CN111593021B - Ovarian follicular membrane stem cell, and separation method, culture method and application thereof - Google Patents

Ovarian follicular membrane stem cell, and separation method, culture method and application thereof Download PDF

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CN111593021B
CN111593021B CN202010214438.5A CN202010214438A CN111593021B CN 111593021 B CN111593021 B CN 111593021B CN 202010214438 A CN202010214438 A CN 202010214438A CN 111593021 B CN111593021 B CN 111593021B
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ovarian
stem cells
follicular membrane
membrane stem
ovarian follicular
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CN111593021A (en
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项鹏
柯琼
陈洪
李伟强
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Sun Yat Sen University
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Abstract

The invention provides an ovarian follicular membrane stem cell, a separation method, a culture method and application thereof, and relates to the technical field of stem cells and tissue engineering. The separation method of the ovarian follicular membrane stem cells comprises the following preparation methods: obtaining ovarian tissue: obtaining ovarian tissues from female cynomolgus monkeys with normal fertility and ovary function and ages of 4-10 years, peeling off fat around the ovarian tissues, retaining ovarian cortex matrixes, cleaning, and cutting the ovarian cortex matrixes into tissue fragments; culturing: adding culture medium into the tissue fragments, centrifuging, removing supernatant, adding culture solution for resuspension, incubating, centrifuging, removing supernatant, adding culture solution for resuspension to obtain cell suspension, and inoculating and culturing to obtain the ovarian follicular membrane stem cells. The method can successfully separate the ovarian follicular membrane stem cells, can differentiate the ovarian follicular membrane stem cells into the ovarian follicular membrane cells, can improve the serum AMH level and improve the ovarian function.

Description

Ovarian follicular membrane stem cell, and separation method, culture method and application thereof
Technical Field
The invention relates to the technical field of stem cells and tissue engineering, in particular to an ovarian follicular membrane stem cell, and a separation method, a culture method and application thereof.
Background
With the delay of the childbearing age, the influence of life pressure and environmental factors, the infertility caused by gonadal aging is obviously increased. In the past ten years, the infertility rate of the people of childbearing age in China reaches about 15 percent, and 1 pair of infertility patients exist in every 8 pairs of couples of childbearing age. The release of the two-child policy also presents new challenges to reproductive health: women 50% of which are fertile to two children are older than 40 years old, resulting in serious decline in fertility. At the same time, the aging speed of human body is directly aggravated by the aging of the ovary. Therefore, how to effectively treat the ovarian aging and delay the body aging is an important issue facing us. One of the cores of fertility is the germ cells, which are also the most important seeds in the fertility segment. Meanwhile, the germ cells are not isolated, and the functions of proliferation and differentiation of gonadal somatic cells, hormone secretion and the like are important guarantees for maintaining normal development of the germ cells, so that the aim of delaying senescence and reconstructing fertility is also an important link for providing a soil microenvironment suitable for development for the germ cells.
The diseases of low ovary function and the like are manifested by problems of low ovum quantity and/or quality, ovulation disorder, infertility, reproductive endocrine disturbance and the like, and the diseases of ovary function mainly comprise premature ovarian failure (premature ovarian failure, POF), reduced ovarian reserve function (diminished ovarian reserve, DOR), premature ovarian failure (premature ovarian insufficiency, POI), low ovary reaction (poor ovarian response, POR) and the like. Taking premature ovarian failure as an example, the cause of the disease is complex, and it is not completely clear at present, and known influence factors mainly include: genetic factors (such as fragile X chromosome, etc.), deficiency of metabolic enzymes related to hormone synthesis, protein and receptor deficiency, immune factors, infectious factors, and iatrogenic factors such as chemotherapy and radiotherapy, which often accelerate follicular locking. The treatment modes of premature ovarian failure mainly comprise hormone replacement therapy, cryopreservation of ova, ovarian transplantation, immunotherapy, psychological therapy and the like, and certain risks and side effects exist in the treatment modes, so that no specific treatment mode exists at present, and a need exists for searching for a new treatment mode.
Ovarian follicular membrane cells (TC), which are a population of cell types similar to testosterone mesenchyme cells secreting androgens in the male testes, are the primary source of synthetic androgens in the ovaries, and the produced androgens are transported to granulosa cells as substrates for estrogen synthesis, so that the cells are likely to become seed cells for the treatment of a series of diseases such as hypoovarial dysfunction. Ovarian follicular membrane stem cells (TSCs) are a group of cell types with self-renewing capacity, which can differentiate into TCs. There is currently little research on TSC, and due to the ambiguous source of TSC in organisms, the limited number of TSCs in the ovaries, it has been reported that TSC can be isolated from whole ovaries of mice and pigs. Primate and human relatives are closer, the ovary structure and physiological cycle of primate are similar to those of human, but separation of TSC has not been reported yet. The existing TSC is obtained by adopting a whole ovary separation mode, so that the damage is large, and the autologous transplantation treatment is not facilitated.
The method can adopt a mode of minimally invasive acquisition of small amount of tissue of the monkey ovary cortex, the TSC is obtained through separation, and the cell is greatly expanded and the function of the cell is verified through improvement of culture conditions, so that a research foundation is provided for subsequent basic research and clinical transformation application research of the cell, and the method has important significance for further promoting treatment of diseases such as human ovary hypofunction and the like.
Disclosure of Invention
In view of the above, it is desirable to provide a method for separating ovarian follicular membrane stem cells, which can successfully separate monkey ovarian follicular membrane stem cells having the function of regenerating and repairing damaged tissues.
The separation method of the ovarian follicular membrane stem cells comprises the following preparation methods:
obtaining ovarian tissue: selecting a female cynomolgus monkey with the age of 4-10 years old, acquiring ovarian tissues from ovaries of the cynomolgus monkey with normal fertility and ovaries, peeling off fat around the ovarian tissues, retaining an ovarian cortical matrix, cleaning, and cutting the ovarian cortical matrix into tissue fragments;
culturing: adding culture medium into the tissue fragments, centrifuging, removing supernatant, adding culture solution for resuspension, incubating, centrifuging, removing supernatant, adding culture solution for resuspension to obtain cell suspension, and inoculating and culturing to obtain the ovarian follicular membrane stem cells.
The inventor adopts the method to obtain the ovarian follicular membrane stem cells in primate animals for the first time. The cells obtained by the separation method of the invention can express P75, nestin and PDGFR-alpha from molecular level analysis, but not 3 beta-HSD and LHR, and accord with the molecular biological characteristics of ovarian follicular membrane stem cells, and the cells also have self-renewal capacity and can successfully differentiate into ovarian follicular membrane cells, which indicates that the method of the invention can successfully obtain the ovarian follicular membrane stem cells from the ovaries of the cynomolgus monkeys.
In one embodiment, in the step of obtaining ovarian tissue, a laparoscopic minimally invasive manner is used to cut out square ovarian cortex tissue with a side length of 1-5mm from ovaries of cynomolgus monkeys with normal fertility and ovaries. A small amount of tissues are taken in a minimally invasive mode, most of the structures of the ovaries are reserved, a large amount of cells for research and treatment can be obtained, the influence of the ovaries on the body is avoided, and a foundation is provided for the development of subsequent autologous cell transplantation treatment.
In one embodiment, the culturing step is specifically: centrifuging the tissue fragments at 1400-1600 rpm for 4-6 min, retaining the precipitate, adding culture solution, re-suspending, and adding 5+ -0.5% CO at 37+ -0.5 deg.C 2 The incubation time is 20-30 h, the centrifugation speed is 1400-1600 rpm, the centrifugation time is 4-6 min, the sediment is reserved, the culture solution is added for re-suspension to obtain cell suspension, and the cell suspension is inoculated for culture to obtain the ovarian follicular membrane stem cells.
In one embodiment, the culture solution DMEM-F12 in the culture step is a base solution, and comprises the following components in concentration: 0.8-1.2 nM dexamethasone, 0.5-1.5 ng/ml LIF, 45-55 ng/ml insulin, 45-55 ng/ml transferrin, 45-55 pg/ml sodium selenite, 15-25 ng/ml Oncoinhibin M (OSM), 1+ -0.1% V/V non-essential amino acids, 1+ -0.1% V/V N2, 2+ -0.1% V/V B27 serum-free additives, 15-25 ng/ml bFGF (fibroblast growth factor), 15-25 ng/ml EGF (epidermal growth factor), 15-25 ng/ml PDGF-BB (platelet derived growth factor), 0.05-0.15 mM beta-mercaptoethanol. The dexamethasone, insulin, bFGF and EGF in the culture solution can provide hormone and growth factors for cells, promote cell proliferation, inhibit cell differentiation by OSM, and the optional amino acid is a nutrient substance necessary for cell growth, sodium selenite and the like can provide microelements, so that the culture solution is an important role in the cell metabolism detoxification process, and transferrin is taken as a binding protein and has an important role in the cell metabolism process.
In one aspect, the invention also provides an ovarian follicular membrane stem cell obtained by the method.
The expression of the ovarian follicular membrane stem cells is not limited to specific marker proteins such as Nestin, P75, PDGFR-alpha and the like, but is not limited to specific marker proteins such as Star, LHR,3 beta-HSD and the like; the ovarian follicular membrane stem cells have self-renewal capacity, can differentiate into ovarian follicular membrane cells, secrete androgens, and have the function of repairing lesion tissues.
The invention also provides a method for culturing the ovarian follicular membrane stem cells, which comprises the following steps:
adding a culture solution into the ovarian follicular membrane stem cells for subculture, wherein the culture solution takes DMEM-F12 as a base solution and comprises the following components in concentration: 0.8 to 1.2nM dexamethasone, 0.5 to 1.5ng/ml LIF, 45 to 55ng/ml insulin, 45 to 55ng/ml transferrin, 45 to 55pg/ml sodium selenite, 15 to 25ng/ml Oncoinhibin M, 1+ -0.1% V/V nonessential amino acids, 1+ -0.1% V/V N2, 2+ -0.1% V/V B27 serum free additives, 15 to 25ng/ml bFGF, 15 to 25ng/ml EGF, 15 to 25ng/ml PDGF-BB, 0.05 to 0.15mM beta-mercaptoethanol.
The invention also provides a differentiation method of the ovarian follicular membrane stem cells, which comprises the following steps:
adding an induction culture medium into the ovarian follicular membrane stem cells for differentiation culture, wherein the induction culture medium takes DMEM-F12 as a base solution and comprises the following components in concentration: 1.5-2.5% (V/V) FCS (calf serum), 0.5-1.5 nM T3 (sheep embryo factor), 0.5-1.5 ng/ml LH (luteinizing hormone), 65-75 ng/ml IGF-1 (growth factor), 5-15 ng/ml PDGF-BB, 45-55 ng/ml insulin, 45-55 ng/ml transferrin, 45-55 pg/ml sodium selenite.
In the differentiation method, LH in the induction culture medium can act on LHR receptor of the ovarian follicular membrane cells to promote the differentiation of TSC to TC, and sheep embryo factors, growth promoting factors, insulin and the like can provide necessary growth factors and hormones for cell proliferation and differentiation, and the components promote cell differentiation while promoting cell proliferation, so that the ovarian follicular membrane stem cells can be induced to differentiate into the ovarian follicular membrane cells.
In one aspect, the invention also provides an ovarian follicular membrane cell obtained by the above differentiation method.
In one aspect, the invention also provides an application of the follicular membrane stem cells in preparing medicines for treating ovarian function diseases. The ovarian follicular membrane stem cells of the invention can improve the serum AMH level and promote the ovarian function.
Compared with the prior art, the invention has the following beneficial effects:
the separation method can successfully obtain the ovarian follicular membrane stem cells from the ovaries of the cynomolgus monkeys, and the obtained cells can express P75, nestin and PDGFR-alpha but not 3 beta-HSD and LHR according with the molecular biological characteristics of the ovarian follicular membrane stem cells, and the cells have self-renewal capacity and can successfully differentiate into the ovarian follicular membrane cells; the ovarian follicular membrane stem cells of the invention can improve the serum AMH level and promote the ovarian function.
The differentiation method can induce the ovarian follicular membrane stem cells to differentiate into the ovarian follicular membrane cells.
Drawings
FIG. 1 is a morphology of ovarian follicular membrane stem cells cultured in vitro;
FIG. 2 shows the expression of specific marker proteins by ovarian follicular membrane stem cells;
FIG. 3 is a morphology of single ovarian follicular membrane stem cell proliferation cells;
FIG. 4 is a graph of the number of ovarian follicular membrane stem cell amplifications versus time;
FIG. 5 is a graph showing the directed differentiation expression of ovarian follicular membrane stem cells;
FIG. 6 is a graph showing the number and time relationship of androstenedione expression after differentiation of ovarian follicular membrane stem cells;
FIG. 7 is a graph of hormonal changes following ovarian follicular membrane stem cell transplantation;
FIG. 8 shows the intra-ovarian positioning differentiation of ovarian follicular membrane stem cells;
FIG. 9 is a comparative cell morphology.
Detailed Description
In order that the invention may be understood, a more complete description of the preferred embodiments of the invention will be presented below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the following examples, AMH represents Anti-mullerian hormone serum Anti-Mueller hormone; FSH represents follicle-stimulating hormone follicle stimulating hormone; LH represents Luteinizing hormone luteinizing hormone; e2 represents Estradiol Estradiol.
Example 1
Isolation of monkey ovarian follicular membrane stem cells.
1) Female cynomolgus monkeys aged 4-10 years old are selected, and fertility and ovarian function of the female cynomolgus monkeys are detected to be normal, and the serum hormone (AMH, FSH, LH, E2) level is detected to be normal. In a sterile operating room, a Shutai preliminary anesthesia experimental monkey is adopted, an anesthesia machine is used for providing isoflurane to maintain an anesthesia state, and an electrocardiograph monitor is used for monitoring animal states. Under the mediation of laparoscope, the ovary ligament part is clamped by using a fixed grasping forceps, the ovary is fixed by using celiac scissors, a square ovary tissue cortex sample with a side length of about 2mm is sheared, and the tissue sample is clamped out and transferred into a fresh constant temperature medium at 37 ℃. The abdominal cavity of the cynomolgus monkey was flushed with normal saline at a constant temperature of 37 ℃ and the normal saline was withdrawn and repeated 3 times. The small wound is sutured, and the cynomolgus monkey is resuscitated and then observed until its state returns to normal.
2) The sheared ovarian cortical tissue was collected into 5ml of medium, centrifuged at 1500rpm for 5min, the supernatant was discarded, resuspended in 2ml of ovarian follicular membrane stem cell culture medium, and transferred into a 12-well plate. At 37℃with 5% CO 2 After 24 hours incubation, the cell suspension was collected in a centrifuge tube, centrifuged at 1500rpm for 4min, the supernatant was removed, the resulting cell pellet was resuspended in 1-3ml of medium and the cells were cultured in 6-or 12-well plates.
The culture solution takes a DMEM-F12 culture medium as a base solution and comprises the following components in concentration: 1nM dexamethasone, 1ng/ml LIF, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 20ng/ml Oncostatin M, 1%V/V nonessential amino acids, 1%V/V N2, 2%V/V B27 without serum additives, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
The cells obtained by the separation method of the embodiment can be cultured in vitro for a long time (more than 15 generations) to maintain the characteristics of stem cells, and meanwhile, the separation method meets the welfare ethics requirements of animals.
Example 2
Culturing ovarian follicular membrane stem cells and identifying biological characteristics.
1) Culturing of ovarian follicular membrane stem cells and expression of specific markers:
a, TSCs isolated in example 1 were placed in individual wells of a 6-well plate for cultivation. The culture solution takes a DMEM-F12 culture medium as a base solution and comprises the following components in concentration: 1nM dexamethasone, 1ng/ml LIF, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 20ng/ml Oncostatin M, 1%V/V nonessential amino acids, 1%V/V N2, 2%V/V B27 without serum additives, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
Observing the cloning condition of the cells, when the cloning size of the cells reaches more than 50 mu m and the density reaches 70%, washing the cells by using PBS, then treating the cells for about 20-60 seconds by using a constant temperature cell separation liquid (such as trypsin-EDTA or ACCUTASE commercialized by STEMCELL company, and the like), and carrying out cell passage according to the experiment requirement according to the proportion of 1:2-1:4. Primary cells grew in the culture dish by adherence, the cells were spindle-shaped, and gradually formed clone balls with time, while the cells were passaged to expand and maintain their spindle-shaped morphology (as shown in fig. 1).
b, detecting the expression of TSC specific markers by adopting an immunofluorescence staining method, wherein the TSC specific markers comprise TSC specific markers such as P75, nestin, PDGFR-alpha and TC specific markers such as LHR,3 beta-HSD. Cell clones were collected and embedded with OTC, fixed with 4% paraformaldehyde for 20min after sectioning, followed by 5min of immersion in PBS buffer, repeated 3 times, 0.2% triton-X-100 was penetrated for 30min, then goat serum was added for incubation for 20min at room temperature, primary antibody was added overnight in a wet box at 4 ℃, followed by 5min of immersion in PBS buffer, repeated 3 times, the corresponding secondary antibody was added for incubation for 60min at room temperature, 5min of immersion in PBS buffer, repeated 3 times, DAPI staining was added, followed by 5min of immersion in PBS buffer, repeated 3 times, and the results were observed under a fluorescence microscope and photographed. The experimental results are shown in FIG. 2, green fluorescence in FIGS. 2-1 to 2-3 represents Nestin, P75, PDGFR-alpha, respectively, blue fluorescence represents DAPI, and only blue fluorescence in FIGS. 2-4 and 2-5, respectively. The results in FIG. 2 demonstrate that the TSCs of the present invention express Nestin, P75, PDGFR-alpha, and do not express 3β -HSD, LHR.
2) Identification of proliferation capacity of ovarian follicular membrane stem cells:
to verify the proliferation potency of TSC, P1 generation TSC was digested to give single cells, which were subjected to a single cell pelleting experiment, as shown in FIG. 3, and after 10 days of single cell culture, clones were formed. The results show that TSC has proliferation capacity, single cells can form multicellular clones by culture, and cells have faster and more stable multiplication speed and better expansion capacity (shown in FIG. 4).
3) Identification of ovarian follicular membrane stem cell differentiation capability:
to verify the differentiation capacity of TSC, TSC was cultured in a TSC differentiation medium using DMEM-F12 as a base fluid comprising the following concentrations of the components: 2% (V/V) FCS, 1nM T3, 1ng/ml LH, 70ng/ml IGF-1, 10ng/ml PDGF-BB, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite; after 10 days, cells are collected and subjected to immunofluorescence staining, ELISA and RT-PCR detection, the detection results are shown in FIG. 5, the results show that the cells express specific markers of mature ovarian follicular membrane cells such as STAR, LHR, CYP A1, CYP17A1, 3β -HSD, SF-1 and the like after TSC differentiation, the RT-PCR detection shows that differentiated cells (TC group) express Gli2, ptch2, star, CYP11A1, 3β -HSD, SF-1 and CYP17A1 similar to ovarian tissues, and undifferentiated TSC does not express the RNA; ELISA detection results revealed that a large amount of Androstenedione (Androstenedione) was secreted after the differentiation of TSC, indicating that under this differentiation condition, TSC can differentiate into mature TC (as shown in FIG. 6).
Example 3
Repair of ovarian follicular membrane stem cells in vivo tissues.
The animal model is used to verify that the damaged tissue in the stem cell regeneration repair body is an important index for evaluating the function of the stem cells and is also an important evaluation standard for the clinical transformation application potential of the stem cells.
Selecting a group of female cynomolgus monkeys about 15 years old, screening the cynomolgus monkeys with fertility decline, menstrual cycle disorder, serum AMH (advanced meyer disease), estrogen hypofunction and gonadotrophin secretion normal or excessive as a cynomolgus monkey model for treating the ovarian dysfunction, and carrying out TSC transplantation repair experiments.
To facilitate the in vivo tracking of stem cells, TSCs were transfected with GFP or RFP expressing lentiviruses to obtain GFP or RFP expressing TSCs, to verify their self-renewal and differentiation potential, and to obtain large amounts (about 2.0x10) by in vitro expansion 8 ) TSCs expressing GFP or RFP were used in vivo cell transplantation experiments.
GFP/RFP positive TSCs were transplanted in situ under the ovarian cortex of the ovarially incompetent cynomolgus monkey using a microinjection needle under laparoscopic mediation. On day 15 after TSC transplantation, serum levels of hormones such as AMH and FSH were measured. The results indicate that TSC transplantation can increase AMH expression in cynomolgus monkey model serum while decreasing FSH levels (as shown in fig. 7); a small amount of ovarian tissue is cut for embedding and slice staining, the detection result is shown in figure 8, TSC transplanted into the ovary can express specific markers Star, 3 beta-HSD and P450scc of mature ovarian follicular membrane cells in vivo, ki67 can be expressed in vivo, the transplanted TSC can proliferate and directionally differentiate in vivo, so that GFP/RFP positive TSC transplanted by us can be well planted in the ovary and participate in the development process of follicular structure, CX43 (connexin) expression is identified through an immunohistochemical method, and good gap connection can be established between the transplanted TSC and TC in the ovary after the transplanted TSC is differentiated.
Comparative example 1
The method for isolating monkey ovarian follicular membrane stem cells differs from example 1 in that step 2) is replaced with:
adding 1mg/ml type IV collagenase solution into sheared ovarian cortex tissue, and heating at 37deg.C and 5% CO 2 Incubating for 30min, adding 3 times volume of PBS to dilute collagenase solution, and centrifuging at 1500rpm for 5min; discarding supernatant, adding 15ml PBS, filtering cell suspension with nylon mesh with pore size of 40 μm, centrifuging the collected filtrate at 1500rpm for 5min, and retaining the sedimentAdding culture solution to resuspend to obtain cell suspension, and inoculating to 6-well plate or 12-well plate for culturing. The primary cells obtained in this comparative example are hardly formed into clonal growth cells, almost all are adherent fibroblasts, and are unfavorable for the maintenance of the subsequent cell stem property and cell expansion.
Comparative example 2
The method for separating monkey ovary follicular membrane stem cells is different from example 1 in that the culture medium used is replaced with:
the DMEM-F12 culture medium is used as a base solution, and contains the following components in concentration: 1nM dexamethasone, 1ng/ml LIF, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 1%V/V nonessential amino acids, 1%V/V N2, 2%V/V B27 serum free additives, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
As shown in FIG. 9 (-OSM), the cells obtained by the culture of this comparative example were observed to be easily aggregated and to be significantly blackened to death, indicating that the culture system without OSM was unfavorable for proliferation and expansion of cells.
Comparative example 3
The method for separating monkey ovary follicular membrane stem cells is different from example 1 in that the culture medium used is replaced with:
the DMEM-F12 culture medium is used as a base solution, and contains the following components in concentration: 1nM dexamethasone, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 20ng/ml Oncoinhibin M, 1%V/V nonessential amino acids, 1%V/V N2, 2%V/V B27 serum free additives, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
As shown in FIG. 9 (-LIF), the cells obtained by the culture of this comparative example were observed to have a slow proliferation rate, a large cell width, a clear nucleus, and a differentiation or aging state, which is detrimental to the maintenance of cell stem properties and cell proliferation, indicating that the LIF-deficient culture system is detrimental to the maintenance of cell stem properties and cell proliferation.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (3)

1. The separation method of the ovarian follicular membrane stem cells is characterized by comprising the following preparation methods:
obtaining ovarian tissue: selecting female cynomolgus monkeys with the age of 4-10 years old, obtaining cortex parts of ovarian tissues, peeling off fat around the ovarian tissues, retaining an ovarian cortex matrix, cleaning, and cutting the ovarian cortex matrix into tissue fragments;
culturing: adding culture solution into the tissue fragments, centrifuging at 1400-1400 rpm for 4-6 min, retaining precipitate, adding culture solution, re-suspending at 37+ -0.5deg.C and 5+ -0.5% CO 2 Incubating for 20-30 h, centrifuging at 1400-1400 rpm for 4-6 min, retaining sediment, adding culture solution to resuspend to obtain cell suspension, and inoculating and culturing to obtain ovarian follicular membrane stem cells; the culture solution DMEM-F12 in the culture step is taken as a base solution, and comprises the following components in concentration: 1nM dexamethasone, 1ng/ml LIF, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 20ng/ml Oncostatin M, 1%V/V nonessential amino acids, 1%V/V N2, 2% V/V B27 serum free additive, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
2. A method for culturing ovarian follicular membrane stem cells, comprising the steps of:
adding a culture solution to the ovarian follicular membrane stem cells obtained by the separation method of claim 1 for subculturing, wherein the culture solution takes DMEM-F12 as a base solution and comprises the following components in concentration: 1nM dexamethasone, 1ng/ml LIF, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite, 20ng/ml Oncostatin M, 1%V/V nonessential amino acids, 1%V/V N2, 2% V/V B27 serum free additive, 20ng/ml bFGF, 20ng/ml EGF, 20ng/ml PDGF-BB, 0.1mM beta-mercaptoethanol.
3. A method for differentiating ovarian follicular membrane stem cells, comprising the steps of:
adding an induction medium to the ovarian follicular membrane stem cells obtained by the separation method of claim 1 for differentiation culture, wherein the induction medium takes DMEM-F12 as a base solution, and comprises the following components in concentration: 2%V/V FCS, 1nM T3, 1ng/ml LH, 70ng/ml IGF-1, 10ng/ml PDGF-BB, 50ng/ml insulin, 50ng/ml transferrin, 50pg/ml sodium selenite.
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