 0.05% trypsin was added to the centrifuge tube, and the centrifuge tube was slowly shaken for 2-3 minutes in a water bath at 37 C. After the cells were substantially dispersed, 10% fetal bovine serum was added to stop the action of trypsin. The solution was centrifuged at 300 g for 5 minutes and then the supernatant was carefully removed. The cells were resuspended by adding the culture medium and inoculated on the mitomycin C-inactivated feeder layer of STO single cells. The cells were cultured in a stem cell culture medium for two weeks and subjected to magnetic bead sorting. During magnetic bead sorting, goat anti-rabbit IgG-conjugated magnetic beads (Dynabeads, 112.03 D) were incubated with the rabbit polyclonal DDX4 antibody (Abcam; ab13840) according to the instructions, and then the coated magnetic beads were incubated with the cells. The positive cells were sorted by using a magnetic bead rack.

[0086] 3. Culturing FGSCs

[0087] The culture system of FGSCs requires the use of the inactivated STO feeder layer. The culture medium for FGSCs was MEM containing the following components: 10% fetal bovine serum (Front), 1 mM sodium pyruvate (Sigma), 1 mM non-essential amino acids, 2 mM L-glutamine (Sigma), 0.1 mM -mercaptoethanol (Sigma), 10 ng/mL human leukemia inhibitory factor (LIF; Santa Cruz Biotechnology, sc-4377), 10 ng/mL human epidermal growth factor (EGF; Peprotech, AF-100-15), 40 ng/mL human glial cell-derived neurotrophic factor (GDNF; Peprotech, 450-10), 10 ng/mL human basic fibroblast growth factor (bFGF; Peprotech, AF-100-18B) and 6 mg/L penicillin.

[0088] Magnetic beads-sorted cells (approximately 500 cells) were inoculated into a well of a 24-well plate containing the STO feeder layer, and the stem cell culture medium was added for culture.

[0089] The culture medium was exchanged every 2-3 days, and the cells were passaged at a ratio of 1:1 to 1:3 with dispase II (Roche) every 5-8 days. All cells were cultured in a constant incubator containing 5% CO.sub.2 at 37 C.

[0090] 4. Immunofluorescence Staining

[0091] The cells were fixed with 4% PFA at room temperature for 20 minutes, washed twice with PBS, and blocked with a blocking solution (PBS containing 10% goat serum) at 37 C. for 10 minutes. The cells were then incubated overnight at 4 C. in one of the following diluted primary antibodies [1:500 diluted DDX4 antibody (Abeam; ab13840); 1:100 diluted OCT4 antibody (Santa Cruz Biotechnology; sc-9081); 1:100 diluted IFITM3 antibody (Abcam; ab15592); 1:100 diluted BLIMP-1 antibody (ABGENT Crown; AP14521a); 1:100 diluted DAZL antibody (Abcam; ab128238); or 1:150 diluted BrdU (Thermo scientific; clone BRD.3)].

[0092] The next day, the cells were washed twice with PBS, and then incubated in a 1:150 diluted secondary antibody [TRITC-conjugated goat anti-rabbit IgG (ProteinTech, recognizing DDX4, OCT4, IFITM3, BLIMP-1 and DAZL) or goat anti-mouse IgG (ProteinTech, recognizing BrdU)] at 37 C. in dark for 30 minutes, and then counterstained with DAPI for 20 minutes. A fluorescent anti-quencher was added and photos were taken.

[0093] 5. RT-PCR

[0094] Total RNAs were extracted from the tissues and cells and reverse-transcribed using the HiScript II Q RT SuperMix (+ gDNA wiper) kit (Vazyme, R223-01) according to the instructions. RT-PCR was performed using Taq polymerase (Takara, R10T1M) with primers shown in Table 1 for 35 cycles. The PCR-amplified GAPDH gene was used as an internal reference. Electrophoresis was performed using 2% agarose gel and the DNA bands were stained with ethidium bromide (EB). The PCR products were obtained and sequenced to determine whether the bands were the corresponding genes.

TABLE-US-00001 TABLE1 PCRprimersusedforanalyzingthegeneexpressionsinhuman FGSCsandovariantissues Product SEQ Accession length ID Gene number (bp) Primersequence(5.fwdarw.3).sup.# NO: DDX4 NM_024415 139 F:CGTTGAAATTCTGCGAAACA 1 R:TCTCTGTTCCCGATCACCAT 2 IFITM3 NM_021034 214 F:ACCATGTCGTCTGGTCCCTGT 3 R:AGCACTGGGATGACGATG 4 AGCAGA OCT4 NM_002701 564 F:TGAGGGCGAAGCAGGAGT 5 R:TGGCGCCGGTTACAGAA 6 STELLA NM_199286 168 F:GTTACTGGGCGGAGTTCGTA 7 R:TGAAGTGGCTTGGTGTCTTG 8 DAZL NM_001190811 163 F:GCCCACAACCACGATGAATC 9 R:CGGAGGTACAACATAGCTC 10 CTTT BLIMP-1 NM_001198 300 F:GGGTGCAGCCTTTATGAGTC 11 R:CCTTGTTCATGCCCTGAGAT 12 STRA8 NM_182489 220 F:ACTCTCAGTCTGATCTCAT 13 AGCC R:TACCAAGGGGAGGAACCA 14 TTC SYCP3 NM_001177949 136 F:TCAGAGCCAGAGATTGAA 15 AACA R:TTGCAACATAGCCATTT 16 CTTTTT C-KIT NM_000222 291 F:GGCATGCTCCAATGTGTGG 17 R:GGTGTGGGGATGGATTTGC 18 FIGLA NM_001004311 155 F:GGCAAGACAGCTGTCAAGA 19 R:TTGGGGAGATAATTTCAGT 20 CGT GDF9 NM_005260 118 F:ATGGCACGTCCCAACAAA 21 TTC R:ACTCAGCACTAGCAGCAA 22 TCT GJA4 NM_002060 119 F:TGCAAGAGTGTGCTAGA 23 GGC R:ACAAAGCAGTCCACGAGG 24 TAG ZP1 NM_207341 219 F:CGCCATGTTCTCTGTCTCAA 25 R:CGTTTGTTCACATCCCAGTG 26 ZP2 NM_003460 245 F:GCCTCCCAGGACCCATTCTC 27 R:CAGGTAGCAGATGGAGCCT 28 A ZP3 NM_001110354 273 F:AGCAGGACCCAGATGAAC 29 TCAACA R:AAGCCCACTGCTCTACTTC 30 ATGGT NANOG NM_024865 215 F:CAGAAGGCCTCAGCACCTAC 31 R:CTGTTCCAGGCCTGATTGTT 32 SOX-2 NM_003106 437 F:ATGCACCGCTACGACGTGA 33 R:CTTTTGCACCCCTCCCATTT 34 REX-1 NM_174900 306 F:CAGATCCTAAACAGCTCGC 35 AGAAT R:GCGTACGCAAATTAAAGTC 36 CAGA GAPDH NM_002046 210 F:GACATCAAGAAGGTGGTGA 37 AGC R:GTCCACCACCCTGTTGCTG 38 TAG .sup.#F: forward primer; and R: reverse primer.

[0095] 6. Cryopreserving and Resuscitating FGSCs

[0096] FGSCs were digested with trypsin, mixed with FGSC culture medium containing 10% DMSO (Sigma) and then placed in a 2 ml cryotube. The cryotube was placed in an isopropanol cryopreserving box (Mr. Frosty) and placed in a freezer at 80 C. overnight. The next day, the cryotube was placed in liquid nitrogen for long-term storage. For cell resuscitation, the cryotube was quickly placed and thawed in a water bath at 37 C. The cryotube was centrifuged at 300 g for 5 minutes and the supernatant was discarded. The cells were cultured in the FGSC culture medium.

[0097] 7. Results

[0098] The small amount of ovarian tissues described above were digested into single cells using two-step enzymatic digestion, and then the cells were inoculated on the mitomycin C-inactivated STO feeder layer and cultured in vitro. Two weeks later, magnetic bead sorting was performed using a C-terminal DDX4 antibody. The sorted cells were round or oval, with a diameter of 8-15 m and a relatively large nucleus, and each cell was bound with 1-3 unequal magnetic beads around (FIG. 4A).

[0099] In order to identify whether these cells express the germline markers, the present inventors immunocytochemically detected the expression of DDX4. As shown in FIG. 4B, the sorted cells express DDX4.

[0100] The DDX4.sup.+ cells were subsequently inoculated on the feeder layer for subsequent culture. Early in the culture, the present inventors could observe the aggregated growth of the cells, and that the cells grew slowly in clusters (FIG. 5A). The cells then grew quickly and stably, requiring one passage every 5-7 days (FIG. 5B). With culturing according to the procedure described above, the cells could grow stably after cryopreserving-thawing (FIG. 5C).

[0101] The present inventors detected the gene expressions of two FGSC cell lines (1# and 2#) and FGSC cell lines obtained from surgically removed ovaries (srFGSCs) by RT-PCR at the mRNA level, in order to determine the cell characteristics. The expressions of DDX4, IFITM3, OCT4, STELLA, DAZL, BLIMP-1, STRA8, SYCP3, C-KIT, FIGLA, GDF9, GJA4, ZP1, ZP2, ZP3, NANOG, SOX2, and REX-1 in the above-mentioned cells/tissues were detected by using the ovarian total cDNAs of a 28-year-old woman of childbearing age (a patient with ovarian cysts) as a positive control and the non-reverse-transcribed mRNA as a negative control. As shown in FIG. 6, these two FGSC cell lines (1# and 2#) and srFGSCs express DDX4, IFITM3, OCT4, STELLA, DAZL, BLIMP-1, and REX-1. However, they do not express the meiosis specifically expressed genes STRA8 and SYCP3; the oocyte-associated genes C-KIT, FIGLA, GDF9, GJA4, ZP1, ZP2, and ZP3; and the pluripotent genes NANOG and SOX2. Therefore, it can be determined that the cell lines established by the present inventors are undifferentiated germline stem cells.

[0102] Subsequently, the present inventors detected germline cell markers at the protein level. As shown in FIG. 7, FGSCs express the germline-specific markers DDX4, OCT4, IFITM3, BLIMP-1, and DAZL proteins. It is confirmed at the protein level that the cell lines cultured by the present inventors are of germ cell origin.

[0103] BrdU (5-bromodeoxyuridine) is an analogue of thymidine T, and may be incorporated into the genome during DNA replication. The antibody antigen reaction can be used to detect its incorporation into cells, thereby reflecting its proliferative capacity. DDX4 is a germ cell-specific marker. Proliferating germ cells can be identified by dual-labelled immunofluorescence cytochemistry with BrdU and DDX4. As shown in FIG. 8, the cultured cells have proliferation activity.

[0104] Then, the present inventors performed G-banding karyotype analysis on FGSCs to determine whether their karyotypes were normal. As shown in FIG. 9, 83% of FGSCs have 46 chromosomes and belong to diploid cells.

[0105] In summary, it is demonstrated that FGSCs can be obtained from follicular aspirates.

[0106] All the documents mentioned in the present invention are incorporated by reference in the present application, as if each document was incorporated by reference alone. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art would be able to make various modifications or amendments to the present invention, and these equivalents likewise fall within the scope defined by the appended claims of the present application.