Strategy for treating premature ovarian failure based on extracellular vesicles derived from embryonic stem cells

Abstract

The premature ovarian failure is caused by ovarian function decline and is one of important factors affecting physical and psychological health of women. The invention relates to a technology for reversing premature ovarian failure by using embryonic stem cells (ESCs)-derived extracellular vesicles (ESC-EVs). The ESCs in the early development stage have self-renewal capacity and multidirectional differentiation potential, and have great application prospects in degenerative disease treatment and regenerative medicine. The ESC-EVs is formed by wrapping tiny vesicles with a phospholipid bilayer, carries nucleic acid, protein and other components secreted by the embryonic stem cells, and has a regeneration promoting effect similar to that of the embryonic stem cells; the problems of immunological rejection, ethical restriction, safety and the like are avoided, and compared with stem cells, the extracellular vesicles are more stable and easier to store, so that more and more extracellular vesicles are applied to the field of regenerative medicine. The invention relates to a new technology for treating premature ovarian failure by using embryonic stem cell-derived extracellular vesicles, which can improve the ovarian morphology and ovarian microenvironment, increase the number of primary follicles, anterior sinus follicles and sinus follicles, reduce the number of atresia follicles, reduce the expression of oxidative stress and senescence-related proteins, promote the recovery of ovarian functions and estrus cycle, and improve the curative effect of premature ovarian failure. The technical invention expands the application of the embryonic stem cell source exosome in the field of reproduction.

Description

Technical Field

[0001] The present invention relates to a technical means for treating premature ovarian failure and restoring reproductive function by utilizing extracellular vesicles secreted by embryonic stem cells, and belongs to the field of tissue engineering and new medical technology. Background Art

[0002] Embryonic stem cells (ESCs) are pluripotent cells isolated from mammalian blastocysts. Embryonic stem cells were originally derived from mice, and ESC lines or similar lines have subsequently been isolated from other rodents, domesticated animal species, and non-human primates. ESCs possess high self-renewal and multipotential differentiation abilities, allowing them to differentiate into various cell types under certain conditions. Studies have reported significant therapeutic benefits in treating neurodegenerative diseases, diabetes, repairing myocardial necrosis, and liver regeneration. However, the use of ESCs in clinical treatment is limited by immune rejection and ethical considerations. Therefore, scientists are searching for alternatives to ESCs that leverage their beneficial properties. In recent years, several alternative therapies have been reported, including therapeutic cloning, parthenogenetic ESCs, and induced pluripotent stem cells (iPSCs) for disease treatment or tissue repair. However, their tumorigenic potential still limits the clinical application of pluripotent stem cells.

[0003] Extracellular vesicles (EVs) are phospholipid bilayer-encapsulated vesicles produced by invagination of the endosomal membrane. With an average diameter of approximately 100 nm, EVs contain RNA, lipids, and proteins. The effects of EVs depend on the delivery of their contents to recipient cells, thereby altering the biological processes of target cells. The trafficking and transport of EVs influence various physiological functions of their target cells and play a vital role in intracellular and intercellular communication, including regulating immune responses, cell proliferation, cell migration, angiogenesis, and cancer progression. EVs also remove excess components from cells to maintain cellular homeostasis and have been implicated in radiation-induced tissue injury, cardiovascular disease, viral pathogenicity, central nervous system diseases, and cancer progression. EVs are secreted by a variety of cells and can promote or limit disease progression. Therefore, given these intrinsic properties, EVs may have potential applications in the prevention and treatment of numerous diseases.

[0004] Multiple studies have reported that embryonic stem cells can also secrete extracellular vesicles, and these extracellular vesicles derived from embryonic stem cells have been shown to play an important role in various disease recovery models. Extracellular vesicles derived from embryonic stem cells can promote angiogenesis and cardiomyocyte proliferation by delivering miR-294, thereby promoting cardiac function repair after myocardial infarction. In recent years, studies have found that extracellular vesicles secreted by various cells have the effect of delaying aging. Extracellular vesicles derived from young human fibroblasts can improve aging-related tissue damage. Extracellular vesicles derived from mesenchymal stem cells promote skin regeneration by improving the aging of skin fibroblasts. Studies have also reported that extracellular vesicles derived from embryonic stem cells can also improve the aging-related phenotype of aged mesenchymal stem cells and promote their effectiveness in treating skin damage.

[0005] Furthermore, extracellular vesicles successfully circumvent the aforementioned problems faced by embryonic stem cell therapy. First, extracellular vesicles derived from embryonic stem cells can avoid ethical issues because they are derived from the supernatant of embryonic stem cells, and since embryonic stem cells can proliferate indefinitely in vitro, extracellular vesicles derived from embryonic stem cells can be continuously isolated without destroying the new embryo. Second, compared with cell therapy, extracellular vesicles are more stable, more convenient to store, and easier to manage and control. Third, extracellular vesicles derived from embryonic stem cells are less tumorigenic than embryonic stem cells. Therefore, extracellular vesicles derived from embryonic stem cells are considered a good alternative to embryonic stem cells in cell therapy, avoiding the shortcomings of embryonic stem cells in clinical applications and greatly leveraging the excellent properties of embryonic stem cells.

[0006] Premature ovarian failure, also known as primary ovarian insufficiency, refers to a significant decline in ovarian function in women before the age of 40. Clinically, it manifests as amenorrhea, elevated gonadotropin levels, and decreased estradiol levels. It is one of the main causes of female infertility and menstrual irregularities. Chemotherapy, particularly treatment with alkylating agents, is a major cause of premature ovarian failure. Because the primordial follicle reservoir is extremely sensitive to reproductive toxicity, it often causes irreversible damage. Epidemiological data show that the incidence of premature ovarian failure is increasing worldwide, which is closely related to the increase in the incidence of various cancers in recent years. Current strategies for fertility preservation and restoration include hormone replacement therapy, ovulation induction, and gamete or embryo cryopreservation, but these methods rely on functional oocytes and an intact reproductive system. Therefore, there is an urgent need to develop more effective treatments to combat chemotherapy-induced reproductive toxicity and improve patients' quality of life and reproductive prognosis.

[0007] To address this therapeutic gap, this study investigated the role of extracellular vesicles (EVs) derived from embryonic stem cells (ESCs) in a mouse model of premature ovarian failure. We evaluated the regenerative potential of EVs derived from ESCs and preliminarily elucidated the mechanisms underlying their ability to restore ovarian function. Our results suggest that EVs derived from ESCs may represent a novel and promising strategy to mitigate ovarian damage and the resulting infertility. Summary of the Invention

[0008] The present invention is a technical means for rescuing premature ovarian failure in mice by utilizing extracellular vesicles secreted by embryonic stem cells.

[0009] The present invention utilizes extracellular vesicles derived from embryonic stem cells to treat premature ovarian failure mice, thereby rescuing the premature ovarian failure of the mice.

[0010] The present invention can effectively inhibit the expression of aging-related genes and proteins in the ovaries of mice with premature ovarian failure and increase the number of follicles in various stages.

[0011] The present invention can effectively reverse the aging of the ovaries of mice with premature ovarian failure, improve the morphology of the ovaries, promote the proliferation of oocytes in various stages, and reduce the degree of oxidative damage in oocytes and ovaries. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The invention relates to a characterization of extracellular vesicles, wherein the extracellular vesicles are extracellular vesicles derived from embryonic stem cells.

[0013] Figure 2 The results demonstrate the successful establishment of a mouse model of premature ovarian failure, which results in weight loss and disrupted estrous cycles.

[0014] Figure 3 When mice with premature ovarian failure were treated with extracellular vesicles derived from embryonic stem cells, the expression of aging-related genes and proteins was significantly decreased, cell proliferation was significantly increased, and the degree of DNA damage was reduced.

[0015] Figure 4 Treatment of mice with premature ovarian failure with extracellular vesicles derived from embryonic stem cells increased the number of follicles at each stage and improved ovarian morphology. DETAILED DESCRIPTION

[0016] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used can be obtained from commercial sources.

[0017] Example 1. The present invention provides a method for extracting extracellular vesicles derived from embryonic stem cells.

[0018] Cell basal culture medium and serum were purchased from Hyclone; double antibodies, trypsin and other reagents were purchased from Gibco; and cell culture consumables were purchased from NEST.

[0019] The serum required for cell culture to extract extracellular vesicles is fetal bovine serum from which extracellular vesicles have been removed. The processing steps are as follows: place the fetal bovine serum in an ultracentrifuge tube, balance it, and centrifuge it at 120,000g at 4°C for 2 hours. After centrifugation, take the supernatant in an ultraclean bench, discard the precipitate, and filter the supernatant with a 0.22μm needle filter and store it in a -80°C refrigerator until use.

[0020] For detailed procedures of cell biology experiments such as cell recovery, subculture, and cryopreservation, please refer to "Animal Cell Culture (Sixth Edition)".

[0021] Collecting embryonic stem cell culture supernatant containing extracellular vesicles: When embryonic stem cells have just recovered, culture them in a medium prepared with normal serum. When the cell confluence reaches 80%, discard the medium and gently wash twice with PBS. After passage, use a medium prepared with 15% extracellular vesicle-free serum. After continuing to culture for 24 hours, collect the cell culture supernatant into a 50ml centrifuge tube. This supernatant is rich in extracellular vesicles derived from embryonic stem cells.

[0022] Ultracentrifugation was used to separate and extract extracellular vesicles: ① The cell culture supernatant obtained in the above steps was centrifuged at 4°C, 500g for 10 min, and the supernatant was removed to remove cell debris; ② The supernatant was centrifuged at 4°C, 2000g for 30 min, and the supernatant was removed to remove large cell debris such as apoptotic bodies; ③ The supernatant was filtered through a 0.22μm needle filter to remove microvesicles with a diameter greater than 200nm; ④ The filtered supernatant was placed in an ultracentrifuge tube and centrifuged at 4°C, 120,000g for 2h. The supernatant was discarded, and an appropriate amount of PBS was added to resuspend the pellet at the bottom of the tube. The aliquots were stored at -80°C.

[0023] Example 2: The present invention provides a method for identifying extracellular vesicles derived from embryonic stem cells.

[0024] 1. Detection of extracellular vesicle marker proteins Alix, TSG101 and CD63 by Western Blot ( Figure 1 ).

[0025] 1) Protein sample preparation: Prepare protein lysis buffer at a ratio of PMSF:RIPA = 1:100. Add protein lysis buffer to the extracellular vesicle pellet obtained after ultracentrifugation to lyse the extracellular vesicles. Lyse the sample on ice for 30 minutes, shaking on a vortex shaker every 5 minutes to ensure thorough lysis. After 30 minutes, centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the supernatant to a new EP tube. Determine the extracellular vesicle protein concentration using the BCA assay. Add 5× SDS loading buffer to the remaining protein solution and denature it in boiling water for 5-10 minutes. After denaturation, immediately freeze the sample in liquid nitrogen and store at -80°C until use.

[0026] 2) Glue Preparation: Carefully clean the glass plates used in Western Blot experiments with a sponge. After washing with detergent, rinse thoroughly with tap water, and then rinse three times with ultrapure water. Clamps, sponges, glue preparation bottles, electrophoresis tanks, and transfer tanks used subsequently should also be cleaned thoroughly. Place the clean, air-dried glass plates on the glue preparation rack, ensuring a tight fit at the bottom edges. Check for leaks with distilled water. Prepare a 10% separation gel solution according to the separation gel recipe, mix thoroughly, and add all at once to the glass plates secured with clamps in advance using a 5ml pipette, taking care to avoid air bubbles. Add the separation gel to approximately 1.5cm from the top edge. Gently add water to seal the gel to avoid damaging the gel surface. After 40 minutes, a clear dividing line will be visible between the water and the gel, indicating that the separation gel has solidified. Use absorbent paper to remove any water from the separation gel seal. Prepare a 5% concentrated gel solution according to the recipe, add 1.5ml of this solution to the top of the separation gel, and immediately insert a comb. Once the concentrated gel has solidified, it is ready for use.

[0027] 3) Polyacrylamide gel electrophoresis: Place the prepared gel plates into the electrophoresis tank, with the shorter plate facing inward. Add freshly prepared 1x Running buffer between the two plates. Remove the comb and load the protein sample using equal mass (20 μg-50 μg). Calculate the loading volume based on the measured concentration and adjust the total loading volume to 20 μl with 1x SDS. Add the protein sample to the loading well and add electrophoresis solution to the marked area of the electrophoresis tank. Secure the tank lid, ensuring that the positive and negative electrodes are aligned. Run the stacking gel at 80 V. Once the bromophenol blue has reached the separating gel and the protein markers have separated, adjust the voltage to 110 V and stop the electrophoresis when the bromophenol blue approaches the bottom of the plate. The entire electrophoresis run takes 1.5 hours.

[0028] 4) Transfer: Prepare pre-chilled transfer buffer (1x Transfer buffer) containing 20% methanol in advance. Soak the transfer cassette, sponge, and filter paper in pre-chilled transfer buffer. Carefully pry open the gel plates and gently remove the concentrated gel along the dividing line. Place the resolving gel after electrophoresis on one side of the black cassette. Cut a PVDF membrane of appropriate size and activate it in methanol for 60 seconds. Place it on the gel to remove all bubbles between the gel and membrane. Gently cover the membrane with filter paper and sponge. Clamp the transfer cassette in the order of negative electrode (black) of transfer cassette - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - positive electrode (white) of transfer cassette. Place it in the transfer tank, add transfer buffer, and place the entire transfer tank in a foam box filled with ice. Transfer the membrane at a constant voltage of 100V in an ice bath for 2 hours.

[0029] 5) Blocking: The transferred PVDF membrane was taken out and placed in 5% skim milk (blocking solution) with the protein side facing up, and blocked at room temperature for 2 h on a horizontal shaker at 60 rpm.

[0030] 6) Antibody incubation: ① Primary antibody incubation: Dilute the primary antibody in blocking buffer according to the instructions (Alix, 1:2000 dilution; TSG101, 1:2000 dilution; CD63, 1:2000 dilution). Pipette 2 ml of primary antibody into the antibody incubation box, cut the membrane using the control protein marker, incubate the membrane with the corresponding primary antibody, and incubate overnight at 4°C. ② Secondary antibody incubation: Place the strips in TBST and wash three times for 10 minutes on a horizontal shaker at 120 rpm. Then add the secondary antibody of the corresponding species and incubate at room temperature for 2 hours on a horizontal shaker at 60 rpm. After incubation, wash three times with TBST for 10 minutes each.

[0031] 7) Exposure: Mix luminescent liquid A and luminescent liquid B in a 1:1 ratio to form a working solution, and expose through an exposure device.

[0032] 2. Characterization of Extracellular Vesicle Morphology and Size

[0033] Transmission electron microscopy (TEM) was used to observe the morphology of extracellular vesicles. Samples were deposited on carbon-coated copper grids and dried at room temperature for 2 minutes. Excess liquid was removed using a filter, and the samples were negatively stained with 2% uranyl acetate for 30 seconds. After air-drying for 60 minutes, the samples were imaged using a transmission electron microscope. The size of the extracellular vesicles was determined using nanoparticle tracking analysis (NTA).

[0034] Example 3: The present invention provides a method for establishing a premature ovarian failure mouse model.

[0035] Female mice aged 6–8 weeks received four intraperitoneal injections of cyclophosphamide at a dose of 60 mg / kg in a total volume of 200–300 μl, administered every three days. Previous studies have reported that the number of primordial follicles in mice is significantly reduced on day 13 after cyclophosphamide treatment compared to day 7. Therefore, this study established the Cy-POF mouse model by inducing cyclophosphamide for nine consecutive days, followed by a five-day interval, to evaluate the therapeutic efficacy of ESC-EVs in protecting primordial follicles. During the induction process, weight changes and estrous cycles were continuously recorded for 20 days. While the estrous cycle of normal mice exhibits an N-shaped pattern, the estrous cycle of the Cy-POF model mice exhibits severe disruption. Changes in Ki67 and γH2AX protein expression were used to assess cellular senescence.

[0036] Example 4. The present invention provides a technology for reversing premature ovarian failure in mice using extracellular vesicles.

[0037] Mice with premature ovarian failure were treated with extracellular vesicles derived from embryonic stem cells (ESCs) isolated by ultracentrifugation. After a 5-day interval, the mice received three injections of ESCs or an equal volume of control solution (200 μg each, injected via the tail vein every two days). It should be noted that the dosing interval was shortened to two days because, in this acute injury model, increased drug exposure is beneficial for cell survival and free ESCs are cleared more quickly in vivo. Ovarian H&E staining results showed that ESCs significantly alleviated follicular damage in mice with premature ovarian failure and promoted normal follicular development. Quantitative analysis showed that ESCs treatment increased the total number of ovarian follicles, the number of primordial follicles, and preantral follicles, reduced the number of atretic follicles and ovarian morphological damage, and improved the arrangement of granulosa cells, suggesting that ESCs have a beneficial repair effect on both folliculogenesis and development.

Claims

1. The active ingredient of this treatment is extracellular vesicles derived from embryonic stem cells, which are isolated and extracted from the supernatant of cells cultured in vitro. They carry a variety of bioactive molecules such as proteins and RNA in donor cells and are paracrine components with damage treatment functions.

2. The extracellular vesicle according to claim 1, wherein: The extracellular vesicles are derived from embryonic stem cells.

3. A technical means for delivering extracellular vesicles and delaying body aging, which is to treat mice with premature ovarian failure with the extracellular vesicles described in claim 1 to inhibit the occurrence of premature ovarian failure.

4. The technical means according to claim 3, characterized in that: The technology can reverse premature ovarian failure in mice through extracellular vesicles derived from embryonic stem cells, including improving ovarian morphology and inhibiting aging-related proteins.

Images