Improved msc-ses for ovarian function and methods of making and using the same

Abstract

The application discloses application of MSC-sEVs in treatment of reduced ovarian reserve. By targeting and activating DNA repair genes (such as PARP1) and combining NAD+ metabolism regulation, the ovarian function and reproductive potential of DOR are significantly improved. The method has high safety and unique advantages, and provides an innovative treatment scheme for patients with ovarian reserve dysfunction.

Description

Technical Field

[0001] This invention relates to the fields of reproductive medicine and cell therapy, and in particular to the application of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) in the preparation of a drug that improves ovarian function in a cyclophosphamide-induced ovarian reserve reduction (Cy-DOR) mouse model. Background Technology

[0002] Primary ovarian insufficiency (POI) or premature ovarian failure (POF) is a heterogeneous gynecological endocrine disorder caused by follicular dysfunction or failure to function properly. It is characterized by elevated serum gonadotropin levels, absence of residual follicles in the gonads, and is associated with oligomenorrhea, amenorrhea, insufficient fertility, and even infertility. Based on the progression and severity of the disease, the National Institutes of Health (NIH) and the American Society for Reproductive Medicine (ASRM) have proposed that POI can be divided into three progressive stages: the occult stage, the biochemical abnormality stage (bPOI), and the ovarian failure stage. Patients in the first two stages of POI can still conceive using their own oocytes, while patients in the ovarian failure stage often require donor oocyte in-vitro fertilization-embryo transfer (IVF-ET) to resolve their fertility issues. Diminished ovarian reserve (DOR) refers to a decrease in the number, quality, or growth capacity of remaining eggs in a woman's ovaries, which may affect fertility. It is usually related to aging, but can also be caused by genetic factors, medical treatments (such as chemotherapy or radiation therapy), surgical removal of ovarian tissue, or other unknown causes. Decreased ovarian reserve means a reduced chance of conceiving naturally, but it is still possible.

[0003] Diminished ovarian reserve (DOR) and POI differ significantly in their pathological mechanisms and treatment goals: POI, due to complete ovarian failure, is primarily treated with hormone replacement therapy or donor egg assisted reproduction; DOR, however, involves a decline in the number and quality of follicles, but still retains reproductive potential, requiring intervention to slow ovarian decline by improving the ovarian microenvironment. Hormonal imbalances associated with DOR can adversely affect metabolic health, leading to insulin resistance and metabolic syndrome, which are linked to an increased risk of neurodegenerative diseases such as Alzheimer's. Current treatments for POI have failed to effectively restore ovarian function, and treatment strategies for DOR are similarly limited. Summary of the Invention

[0004] In light of the above background, this invention provides a novel treatment method that targets and activates DNA repair genes (such as PARP1) through MSC-sEVs and combines this with NAD+ metabolic regulation, aiming to significantly improve ovarian function and reproductive potential in a DOR mouse model. This method has high safety and unique advantages, providing an innovative treatment option for patients with ovarian reserve dysfunction.

[0005] Our study employed a scalable isolation method to isolate and characterize mesenchymal stem cell-derived exosomes (MSC-sEVs) and discovered their protective effect on the ovaries in a mature cyclophosphamide-induced decreased ovarian reserve (Cy-DOR) mouse model. Specifically, in the Cy-DOR mouse model, MSC-sEVs enhanced the DNA repair capacity of ovarian cells by upregulating DNA repair genes such as PARP1, thereby protecting them from damage, alleviating oxidative stress, and restoring ovarian function by enhancing DNA repair pathways.

[0006] On the one hand, the present invention provides the application of MSC-sEVs in the preparation of drugs for treating decreased ovarian reserve.

[0007] Furthermore, the treatment of reduced ovarian reserve aims to improve estrous cycles and / or follicular development.

[0008] Furthermore, the improvement in follicle development is characterized by a significant increase in the number of primordial follicles and the total number of follicles, and a decrease in the number of atretic follicles.

[0009] Alternatively, the treatment of reduced ovarian reserve may aim to improve the number of oocytes and / or fertility outcomes.

[0010] Furthermore, the improvement in the number of oocytes refers to an increase in the number of oocytes and a decrease in the number of abnormal oocytes.

[0011] Furthermore, the improved reproductive results include an increase in the number of offspring and an increase in the weight of the offspring.

[0012] Alternatively, the treatment for decreased ovarian reserve may aim to enhance ovarian function and / or serum hormone levels.

[0013] Furthermore, the enhanced ovarian function is achieved by increasing AMH levels and increasing DDX4 expression.

[0014] Furthermore, the enhancement of ovarian function is achieved by decreasing the expression of caspase-3 and total caspase-3.

[0015] Furthermore, the enhancement of ovarian function is described as a decrease in the apoptosis index.

[0016] Furthermore, the enhanced serum hormone levels are characterized by a significant increase in serum E2 levels and a significant decrease in FSH levels.

[0017] Preferably, the aforementioned treatment for decreased ovarian reserve improves symptoms of decreased ovarian reserve by maintaining NAD+ levels and DNA.

[0018] On the other hand, the present invention provides the application of MSC-sEVs in the preparation of drugs that improve behavior and cognitive function.

[0019] Furthermore, the improved behavioral and cognitive functions refer to enhanced learning and memory abilities.

[0020] On the other hand, the present invention provides a method for improving follicular development through non-disease treatment, characterized by using MSC-sEVs or drugs prepared based on MSC-sEVs to improve follicular development.

[0021] Furthermore, the improvement in follicle development is characterized by a significant increase in the number of primordial follicles and the total number of follicles, and a decrease in the number of atretic follicles.

[0022] On the other hand, the present invention provides a method for improving the number of ovulations through non-disease treatment, characterized in that MSC-sEVs or drugs prepared based on MSC-sEVs are used to increase the number of ovulations in animals.

[0023] Furthermore, the improvement in the number of oocytes refers to an increase in the number of oocytes and a decrease in the number of abnormal oocytes.

[0024] On the other hand, the present invention provides a method for improving reproductive numbers through non-disease treatment, characterized in that MSC-sEVs or drugs prepared based on MSC-sEVs are used to improve animal reproductive numbers.

[0025] Furthermore, the improvement in reproductive output refers to an increase in the number of offspring produced and an increase in the weight of the offspring.

[0026] Preferably, the aforementioned method can be applied in scientific research experiments or animal husbandry production. For example, it can be used as a positive control in scientific experiments or to increase yields in animal husbandry production.

[0027] Preferably, the target of the aforementioned drug or method is a mammal.

[0028] Finally, the present invention also provides a method for preparing engineered exosome MSC-sEVs, the method comprising the following steps:

[0029] 1) Culture human induced pluripotent stem cells in intact cell culture medium (CCM) for 2-3 days. When the cells have grown to about 80% confluence, expand to a 5-layer cell factory.

[0030] 2) After washing the cells with phosphate-buffered saline (PBS), the CCM was replaced with chemically defined protein-free (CDPF) medium.

[0031] 3) After 6 hours, replace with fresh CDPF medium, collect about 600 ml of supernatant, and centrifuge at 2,000 g for 20 minutes to remove cell debris.

[0032] 4) Infect iPSC-MSCs with a recombinant lentivirus carrying the luciferase gene. Plant the cells at a rate of 3 × 10⁶ cells / year. 4 Cells were seeded at a density of 1 / ml in 12-well plates. After 6 hours of infection, the medium was replaced with fresh medium. After 72 hours, the cells were selected using puromycin to obtain iPSC-MSCs that stably expressed luciferase, and then cryopreserved to form a working cell bank.

[0033] 5) Purification of MSC-derived small exosomes (MSC-sEVs) by anion exchange chromatography ( Figure 1 A).

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. Low immunogenicity and high non-tumorigenicity: Compared with cells, exosomes have the advantages of low immunogenicity, high non-tumorigenicity, high clinical safety, and low ethical risk.

[0036] 2. High safety: Since engineered MSC-sEVs do not use live cells but are in the form of exosomes, their clinical safety is higher, avoiding potential risks such as tumor transformation.

[0037] 3. Highly Efficient DNA Repair: By enhancing DNA repair pathways, engineered exosomes exhibit more powerful functions in ovarian cell damage repair than ordinary MSC-sEVs, and are expected to significantly improve ovarian function and reproductive capacity. Furthermore, because NAD+ is extremely unstable and easily degraded in vitro, the closed structure of MSC-sEVs significantly prolongs the half-life of NAD+ in the extracellular microenvironment, enabling effective delivery of NAD+ to cells with low NAD+ levels, further promoting their functional recovery. Attached Figure Description

[0038] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, illustrates the method of the present invention for regulating plant resistance to salt stress and / or cultivating salt-stress-resistant plant varieties, and its beneficial effects.

[0039] Figure 1The following diagram illustrates the characterization of iPSC-MSC-SEV: A. Schematic diagram of the MSC-SEV separation process, including cell culture, culture medium changes, and anion exchange chromatography. B. Nanoparticle tracking analysis showing the size distribution of iPSC-MSC-SEV. C. Western blots showing sEV markers (ALIX, CD63, CD81) positive and Calnexin negative. D. Transmission electron microscopy of iPSC-MSC-SEV. Scale bar: 200 nm. E. Nanoparticle- MSC-SEV labeled by the luciferase detection system reached the ovary after 1.5 hours.

[0040] Figure 2 The following diagram illustrates the improvement of ovarian involution and abnormal estrous cycles in the Cy-DOR model using msC-sev: A. Experimental timeline and procedures for Cy-DOR mouse induction, MSC-sEV treatment, and sample collection. B. Representative estrous cycles of mice in the control, Cy, and Cy.MSC.sEVs groups. C. Box plot showing the duration of each estrous phase. D. Representative images of ovarian HE staining. Scale bar, 200 μm. E. Quantification of primitive and atretic follicles. F. Total number of follicles and corpus luteum quantification. All experiments were repeated at least three times. Data are presented as mean ± scanning electron microscopy. Statistical analysis was performed using one-way ANOVA (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0041] Figure 3 The following images illustrate how MSC-sev improved oocyte count and fertility: A. Representative micrographs of oocytes from each group. Black arrows indicate oocyte abnormalities. B. Quantitative analysis of the total number of ovulated oocytes and the ratio of abnormal oocytes to total abnormal oocytes per mouse. C. Representative images of embryo implantation sites in each group. Mice were sacrificed 9.5–10.5 days after vaginal plug testing. Red arrows indicate embryos implanted in the uterus. D. Quantitative analysis of the number of embryos implanted per mouse. E. Representative images of litter size in the initial mating round in each group. F. Quantitative analysis of fertility parameters for the first litter. Total body size and weight. G. Representative images of litters from the second mating round in each group. H. Size of pups from the second mating round, measured 4 weeks after the first round of birth. All experiments were repeated at least 3 times.

[0042] Figure 4The following images illustrate how MSC-sEVs alleviate ovarian defects and gonadal toxicity: A. Immunohistochemical images of ovaries stained with AMH and DDX4 antibodies. Scale bars: 50 μm and 20 μm. B. Representative Western blot and quantitative analysis of AMH expression. GAPDH was used as a loading control. C. Quantitative analysis of AMH and DDX4 expression levels. D. Immunofluorescence images of ovaries stained with lysed caspase-3 antibody. Scale bars: 50 μm: 50 μm. E. Quantification of the apoptosis index, expressed as the ratio of lysed caspase-3 fluorescence to total ovarian fluorescence. F. Representative Western blots of caspase-3 and lysed caspase-3 expression. G. Quantification of the ratio of lysed caspase-3 to total caspase-3. H. Serum estrogen and FSH levels detected by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). FSH: Follicle-stimulating hormone.

[0043] Figure 5 The following images illustrate the enhancement of learning and memory abilities of the Cy-DOR model by MSC-sEVs: A. Representative images of the behavior of each group in the Barnes maze. B. Quantitative analysis of escape latency. C. Quantitative analysis of the number of safe holes explored, the latency to reach a safe hole, the distance traveled before reaching a safe hole, and the major error. The "major error" refers to the mouse's first entry into a hole other than a safe hole. D. Representative images of the behavior of each group of mice in the elevated maze. E. Quantitative analysis of the number of mice entering open and closed arms. F. Quantitative analysis of the distance traveled in open and closed arms, the time spent in open and closed arms, and the total distance traveled. G. Representative Western blot images of NeuN, PSD95, cleaved PARP1, γ-H2AX, XRCC1, and GAPDH. H. Quantitative analysis of NeuN, PSD95, cleaved PARP1, γ-H2AX, and XRCC1.

[0044] Figure 6The following images illustrate how restoring NAD+ levels and increasing DNA repair are dynamic processes in MSC-sEVs that improve the Cy-DOR model: A. Levels of NAD+, NADH, total NAD+ plus NADH, and the NAD+ / NADH ratio in the short-term Cy-DOR model. BC. Representative Western blot and quantitative analysis of cleaved PARP1 and PAR expression with GAPDH as a loading control (short-term Cy-DOR). D. Levels of NAD+, NADH, total NAD+ plus NADH, and the NAD+ / NADH ratio in the long-term Cy-DOR model. EG. Representative Western blot and quantitative analysis of PAR, γ-H2AX, and XRCC1 expression with GAPDH as a loading control (long-term Cy-DOR). H. Immunofluorescence image of ovaries stained with BRCA2 antibody. Zoom bar: 40 μm. I. Immunofluorescence image of ovaries stained with γ-H2AX antibody. Scale bar: 200 μm. J. Quantitative analysis of BRCA2 and γ-H2AX fluorescence intensity. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0047] Example 1: Preparation of MSC-SEV

[0048] MSCs were generated from human induced pluripotent stem cells (iPSCs) (urine cell-derived iPSCs (UC-IPSCs) were donated by the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Guangzhou, Guangdong, China)). The specific method is described in CN201610107276.9. Cells were thawed from the working cell bank, seeded in culture dishes, and cultured in intact cell culture medium (CCM) for 2-3 days. When the cells reached approximately 80% confluence, they were expanded to a 5-layer cell factory. After washing the cells with phosphate-buffered saline (PBS), the CCM was replaced with chemically defined protein-free (CDPF) medium. After 6 hours, fresh CDPF medium was added, and approximately 600 ml of supernatant was collected and centrifuged at 2,000 g for 20 minutes to remove cell debris.

[0049] Next, to load luciferase into MSC-derived exosomes, iPSC-MSCs were infected with a recombinant lentivirus carrying the luciferase gene. Cells were cultured at 3 × 10⁶ cells / year. 4 Cells were seeded at a density of 1 / ml in 12-well plates. After 6 hours of infection, the medium was replaced with fresh medium. After 72 hours, the cells were selected using puromycin to obtain iPSC-MSCs that stably expressed luciferase, and then cryopreserved to form a working cell bank.

[0050] Subsequently, MSC-derived small exosomes (MSC-sEVs) were purified by anion exchange chromatography. Figure 1 A) Using an anion exchange chromatography kit provided by Mylab, each Econo-Pac column containing anion exchange resin was pre-equilibrated with equilibration buffer. MSC supernatant was loaded onto the column, eluted in eight fractions with elution buffer, and the concentration of sEVs in each fraction was estimated using the Bradford protein quantification method and CD63 enzyme-linked immunosorbent assay (ELISA). Fractions with peak sEV concentrations were collected, dialyzed against PBS at 4°C, and purified using Pierce chromatography. TM The protein was further concentrated using a 30 kDa protein concentrator (Thermo Fisher Scientific). The final concentration of MSC-sEVs was determined using the Bradford protein quantification method and nanoparticle tracking analysis (Nanosight NS300, Malvern).

[0051] Through nanoparticle tracking analysis, we determined their size distribution, ranging from 50 to 150 nm, with a peak at approximately 100 nm. Figure 1 B). Using transmission electron microscopy (TEM), we determined the morphology of sEVs as lipid bilayers and nanoscale particles. Figure 1 C). Strong fluorescence was detected in the ovary within 1.5 hours, indicating that sEVs can reach the ovary ( Figure 1 D). Western blot results showed high levels of positive markers CD9, CD63, CD81, and ALIX, and weak calnexin expression in MSC-sEVs, confirming that they are typical sEVs. Overall, the isolated MSC-sEVs showed a single peak around 100 nm, with other peaks being insignificant. All sEV markers were positive, and all negative markers were weakly positive, indicating that the isolated MSC-sEVs were homogeneous, with good quality variation control, and exhibited typical sEV characteristics.

[0052] Example 2: MSC-sev improves estrous cycle and follicular development in Cy-DOR mouse model

[0053] This invention selected 7-8 week old C57BL / 6J mice to establish a chemotherapy-associated degenerative retinopathy (DOR) model. We first evaluated the role of MSC-sEVs in ovarian function within the DOR model. All mice were randomly assigned to a control group (control group), a DOR model group (Cy group), and a DOR model treatment group (Cy.MSC.sEVs). Mice received tail vein injections of 2 × 10⁻⁶ MSC-sEVs from day 1 to day 6. 10 150 μL of either saline or physiological saline. Starting from day 7, collect vaginal smears from mice and observe the estrous cycle over a period of 14 days. On day 21, euthanize the mice and analyze ovarian markers and fertility index. Experimental protocol as follows: Figure 2 As shown in Figure a. The estrous cycle was assessed based on cell types observed in vaginal smears. Similar to the menstrual cycle in women, a normal estrous cycle is one of the main findings of normal ovarian function in female mice. The vertical axis represents proestrus (P), estrus (E), metestrus (M), and diaestrus (D), and the horizontal axis represents the number of days. The control group had a regular estrous cycle, with each cycle lasting 4–5 days. The distribution of mice in each stage corresponded well to the typical duration of each stage. Figure 2 B). Compared with the Cy group, MSC-sEVs treatment increased the average length of phase E by 1.12 days and decreased the duration of phase M / D by 1.88 days. Figure 2 C). These results indicate that msc-sev can restore the disrupted estrous cycle of Cy and increase the proportion of mice in estrus.

[0054] Hematoxylin and eosin (HE) staining revealed significant signs of recovery in follicle distribution at all stages after MSC-sEVs treatment. Figure 2 D). The number of primordial follicles increased significantly by approximately 82.6%, and the total number of follicles increased by approximately 47.8%. Furthermore, the number of atretic follicles decreased by approximately 38.1%. Figure 2 (EF, P<0.0001). Notably, there was no significant difference in the number of corpora lutea among the groups. The improvement in the number of primordial, anterior sinus, and secondary stage follicles, as well as the reduction in atretic follicles, highlights the potential of MSC-sEVs in promoting the early stages of follicle formation.

[0055] Example 3: MSC-sEVs can improve oocyte count and fertility outcomes.

[0056] To investigate the role of MSC-sEVs in DOR model oocytes, we further analyzed the number and quality of oocytes induced by mouse superovulation. We found that compared with the Cy group (P<0.0001), the MSC-sEVs group showed an approximately 86.9% increase in oocyte number and a significantly reduced 40.8% decrease in abnormal oocyte number. Figure 3AB, P<0.01). Since preserving ovarian reserve does not necessarily mean producing better quality oocytes, we conducted a breeding experiment in mice. Male and female mice were allowed to mate freely, and then the embryos implanted in the uterus were analyzed. MSC-sEVs treatment increased the number of implants from an average of 5 induced by Cy to an average of 7 ( ). Figure 3 CD, P<0.01). Subsequently, the number and weight of offspring were recorded in two consecutive breeding cycles to assess the fertility of the mice. In both breeding cycles, the number of live-born pups in the MSC-sev treated Cy mice was significantly higher than that in the Cy group, approximately 75% and 64.2% (P<0.01). Figure 3 EH, P<0.01). Notably, there was no significant difference in body weight among mice born after MSC-sEV treatment. There was also no significant difference in body weight between mice born after MSC-sEV treatment and the normal mouse group. Our results indicate that MSC-sEVs can restore oocyte quality and reproductive potential in Cy-DOR mice.

[0057] Example 4: MSC-sEVs can enhance ovarian function and serum hormone levels

[0058] We assessed the levels of anti-Müller hormone (AMH) and dead-box helicase 4 (DDX4), both markers associated with ovarian reserve. Immunofluorescence imaging showed that MSC-sev treatment increased AMH levels compared to the Cy group (P<0.01). Simultaneously, DDX4 expression was also increased in the MSC-sev group. Figure 4 Similarly, we observed a significant decrease in the expression ratio of caspase-3 cleavage to total caspase-3 (AC). Figure 4 D, F, G, P < 0.0001. Compared with the control group, the apoptosis index in the cyclophosphamide group increased to 12.4%, while the apoptosis index in the MSC-SEV treatment group decreased to 4.2%. Figure 4 E, P<0.0001). Further evaluation of the effects of MSC-sEVs on serum hormones showed that, compared with the Cy treatment group, the MSC-sEVs group had significantly higher serum E2 levels and significantly lower FSH levels (Fig. 4H, P<0.05).

[0059] Example 5: MSC-sEVs can improve behavioral and cognitive functions in Cy-DOR models.

[0060] To investigate whether mesenchymal stem cells (sEVs) can repair ovarian and brain function, we conducted a series of neurobiological experiments to evaluate the effects of sEVs on learning and memory.

[0061] In the Barnes maze experiment, the latency period for escaping to the safe hole was recorded daily. Figure 5 A). Data showed no significant differences between the Cy group and the MSC-sEVs group on days 1, 2, and 4. However, the latency period in the MSC-sEVs group was significantly shortened on days 3 and 5, reaching 36 seconds on day 5. Figure 5 B). On the test day, the number of safety hole explorations increased to 3 in the MSC-sEVs group, significantly higher than in the Cy group (P<0.05). Furthermore, the time to first reach the safety hole was significantly reduced to 9 seconds, and the distance to first reach the safety hole was reduced to 931.9 mm (P<0.05). The number of errors also decreased significantly (P<0.01), but the total travel distance was not different between the groups. Figure 5 C). In the elevated maze test measuring anxiety and fear response, MSC-sEV treatment significantly increased the number of entries into the open arm (P<0.05), but no significant improvement was observed in other parameters. Figure 5 These results indicate that learning and memory abilities were significantly enhanced after MSC-sEV treatment. Mesenchymal stem cell-sEV treatment significantly increased the expression of NeuN and PSD95 in the mouse brain. Figure 5 GH (P<0.05) decreased the expression of cleavage-PARP1 (P<0.0001) and γ-H2AX (P<0.01), and enhanced the expression of XRCC1 (P<0.05). Figure 5 I, P<0.01).

[0062] Example 6: MSC-sEVs alleviate Cy-DOR by maintaining NAD+ levels and DNA repair

[0063] Within two days of treatment with mesenchymal stem cells (sEVs), we observed a significant decrease in the elevation of lysis-PARP1 and PAR levels. Figure 6 BC, P<0.01). However, this early treatment did not significantly alter NAD+ levels, increase NADH, or affect the NAD+ / NADH ratio. Figure 6 A). NADH is the reduced form of NAD+, and the NAD+ / NADH ratio is an important component of cellular redox status, reflecting cellular metabolism. Notably, after 21 days of treatment with mesenchymal stem cells (SEVs), NAD+ content (P<0.0001), the NAD+ / NADH ratio (P<0.01), and the total amount of NAD+ plus NADH all significantly recovered, while NADH levels significantly decreased (P<0.05). Figure 6 D). Furthermore, the PAR initially reduced due to Cy treatment increased after MSC-sEVs treatment, showing an inverse trend compared to two days prior. Figure 6E, P<0.001). Furthermore, the DNA damage marker γ-H2AX was significantly reduced after Cy treatment but increased after MSC-sEVs treatment (E, P<0.001). Figure 6 F, I, J, P < 0.001. The levels of XRCC1 and BRCA2, which are involved in DNA damage repair and respond to PARP1, also increased after MSC-sEVs treatment. Figure 6 G, H, J, P<0.05). These findings indicate that mesenchymal stem cells (sEVs) can not only alleviate early DNA damage and apoptosis, but also restore long-term redox balance and DNA repair, highlighting their role in enhancing cellular metabolic health and DNA repair capabilities.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of MSC-sEVs for the preparation of a medicament for the treatment of reduced ovarian reserve, characterized in that, The MSC-sEVs are derived from human urine cell-induced MSCs, and the treatment of reduced ovarian reserve includes the following symptoms while improving or enhancing: 1) improving the estrous cycle and follicular development; 2) improving the number of oocytes and reproductive outcomes; 3) enhancing ovarian function and serum hormone levels.

2. A method for improving follicular development for non-disease therapeutic purposes, characterized in that, Improving follicular development using MSC-sEVs or drugs prepared based on MSC-sEVs, the MSC-sEVs being derived from human urine cell-induced MSCs.

3. A method of improving the number of ovulations for non-disease treatment purposes, characterized in that, Improving the number of ovulations in animals using MSC-sEVs or drugs prepared based on MSC-sEVs, the MSC-sEVs being derived from human urine cell-induced MSCs.

4. A method for improving the number of offspring for non-disease treatment purposes, characterized in that, Improving the number of reproductions in animals using MSC-sEVs or drugs prepared based on MSC-sEVs, the MSC-sEVs being derived from human urine cell-induced MSCs.

5. Use of the method of any one of claims 2-4 in scientific research experiments or animal breeding production.

Images