Empowered mesenchymal stem cells, empowered mesenchymal stem cell compositions, and uses thereof in the field of regenerative medicine

Abstract

The application belongs to the technical field of biomedicine, and specifically discloses a kind of enabled mesenchymal stem cells, enabled mesenchymal stem cell composition and its application in preparation treatment organ dysfunction drug, and the enabled mesenchymal stem cells are obtained by treating human mesenchymal stem cells with a kind of halogenated salicylic alcohol HEP14, and the enabled mesenchymal stem cell composition includes the enabled mesenchymal stem cells and HEP14 / PLGA microspheres with high HEP14 load and long-acting HEP14 sustained release prepared by using polylactic acid-hydroxyacetic acid copolymer (PLGA) as a carrier.The enabled mesenchymal stem cells of the application have the effects of strengthening mesenchymal stem cell survival viability and stemness, resisting tissue fibrosis, promoting tissue angiogenesis, promoting tissue regeneration and functional recovery;the combined application of the enabled mesenchymal stem cells and HEP14 / PLGA microspheres strengthens the retention and transdifferentiation of the enabled mesenchymal stem cells in the tissue, so the function of the enabled mesenchymal stem cells and the application in the field of regenerative medicine are further strengthened.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an energized mesenchymal stem cell, an energized mesenchymal stem cell composition, and its application in the preparation of drugs for treating ovarian insufficiency or other organ dysfunction. Background Technology

[0002] Ovarian insufficiency is caused by impaired ovarian follicle development and dysfunction due to a reduction or depletion of the ovarian follicle pool. It includes premature ovarian insufficiency (POI) and age-related ovarian aging. POI refers to the failure of ovarian function in women of reproductive age due to various causes, primarily chemotherapy and / or radiotherapy leading to ovarian damage; that is, amenorrhea occurs before the age of 40, and it is a major cause of female infertility. Ovarian aging is a natural process, where normal ovarian function gradually declines with age until it is exhausted. Ovarian insufficiency leads to menopausal syndrome and related degenerative diseases, severely damaging women's health. Currently, there are no effective treatments for ovarian insufficiency, either domestically or internationally. The most commonly used treatment, hormone replacement therapy, only alleviates the symptoms of low estrogen levels and has significant side effects, without any substantial therapeutic effect on improving ovarian reproductive function. Other available treatments, such as cryopreservation and transplantation of ovarian tissue, oocytes, or embryos, have lower efficacy and suffer from a shortage of ovarian tissue resources. Therefore, there is an urgent need in this field to find / explore more effective treatments for ovarian insufficiency, which has consistently been one of the most important research and development topics internationally.

[0003] The application of stem cells, especially various types of mesenchymal stem cells (MSCs), in regenerative medicine, particularly in reproductive medicine, is currently a hot research topic. Treatment strategies based on MSCs and their exosomes are widely recognized as the most promising means of treating ovarian insufficiency. By promoting ovarian tissue regeneration and follicle development, ovarian reproductive and fertility functions can be improved, potentially curing or improving ovarian insufficiency. Compared to MSCs from other sources, adipose-derived stem cells (ADSCs) have many significant advantages and characteristics, including ease of collection, minimal patient trauma, abundant adipose tissue, rapid in vitro expansion, and high biosafety, making autologous stem cell transplantation possible. These have made them one of the most promising stem cell sources and have attracted increasing attention in recent years. Animal transplantation experiments and a recent preliminary clinical trial indicate that ADSCs are effective in treating premature ovarian failure, but the therapeutic effect is insufficient to meet the goal of clinical translation for treatment. The main limitations of using ADSCs or other mesenchymal stem cells to treat ovarian or other tissue and organ dysfunction lie in (ovarian) tissue fibrosis, ischemia at the injection site, and differentiation or aging of the "transplanted" stem cells. To address these obstacles, improved stem cell-based treatment protocols have been under development, including pretreatment of stem cells with cytokines and combining stem cells with collagen scaffolds to enhance therapeutic efficacy. Unfortunately, to date, reported protocols have not made breakthrough progress in promoting the recovery of ovarian and / or other tissue and organ function or / and elucidating the therapeutic mechanisms. To address this clinical challenge, basic research and clinical trials surrounding the application of stem cells in regenerative medicine are ongoing.

[0004] HEP14 is a 5β-O-angeloyl-20-deoxyeuphorbia alcohol compound extracted from the whole herb of Euphorbia pep / us Linn., and is a novel activator of the protein kinase C (PKC) pathway. Currently, besides its reported role in regulating lysosomal formation, other pathways of action for HEP14 have not been reported. In exploring the therapeutic effects and potential mechanisms of HEP14, to overcome its poor water solubility and achieve sustained and stable release during in vivo circulation, we used poly(lactic-co-glycolic acid) (PLGA) as a drug carrier to prepare HEP14-loaded PLGA microspheres, termed HEP14 / PLGA microspheres. PLGA is a biodegradable copolymer composed of polylactic acid and polyethylene glycol acid, widely used as a carrier for delivering therapeutic drugs due to its safety, manageable biodegradability, and excellent drug loading performance. The degradation time of PLGA can be altered by adjusting the monomer ratio of lactic acid and glycolic acid, a characteristic that makes it particularly important in the biomedical field.

[0005] PLGA is a drug carrier approved by the U.S. Food and Drug Administration (FDA). Summary of the Invention

[0006] In response to the lack of breakthroughs in elucidating the mechanisms by which existing mesenchymal stem cell (MSC) therapies, whether used alone or in combination, promote ovarian function and / or the recovery of other tissue and organ functions, the MSCs of this invention exhibit significantly enhanced viability and stemness. They possess anti-fibrotic effects on tissues such as ovarian, liver, lung, brain, heart, muscle, bone, kidney, and skin, and promote tissue angiogenesis, thereby promoting tissue regeneration and functional recovery. They are effective in treating organ dysfunction in the ovaries, liver, lungs, brain, heart, muscles, bones, kidneys, and skin. The combined application of MSCs and HEP14 / PLGA microspheres further enhances the survival and transdifferentiation of MSCs within organ tissues. Therefore, the combined application of this composition further strengthens the anti-fibrotic and angiogenesis-promoting effects of MSCs, further enhancing their role in promoting tissue regeneration and functional recovery, as well as their therapeutic effect on organ dysfunction.

[0007] Especially in the ovary, energized mesenchymal stem cells (MSCs) exhibit significantly enhanced viability and stemness, demonstrating anti-ovarian fibrosis, promotion of ovarian angiogenesis, promotion of ovarian regeneration, follicular development, and improvement of ovarian endocrine function and / or fertility (including improvement of ovarian endocrine function and fertility in women of reproductive age, as well as improvement of ovarian endocrine function in older women). The combined application of energized MSCs and HEP14 / PLGA microspheres further enhances the survival of energized MSCs in ovarian tissue and their transdifferentiation into follicular granulosa cells and theca cells. Therefore, the combined application of this composition further strengthens the anti-ovarian fibrosis, promotion of ovarian angiogenesis and ovarian regeneration, follicular development, and improvement of ovarian endocrine function and / or fertility of energized MSCs.

[0008] The present invention solves the technical problem by adopting the following technical solution:

[0009] This invention provides an energy-enhancing mesenchymal stem cell, wherein the energy-enhancing mesenchymal stem cell is obtained by treating human mesenchymal stem cells with HEP14, wherein HEP14 is a compound with the molecular structure shown below: 5β-O-angeloyl-20-deoxygalactosyl alcohol.

[0010]

[0011] Preferably, the treatment of human mesenchymal stem cells with HEP14 includes the following steps:

[0012] (1) Cultivating and expanding human mesenchymal stem cells;

[0013] (2) Add 1.25-20 μM HEP14 to the culture medium and treat human mesenchymal stem cells for 12-48 hours;

[0014] (3) Human mesenchymal stem cells were digested and prepared into a single-cell suspension;

[0015] (4) The human mesenchymal single cells were eluted with physiological saline to obtain the energized mesenchymal stem cells suspended in physiological saline.

[0016] More preferably, step (2) of treating human mesenchymal stem cells with HEP14 involves adding 2.5 μM HEP14 to the culture medium and treating the human mesenchymal stem cells for 32 hours.

[0017] Preferably, the human mesenchymal stem cell source includes mesenchymal stem cells from different human tissues such as bone marrow, adipose tissue, placenta, umbilical cord, skin, amnion, and dental pulp.

[0018] More preferably, the human mesenchymal stem cells are mesenchymal stem cells derived from adipose tissue.

[0019] Another aspect of the present invention provides a composition for empowering mesenchymal stem cells, the composition comprising:

[0020] Empowered mesenchymal stem cells, wherein the empowered mesenchymal stem cells are obtained by treating human mesenchymal stem cells with HEP14.

[0021] HEP14 / PLGA microspheres, wherein the HEP14 / PLGA microspheres are prepared using polylactic acid-glycolic acid copolymer (PLGA) as a carrier, and are HEP14 / PLGA microspheres with high HEP14 loading and long-term sustained release of HEP14.

[0022] Preferably, the treatment of human mesenchymal stem cells with HEP14 includes the following steps:

[0023] (1) Cultivating and expanding human mesenchymal stem cells;

[0024] (2) Add 1.25-20 μM HEP14 to the culture medium and treat human mesenchymal stem cells for 12-48 hours;

[0025] (3) Human mesenchymal stem cells were digested and prepared into a single-cell suspension;

[0026] (4) The human mesenchymal single cells were eluted with physiological saline to obtain the energized mesenchymal stem cells suspended in physiological saline.

[0027] More preferably, step (2) of treating human mesenchymal stem cells with HEP14 involves adding 2.5 μM HEP14 to the culture medium and treating the human mesenchymal stem cells for 32 hours.

[0028] Preferably, the HEP14 / PLGA microspheres with high HEP14 loading and long-lasting HEP14 sustained release are prepared by the following method:

[0029] In a dark environment, polylactic acid-glycolic acid copolymer (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5–10:1, and stirred with a magnetic stirrer at a speed of 400–500 rpm. Then, the resulting solution was added to an aqueous solution containing 1–2% (w / v) polyvinyl alcohol, and stirred with a magnetic stirrer at a speed of 300–500 rpm at 25°C to allow the dichloromethane to evaporate for at least 12 hours. The microspheres were then collected by centrifugation and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol. Finally, the microspheres were placed in a freeze dryer for freeze drying to obtain HEP14 / PLGA microspheres.

[0030] More preferably, the polylactic acid-hydroxyacetic acid copolymer and HE P14 are dissolved in dichloromethane at a mass ratio of 5:1.

[0031] Preferably, the human mesenchymal stem cell source includes mesenchymal stem cells from different human tissues such as bone marrow, adipose tissue, placenta, umbilical cord, skin, amnion, and dental pulp.

[0032] More preferably, the human mesenchymal stem cells are mesenchymal stem cells derived from adipose tissue.

[0033] In another aspect, the present invention provides the application of the aforementioned empowered mesenchymal stem cells in the preparation of drugs for the treatment of tissue fibrosis.

[0034] In another aspect, the present invention provides the application of the aforementioned empowered mesenchymal stem cells in the preparation of drugs that promote tissue angiogenesis.

[0035] In another aspect, the present invention provides the application of the aforementioned empowered mesenchymal stem cells in the preparation of drugs that promote tissue regeneration and functional recovery.

[0036] Preferably, the tissue is one of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, and skin tissue.

[0037] In another aspect, the present invention provides the application of the aforementioned empowered mesenchymal stem cells in the preparation of drugs for treating organ dysfunction.

[0038] Preferably, the organ dysfunction is caused by damage to the organ's regenerative function or aging, and the organ is one of the following: ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

[0039] More preferably, the organ is an ovary.

[0040] Preferably, the ovary of the recipient is locally injected with a saline solution containing the said energized mesenchymal stem cells, including any of the following applications:

[0041] (1) Application in the preparation of drugs for treating premature ovarian failure in women of childbearing age;

[0042] (2) Application in the preparation of drugs for treating ovarian dysfunction and its complications in elderly women;

[0043] (3) Application in the preparation of drugs to restore ovarian endocrine and / or reproductive function.

[0044] In another aspect, the present invention provides the use of the aforementioned mesenchymal stem cell-enhancing composition in the preparation of a medicament for the treatment of tissue fibrosis.

[0045] In another aspect, the present invention provides the use of the aforementioned mesenchymal stem cell-enhancing composition in the preparation of a medicament that promotes tissue angiogenesis.

[0046] In another aspect, the present invention provides the use of the aforementioned mesenchymal stem cell-enhancing composition in the preparation of a medicament that promotes tissue regeneration and functional recovery.

[0047] Preferably, the tissue is one of ovarian tissue, liver tissue, lung tissue, brain tissue, heart tissue, muscle tissue, bone tissue, kidney tissue, and skin tissue.

[0048] In another aspect, the present invention provides the use of the aforementioned empowered mesenchymal stem cell composition in the preparation of a medicament for treating organ dysfunction.

[0049] Preferably, the organ dysfunction is caused by damage to the organ's regenerative function or aging, and the organ is one of the following: ovary, liver, lung, brain, heart, muscle, bone, kidney, and skin.

[0050] More preferably, the organ is an ovary.

[0051] Preferably, the application includes a local injection of a saline solution containing the energized mesenchymal stem cells into the ovary of the recipient, combined with an injection of a saline solution containing the HE P14 / PLGA microspheres, comprising any of the following applications:

[0052] (1) Application in the preparation of drugs for treating premature ovarian failure in women of childbearing age;

[0053] (2) Application in the preparation of drugs for treating ovarian dysfunction and its complications in elderly women;

[0054] (3) Application in the preparation of drugs to restore ovarian endocrine and / or reproductive function.

[0055] Fibrosis and ischemia in ovarian tissue or other organs are significant causes of transplanted stem cell loss and cell therapy failure. The number of transplanted stem cells and the cytokines they secrete significantly affect treatment efficacy. Local injection of saline solution containing the aforementioned energized mesenchymal stem cells into the ovary, combined with injection of saline solution containing the aforementioned HEP14 / PLGA microspheres into the recipient, offers the following advantages: First, using different routes of administration helps provide more receptive space for locally injected energized mesenchymal stem cells. Second, this route of administration facilitates more direct, timely, and effective interactions between energized mesenchymal stem cells, follicular cells, cytokines, and the extracellular matrix. Therefore, it promotes the survival and viability of transplanted energized mesenchymal stem cells within the ovary and / or the corresponding treated organ tissue. Third, this route of administration allows for multiple doses, and the long-acting sustained-release properties of HEP14 / PLGA microspheres maintain a stable and continuous HEP14 blood concentration, ultimately improving the therapeutic effect of the energized mesenchymal stem cell composition in treating ovarian insufficiency and / or other organ tissue dysfunction, especially improving the treatment efficacy for severe ovarian insufficiency.

[0056] More preferably, the injection route for the physiological saline solution containing the HE P14 / P LGA microspheres includes any one of intraperitoneal injection, subcutaneous injection, or intramuscular injection.

[0057] The treatment of ovarian insufficiency includes promoting the regeneration and functional recovery of damaged ovarian tissue, specifically manifested in at least one of the following: (1) ovarian follicle regeneration and development, with an increase in the number of ovarian follicles at each stage; (2) increased blood estrogen E2 and AMH levels and decreased FSH levels, resulting in improved ovarian endocrine function; (3) significantly improved pregnancy rate and fertility rate.

[0058] The ovarian follicle regeneration and development includes the development of ovarian mesenchymal tissue and follicles at various stages and / or the development of mature follicles.

[0059] This invention has revealed that HEP14 activates the PKC-ERK1 / 2 signaling pathway, stimulating various secretory factors, particularly promoting the secretion of STC1, MMP1, and PDGFD. These factors work synergistically, with MMP1 and PDGFD enhancing the anti-fibrotic and angiogenesis effects of transplanted energized mesenchymal stem cells, thereby improving the organ tissue microenvironment. In the ovary, STC1 stimulates CYP1 9A1 secretion, synergistically stimulating the growth of ovarian follicular granulosa and foam cells, thus promoting follicular development and improving ovarian hormone secretion and endocrine function. In summary, these results demonstrate that the energized mesenchymal stem cells and their compositions of the present invention have excellent effects in promoting the regeneration and functional recovery of damaged organ tissues.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The empowered mesenchymal stem cells of this invention exhibit significantly enhanced viability and stemness, possessing anti-fibrotic effects on tissues such as ovarian, liver, lung, brain, heart, muscle, bone, kidney, and skin, as well as promoting tissue angiogenesis. They promote tissue regeneration and functional recovery, and are effective in treating organ dysfunction in the ovaries, liver, lungs, brain, heart, muscles, bones, kidneys, and skin. The combined application of the empowered mesenchymal stem cells and the HE P14 / PLA microsphere composition further enhances the survival and transdifferentiation of the empowered mesenchymal stem cells within organ tissues. Therefore, the combined application of this composition further strengthens the anti-fibrotic and angiogenesis-promoting effects of the empowered mesenchymal stem cells, further enhancing their role in promoting tissue regeneration and functional recovery, and in treating organ dysfunction.

[0062] Especially in the ovary, energized mesenchymal stem cells (MSCs) exhibit significantly enhanced viability and stemness, demonstrating anti-ovarian fibrosis, promotion of ovarian angiogenesis, promotion of ovarian regeneration, follicular development, and improvement of ovarian endocrine function and / or fertility (including improvement of ovarian endocrine function and fertility in women of reproductive age, as well as improvement of ovarian endocrine function in older women). The combined application of energized MSCs and the HE P14 / PLGA microsphere composition further enhances the survival of energized MSCs in ovarian tissue and their transdifferentiation into follicular granulosa cells and theca cells. Therefore, the combined application of this composition further strengthens the anti-ovarian fibrosis, promotion of ovarian angiogenesis and ovarian regeneration, follicular development, and improvement of ovarian endocrine function and / or fertility of energized MSCs. Attached Figure Description

[0063] Figure 1 H EP14 treatment enhanced the stemness, proliferation, and survival of hADSCs.

[0064] Figure 2 To analyze the gene expression profile of HEP14-induced hADSCs, differentially expressed genes were enriched in signaling pathways that regulate extracellular matrix (ECM) receptor interactions, cell fate determination, angiogenesis, ovarian steroid hormone production, and MAPK activation. These biological functions are crucial for organ tissue remodeling, regeneration, and functional recovery, especially ovarian tissue.

[0065] Figure 3 Morphological and property analysis of HE P14 / PLGA microspheres.

[0066] Figure 4To establish a mouse model of ovarian insufficiency induced by doxorubicin, the results showed that a single intraperitoneal injection of 12 mg / kg of doxorubicin could cause significant damage to the structure and function of the ovaries in mice.

[0067] Figure 5 For the treatment and restoration of premature ovarian failure in women of reproductive age, the combination of adipose-empowered stem cells and adipose-empowered stem cell compositions has shown that adipose-empowered stem cells alone or in combination with HEP14 / PLGA microspheres can significantly reduce ovarian tissue fibrosis, promote ovarian tissue angiogenesis, promote ovarian follicle development in PO1 mice, and restore reproductive and endocrine functions. The combined treatment of the compositions is superior to the therapeutic effect of adipose-empowered stem cells alone.

[0068] Figure 6 To treat ovarian dysfunction and its complications caused by adipose-derived stem cells (ADSCs), AADSCs alone or in combination with adipose-derived stem cells / PLGA microspheres have shown significant effects in reducing ovarian tissue fibrosis, promoting ovarian tissue angiogenesis, and promoting ovarian follicle development and regeneration in aged mice. The combined effect of the combination is significantly better than that of AADSCs alone.

[0069] Figure 7 To study the mechanism of empowering adipose-derived stem cells and the combination of empowering adipose-derived stem cells in treating premature ovarian failure in women of reproductive age, HE P14 can stimulate the secretion of a variety of secretory factors, including STC1, CYP19A1, MMP1 and PDGFD. These factors, through synergistic exocrine and endocrine mechanisms, resist ovarian tissue fibrosis, promote ovarian angiogenesis, improve the ovarian tissue microenvironment, stimulate the growth of ovarian follicle granulosa and foam cells, thereby promoting follicle development and improving the secretion of ovarian hormones. Detailed Implementation

[0070] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. 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.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] Example 1: Preparation and Identification of Empowered Mesenchymal Stem Cells

[0073] The preparation method of empowered mesenchymal stem cells includes the following steps:

[0074] (1) Cultivating and expanding human mesenchymal stem cells;

[0075] (2) Add HEP14 at a concentration of 1.25 to 20 μM to the culture medium and treat human mesenchymal stem cells for 12 to 48 hours.

[0076] (3) Human mesenchymal stem cells were digested and prepared into a single-cell suspension;

[0077] (4) The human mesenchymal cells are eluted with physiological saline to obtain the energized mesenchymal stem cells suspended in physiological saline.

[0078] HEP14 is extracted from the dried whole herb of Euphorbia pep / us Linn. (Takahisa Nakane). Arai, Kazuo Masuda, et al. Fern constituents: six new triterpenOidalcOhols from Adiantum capillus-veneris. Chem. Pharm. Bull. [J]. 1999, 47(4): 543-547.).

[0079] The specific methods for preparing and identifying empowered mesenchymal stem cells are as follows:

[0080] Under the supervision of the medical ethics committee, human adipocyte-derived stem cells (hADSCs) were isolated and extracted from 5-10 ml of fresh adipose tissue obtained from healthy donors. These hADSCs were then cultured and expanded using a serum-free culture system: Ultraculture (Lonza) + commercially available growth factors. The cells were cultured at 37°C in a CO2 incubator containing 5% CO2. After 24 hours, the culture medium was changed, suspended dead cells or cell debris were aspirated, and the cells were washed once with physiological saline, followed by a medium change. When the cells reached 90% confluence, passage and expansion were performed. P2 generation primary cells were cryopreserved as seed cells. In this specification, P2 generation refers to the generation in which primary cells, after two cycles of reaching 90% confluence in a culture flask and digestion, were re-seeded into the culture flask. hADSCs from passages 3 to 5 were used, with passage 4 being preferred. Morphologically, isolated primary hADSCs exhibit a fibroblast-like appearance. Figure 1 A). Flow cytometry analysis showed that over 95% of hADSCs were negative for CD31 and HLA-DR, but positive for CD73, CD90, CD105, and CD29. Figure 1B). Alizarin Red and Oil Red staining results showed that hADSCs have osteogenic ( Figure 1 C) and adipogenic differentiation capacity ( Figure 1 D). These results indicate that the isolated hADSCs are highly pure undifferentiated adipose-derived stem cells. hADSCs were treated with HEP14 at concentrations (1.25–20 μM) and in a time-dependent manner (12 h–48 h). After 32 hours of treatment with 2.5 μM HEP14, the viability of hADSCs ( Figure 1 E) and significantly improved proliferation capacity ( Figure 1 F). This proliferation is characterized by an increase in hADSCs in the S phase and a decrease in the G0 / G1 ratio as determined by flow cytometry. Figure 1 G and 1H). Real-time quantitative PCR results showed that, compared with the control group hADSCs (hADSCs not treated with HEP14, denoted as c-hADSCs), the expression of NANOG, a pluripotent stem cell marker, was significantly increased in HEP14-treated hADSCs, while the expression of NUCLEOSTEM1N, a mesenchymal stem cell proliferation marker protein, was slightly increased. Figure 1 Notably, the Ct values ​​of the real-time qPCR reaction showed that both HEP14-treated and control group hADSCs exhibited high levels of nucleostensin mRNA expression. Immunofluorescence staining and Western blot results further confirmed that HEP14 treatment significantly increased the expression of NANOG and nucleostensin in hADSCs. Figure 1 (J and 1K). These results indicate that HEP14 treatment enhances the stemness, proliferation, and survival of hADSCs. Therefore, we treated hADSCs with 2.5 μM HEP14 for 32 hours, then digested the HEP14-treated hADSCs with digestive enzymes to prepare a single-cell suspension. Finally, we eluted the single cells of the HEP14-treated hADSCs with physiological saline to obtain HEP14-treated hADSCs, namely empowered mesenchymal stem cells, also known as empowered adipose stem cells (referred to as h-hADSCs). The empowered mesenchymal stem cells were suspended in physiological saline for later use.

[0081] Example 2: HEP14-induced changes in the gene expression profile of hADSCs contribute to tissue remodeling and regeneration.

[0082] To gain a deeper understanding of the molecular biological characteristics of h-hADSCs, we performed RNA sequencing on h-hADSCs and c-hADSCs. The results showed that a total of 1003 differentially expressed genes (DEGs) were identified in h-hADSCs, including 573 upregulated genes and 430 downregulated genes. Figure 2A and 2B). Gene Ontology analysis showed that these DEGs were enriched in biological functional processes such as extracellular matrix tissue and tissue remodeling, angiogenesis, cell fate determination, ERK1 and ERK2 cascades, and response to estradiol. Figure 2 C), which is closely related to organ and tissue regeneration and functional recovery, particularly ovarian tissue. We reconstructed sub-heatmaps of key genes enriched in these biological processes ( Figure 2 D). Real-time quantitative PCR confirmed that the expression levels of these genes in h-hADSCs were significantly altered compared to c-hADSCs. Figure 2 (E and 2F). Among them, STC1 is the DEG most highly upregulated by HEP14. STC1 has been reported to be expressed in various tissues of mammals, including liver, kidney, and ovary, and can promote the growth and migration of mesenchymal stem cells. In ovarian tissue, it specifically promotes the production of granulosa cells and the expression of the CYP19A1 gene, the latter being a hallmark gene of ovarian granulosa cells and promoting estrogen production. MMP1 (Matrix metalloproteinase 1) can degrade type I, type II, and type III collagen in the extracellular matrix, promoting extracellular matrix remodeling, thereby leading to changes in the cellular microenvironment. Platelet-Derived Growth Factor D (PDGFD) is a potent angiogenic growth factor, which has been reported to stimulate angiogenesis by binding to its homologous receptor PDGFRβ (Platelet-Derived Growth Factor receptor β). Gene set enrichment analysis (GSEA) results indicate that HEP14 exerts its biological functions by regulating signaling pathways such as extracellular matrix (ECM) receptor interactions, cell fate determination, angiogenesis, ovarian steroid hormone production, and MAPK activity activation. Figure 2 These biological functions are crucial for organ tissue regeneration and functional recovery, particularly in ovarian tissue. In summary, RNA transcriptomics analysis indicates that HEP14-enabled hADSCs contribute to tissue remodeling, regeneration, and functional recovery.

[0083] Example 3: Preparation of HEP14 / PLGA microspheres

[0084] HEP14 / PLGA microspheres were prepared using a single-emulsion solvent evaporation technique. In the dark, poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5:1, and the solution was stirred at 400 rpm using a magnetic stirrer. Next, the resulting solution was added to an aqueous solution containing 1% (w / v) polyvinyl alcohol (PVA), and the dichloromethane was evaporated at 25°C by magnetic stirring (300 rpm) for at least 12 hours. The microspheres were then collected by centrifugation (2000 × g, 5 min) and repeatedly washed with distilled water to thoroughly remove residual PVA. Finally, the microspheres were freeze-dried in a freeze dryer for 24 hours. The resulting HEP14 / PLGA microspheres, formed by PLGA encapsulating HEP14, were obtained.

[0085] Example 4: Preparation of HEP14 / PLGA microspheres

[0086] HEP14 / PLGA microspheres were prepared using a single-emulsion solvent evaporation technique. In the dark, poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 10:1, and the mixture was stirred at 500 rpm using a magnetic stirrer. The resulting solution was then added to an aqueous solution containing 2% (w / v) polyvinyl alcohol (PVA), and the dichloromethane was evaporated at 25°C with magnetic stirring (500 rpm) for at least 12 hours. The microspheres were then collected by centrifugation (2000 × g, 5 min) and repeatedly washed with distilled water to thoroughly remove residual PVA. Finally, the microspheres were freeze-dried for 24 hours. The resulting HEP14 / PLGA microspheres, formed by PLGA encapsulating HEP14, were obtained.

[0087] Example 5: Identification of HEP14 / PLGA microspheres

[0088] For comparative analysis, we also prepared pure PLGA microspheres (HEP14-free PLGA microspheres) using the same method as in Example 3 as an experimental control group.

[0089] To perform detailed morphological and property analysis of the microspheres prepared in Examples 3 and 4, as well as the experimental controls, we used scanning electron microscopy to examine the samples. Simultaneously, we used ImageJ and Origin software for precise quantitative analysis of the microsphere particle size. Furthermore, we used an FTIR system to characterize the chemical structure of the samples. Detection was performed with a scanning range of 400 to 4000 cm⁻¹, a resolution of 4 cm⁻¹, and a scanning speed of 64 scans / min, yielding a total of 1867 data points.

[0090] To further investigate the crystal structure characteristics of the samples, we analyzed the microspheres using X-ray diffraction (XRD). The XRD patterns of the samples were collected using a Rigaku Smartlab X-ray diffractometer. During the experiment, we used a CuKα radiation source (45 kV, 120 mA) and set the 2θ angle range to 5°–100°. The obtained data were collected as transmittance values ​​(%) and processed and analyzed using Oringin software.

[0091] SEM analysis revealed that both PLGA and HEP14 / PLGA microspheres exhibited uniform size distribution, spherical shape, and smooth surface. Figure 3 (A and 3B). Due to the high loading of HEP14, the particle size of 5:1 HEP14 / PLGA microspheres (prepared in Example 3) (0.1688 μm ± 0.0685) and 10:1 HEP14 / PLGA microspheres (prepared in Example 4) (0.100 μm ± 0.0735) were significantly increased compared to pure PLGA microspheres (0.052 μm ± 0.028). To verify whether the HEP14 drug was successfully encapsulated within the PLGA, we analyzed the drug-loaded and unloaded microspheres. From the infrared spectra of the HEP14 / PLGA samples, we did not observe any vibrational peaks of HEP14 (…). Figure 3 C). This suggests that HEP14 may be completely encapsulated within the PLGA matrix, or that the vibrational peaks of the PLGA matrix are similar to those of HEP14, thus masking the HEP14 signal. X-ray diffraction analysis revealed multiple diffraction peaks in HEP14, indicating its inherent crystallinity. In HEP14 / PLGA, the diffraction peaks of HEP14 disappeared, suggesting that the crystallinity of HEP14 decreased during loading. Figure 3 D). This change allows HEP 14 to be gradually released from PLGA microspheres, making it easier for organisms to absorb and utilize it.

[0092] To determine the drug loading rate, encapsulation efficiency, and in vitro release of HE P14 / PLGA microspheres, we used a UV spectrophotometer to measure the drug loading and encapsulation efficiency of the HE P14 / PLGA microspheres and plotted a standard curve of OD value (Y) and concentration (c) for the HE P14 / PLGA microspheres. The detection wavelength was 201 nm, and the sample volume was 1 mL. Drug loading (%) and encapsulation efficiency (%) were calculated using the following formulas, where the theoretical drug loading of the microspheres refers to the initial amount of drug used to prepare the microspheres: Drug loading rate (%) = (weight of drug in microspheres) / (weight of microspheres) × 100%; Retention rate (%) = (actual drug loading in microspheres) / (theoretical drug loading in microspheres) × 100%. The in vitro release rate of HE P14 / PLGA / microspheres was measured at 201 nm using a UV spectrophotometer. HE P14 / PLGA microspheres were placed in PBS solution at pH 7.4 and incubated at 37°C and 100 rpm in a shaker. The microspheres were precipitated by centrifugation (5000×g, 3 min), and the supernatant was collected at 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days for analysis.

[0093] To investigate the drug loading, encapsulation efficiency, and release rate of HEP14, a standard curve was obtained at the maximum absorption peak at 201 nm: y = 0.0785c + 0.1555 (R² = 0.9966, n = 6). The linear correlation coefficient R² reached 0.99853, fully meeting the requirements for determining the drug loading and content of microspheres. The HEP14 / PLGA microspheres prepared in Example 3 had a drug loading and encapsulation efficiency of 3.10% and 18.04%, respectively. The HEP14 / PLGA microspheres prepared in Example 4 had a drug loading and encapsulation efficiency of 2.1% and 26.02%, respectively. The in vitro release kinetics of HEP14 / PLGA microspheres were determined by ultraviolet spectrophotometry, as shown in (…). Figure 3 As shown in E). Initially, rapid release occurred on day 1, followed by sustained and controlled release from day 1 to day 9. Subsequently, additional bursts of release were observed from day 9 to day 17, eventually leading to gradual release until equilibrium was reached on day 17. At the end of the study, the cumulative release of HEP14 / PLGA microspheres was approximately 9.73%.

[0094] In summary, the HEP14 / PLGA microspheres prepared in Examples 3 and 4 have the functions of high HEP14 loading rate, encapsulation efficiency and long-term HEP14 sustained release.

[0095] Example 6: Preparation of a composition for empowering mesenchymal stem cells

[0096] The energized mesenchymal stem cells prepared in Example 1 were combined with the HEP14 / PLGA microspheres prepared in Example 3 or Example 4 to obtain an energized mesenchymal stem cell composition.

[0097] Example 7: Establishment of a mouse model of doxorubicin-induced ovarian insufficiency (POI)

[0098] Doxorubicin (also known as doxorubicin) is a cycle-nonspecific first-line anticancer chemotherapy drug with strong antitumor effects. We established a POI mouse model induced by doxorubicin, characterized by a significant reduction in follicles at different developmental stages. In preliminary experiments, we examined the effects of three different concentrations of doxorubicin administered via a single intraperitoneal injection on ovarian function in 6-7 week old mice.

[0099] Female C57BL / 6N mice aged 6-7 weeks were weighed and randomly divided into two groups (n=18 per group). They were then injected intraperitoneally with different doses (8 mg / kg, 12 mg / kg, and 16 mg / kg) of doxorubicin (Sigma) (n=18 / group) or an equal volume of saline solution as a control. Mice were euthanized by cervical dislocation at 1, 2, and 4 weeks post-treatment to collect samples. Body weight, ovarian size, estrous cycle, serum hormone levels, follicle count and morphology were examined, and a POI model was assessed. Results showed that among the six mice injected intraperitoneally with 16 mg / kg doxorubicin, two mice experienced persistent weight loss and very poor health within 4 weeks and were therefore euthanized according to national regulations for the use of laboratory animals (data not presented). However, this did not occur in mice treated with 8 mg / kg or 12 mg / kg doxorubicin. Hematoxylin and eosin (H&E) staining of ovarian tissue showed that mice treated with 8 mg / kg doxorubicin exhibited minimal changes in ovarian size and follicular structure, while mice treated with 16 mg / kg doxorubicin showed irreversible ovarian atrophy and follicular atresia compared to mice treated with 12 mg / kg doxorubicin. Therefore, we chose to establish a mouse POI model using 12 mg / kg doxorubicin. Figure 4 A). At 1, 2, and 4 weeks after doxorubicin induction, tissue H&E staining results showed that the ovaries of control mice had normal structure, with multiple follicles and corpora lutea at each developmental stage, and all follicles were surrounded by flattened stromal cells. In contrast, the ovaries of doxorubicin-treated mice showed degenerative changes in follicles and mesenchyme, with up to 80% of follicles exhibiting atretic follicles. Figure 4 B). At weeks 1 and 2 of doxorubicin induction, the proportion of follicles in the growth phase to the total number of follicles in the damaged ovaries was slightly increased. By week 4, in stark contrast to the control ovaries, the total number of follicles in the ovaries of doxorubicin-treated mice was further significantly reduced. Figure 4 C). Immunofluorescence (IF) staining showed that the number of Ki67-positive and TUNEL-negative follicles in control ovaries was greater than that in doxorubicin-treated ovaries. Figure 4 These results indicate that doxorubicin treatment leads to a significant loss of ovarian follicular reserve.

[0100] Hormone analysis showed that, within 1-4 weeks after induction, mice treated with doxorubicin had a statistically significant increase in serum FSH levels compared to the control group. Figure 4 F), but E2 and Amh levels were significantly reduced ( Figure 4 G and 4H). Vaginal smear results showed that control group mice had a normal estrous cycle of 4-5 days, while mice treated with doxorubicin exhibited irregular estrous cycles, mainly ceasing during estrus (G and 4H). Figure 4 I). The results showed that a single intraperitoneal injection of 12 mg / kg doxorubicin could cause significant damage to the structure and function of the ovary in mice. Here, we successfully established an doxorubicin-induced POI mouse model (hereinafter referred to as POI mouse).

[0101] Example 8: Treatment of premature ovarian failure in women of reproductive age with adipose-empowered stem cells and a composition thereof.

[0102] To investigate the efficacy of adipose-empowered stem cells and a composition of adipose-empowered stem cells in treating premature ovarian failure in women of reproductive age, adipose-empowered stem cells prepared in Example 1 and a composition of adipose-empowered stem cells prepared in Example 6 were used in POI model mice for the treatment of premature ovarian failure. Specifically, the adipose-empowered stem cell composition used in this example was: adipose-empowered stem cells prepared in Example 1 + HEP14 / PLGA microspheres prepared in Example 3.

[0103] I. Treatment of POI model mice

[0104] Evaluation of treatment, sample collection, and fertility in POI model mice ( Figure 5 A). To track transplanted human adipose-derived stem cells (hADSCs) in vivo, 5 μM CellTracker was used. TM Adipose-derived stem cells (ADSCs-CM-DIL) were pre-labeled and energized with CM-DIL for 30 minutes (defined here as hADSCs-CM-DIL) and observed under a fluorescence microscope (Leica). After washing twice with saline, the labeled cells were maintained at 4°C. 7-8 week old female C57BL / 6N POI mice were used in the experiment and were divided into four groups (n=22 per group): normal control (CTL mice); POI mice were further divided into three groups, including POI mice, h-hADSCs mice, and h-hADSCs / HEP14 mice. Specific treatments are as follows:

[0105] Normal control mice: 15 μl of physiological saline was injected into the ovary (io) and 100 μl of physiological saline was injected into the peritoneum (ip).

[0106] POI mice: POI mice were injected intraovarianly (io) with 15 μl of physiological saline and intraperitoneally (ip) with 100 μl of physiological saline.

[0107] h-hADSCs mice: POI mice were injected intraovarianly with 15 μl of saline containing h-hADSCs-CM-DIL and intraperitoneally (ip) with 100 μl of saline; the h-hADSCs concentration was 5 × 10⁻⁶. 4 Cells / µL

[0108] h-hADSCs / HEP14 mice: POI mice were treated with ovarian injection of 15 μl of saline containing h-hADSCs-CM-DIL and intraperitoneal injection (ip) of 100 μl of saline containing HEP14 / PLGA microspheres; the density of h-hADSCs was 5 × 10⁻⁶. 4 The concentration of HEP14 / PLGA microspheres is 10 micrograms per microliter, with cells per microliter.

[0109] To shorten the operation time and avoid inter-group bias caused by prolonged cell viability, only the right ovary tissue was injected into all four groups of mice. Tissue and blood samples were collected from mice (n=6 per group) at 2 and 4 weeks post-treatment to assess changes in ovarian structure and function. Four weeks post-treatment, the mice (n=10 per group) were mated with confirmed fertile males at a 1:2 ratio for two weeks. Vaginal plugs were examined daily at 9:00 AM the day after mating for two weeks. Fetuses were delivered by cesarean section at 18-19 days of gestation. The number of pregnant mice, the total number of fetuses born, and the average litter size per mouse were recorded for each group to assess fertility.

[0110] II. Promotes the recovery of ovarian tissue structure and follicle regeneration in women with premature ovarian failure during reproductive years.

[0111] Histological and pathological examinations of the ovaries of four groups of mice (CTL-, POI-, h-hADSCs-, and h-hADSCs / HEP14 mice) were conducted at 2 and 4 weeks after treatment. Follicular proliferation, development, and number at each developmental stage were examined using methods. Results showed that, compared with POI mice, both h-hADSCs / HEP14 mice and h-hADSCs mice showed significant recovery in body weight and ovarian size. The recovery was better in h-hADSCs / HEP14 mice than in h-hADSCs mice. Figure 5 (A and 5B). Morphologically, almost all follicles in POI mice were atretic at 2 and 4 weeks post-treatment. Figure 5 B), while the number of healthy follicles at different developmental stages in the ovaries of h-hADSCs mice and h-hADSCs / HEP14 mice was significantly increased (B). Figure 5 C). Detailed follicle counting showed that the total number of ovarian follicles and primordial follicles in h-hADSCs / HEP14 mice was higher than that in h-hADSCs mice ( Figure 5 C). However, there was no statistically significant difference in the number of growing follicles and cavitary follicles between the two groups ( Figure 5 D and 5E). Immunofluorescence (IF) staining showed that the number of Ki67-positive and TUNEL-negative follicles in the control ovaries was higher in h-hADSCs / HEP14 mice than in POI and h-hADSCs mice. The number of follicles in h-hADSCs mice was between that in h-hADSCs / HEP14 mice and POI mice, and significantly higher than that in POI mice. Figure 5 (F-5G). Immunohistochemical staining showed that the expression of FSHR, ERβ, and AMH in the ovaries of h-hADSCs / HEP14 mice was significantly higher than that in h-hADSCs mice and POI mice. The levels in h-hADSCs mice were between those in h-hADSCs / HEP14 mice and POI mice. Figure 5 H and 5I).

[0112] These results indicate that the treatment with adipose-derived stem cells, either alone or in combination with the HE P14 / PLGA microsphere composition, significantly promotes follicle regeneration and development in PO1 mice, and the combined treatment is superior to the treatment effect of adipose-derived stem cells alone.

[0113] III. Promoting the recovery of ovarian endocrine function in women with premature ovarian failure during reproductive years

[0114] Further evaluation of estrous cycles, serum hormone levels, and fertility in the four groups of mice was conducted 4 weeks after treatment. Compared with the normal 4-5 day estrous cycle observed in the control group, 5 out of 6 h-hADSCs / HEP14 mice recovered to a normal estrous cycle, 4 out of 6 h-hADSCs mice recovered to a normal estrous cycle, while the estrous cycles of all PO1 mice were prolonged. Figure 5 J). Serum ovarian hormone detection results showed that, compared with POI mice, h-hADSCs / H EP14 mice and h-hADSCs mice exhibited higher levels of E2 (estradiol) and AMH (anti-Müllerian hormone). Figure 5 K-5L), but with lower FSH (follicle-stimulating hormone) levels. Figure 5 In the h-hADSCs / H EP14 mice, the levels of these hormones in the serum returned to near-normal levels.

[0115] Starting from week 4 post-treatment, the four groups of mice underwent mating experiments at a 1:2 ratio (male:female) for two weeks. To obtain reliable data for assessing fertility in each group, fetuses were removed by cesarean section at 18–19 days of gestation. The pregnancy rate of POI mice was (2 / 10), significantly lower than that of h-hADSCs mice (7 / 10), h-hADSCs / H EP14 mice (10 / 10), and normal control mice (10 / 10). The average number of litters per POI mouse (0 / 2) and the number of healthy litters per POI mouse (1 / 0) were significantly lower than those of empowered h-hADSCs mice (2.4 / 2.0), h-hADSCs / H EP14 mice (4.6 / 3.5), and control mice (5.7 / 5.5). Figure 5 N and 5O). There was one identical twin in the h-hADSCs / H EP14 group ( Figure 5 These results indicate that h-hADSCs / HEP14 mice and h-hADSCs mice have better fertility than POI mice. Both h-hADSCs-enhanced treatment alone and the combination (h-hADSCs-enhanced with HEP14 / PLGA microspheres) promoted ovarian follicle regeneration and restored reproductive endocrine function in POI mice. h-hADSCs / HEP14 treatment was more effective than h-hADSCs-enhanced alone.

[0116] Example 9: Treatment of ovarian dysfunction and its complications in the elderly with adipose-derived stem cells and a composition of adipose-derived stem cells.

[0117] To investigate the efficacy of adipose-derived stem cell (ADSC) empowerment and its combination in treating premature ovarian failure in elderly patients, 73-74 week old female C57BL / 6N mice were used as animal models and randomly divided into three groups: a control group treated with saline (CTL mice), a group treated with adipose-derived stem cells prepared in Example 1 alone (h-hADSCs mice), and a group treated with adipose-derived stem cell combination prepared in Example 6 (h-hADSCs / HEP14 mice). Specifically, the adipose-derived stem cell combination used in this example consisted of adipose-derived stem cells prepared in Example 1 and HEP14 / PLGA microspheres prepared in Example 3.

[0118] The specific grouping and processing are as follows:

[0119] Normal aged control mice (CTL mice): 15 μl of physiological saline was injected into the ovary (io) and 100 μl of physiological saline was injected into the peritoneum (ip).

[0120] Adipose-derived stem cell-enhanced therapy in mice (h-hADSCs mice): Mice were injected intraovarianly with 15 μl of saline containing h-hADSCs-CM-DIL and intraperitoneally (ip) with 100 μl of saline. The density of h-hADSCs was 5 × 10⁻⁶. 4 Cells / µL

[0121] Treatment of mice with adipose-derived stem cell-enhanced composition (h-hADSCs / HEP14 mice): Mice were treated with ovarian injection of 15 μl of saline containing h-hADSCs-CM-DIL and intraperitoneal injection (ip) of 100 μl of saline containing HEP14 / PLGA microspheres. The density of the h-hADSCs was 5 × 10⁻⁶. 4 The concentration of HEP14 / PLGA microspheres is 10 micrograms per microliter, with cells per microliter.

[0122] I. Anti-ovarian tissue fibrosis and promotion of ovarian angiogenesis

[0123] Four weeks after treatment, ovaries from three groups of mice (CTL-, h-hADSCs-, and h-hADSCs / HEP14 mice) were collected for histopathological examination and to assess the proliferation, development, and number of follicles at each developmental stage. The results showed that the ovaries of aged h-hADSCs / HEP14 mice and h-hADSCs mice were significantly larger than those of untreated aged mice. The ovarian recovery in h-hADSCs / HEP14 mice was better than that in h-hADSCs mice. Figure 6 A). Sirius red (PSR) staining results showed that collagen fibers in the ovaries of untreated aged mice were significantly more abundant than those in the ovaries of h-hADSCs mice and h-hADSCs / HEP14 mice. The ovarian tissue of h-hADSCs mice showed significantly more collagen fibers than that of h-hADSCs / HEP14 mice. Figure 6 B). Immunohistochemical (IHC) staining results showed that, compared with the ovaries of h-hADSCs mice and CTL mice, the myofibroblast (MFB) marker protein α-SMA was significantly decreased in the ovaries of h-hADSCs / HEP14 mice, while the endothelial cell-specific marker proteins CD31 and CD31 were significantly increased. The expression of α-SMA and CD31 in the ovaries of h-hADSCs mice was between that in the ovaries of h-hADSCs / HEP14 mice and CTL mice. Figure 6 (C and 6D). The above results indicate that the adipose-derived stem cell-empowering combination, alone or in combination with HEP14 / PLGA microspheres, can reduce ovarian tissue fibrosis and promote ovarian tissue angiogenesis.

[0124] II. Promoting the development and regeneration of ovarian follicles in aged mice

[0125] H&E staining results showed that, morphologically, h-hADSCs / H EP14 mice had more healthy follicles at different developmental stages in their ovaries than h-hADSCs mice, and the latter had more healthy follicles than the control group of aged mice. Figure 6 E). Detailed follicle counting showed that the total number of follicles and the number of primordial follicles in the ovaries of h-hADSCs / HEP14 mice were significantly higher than those in h-hADSCs mice, and the number of follicles in h-hADSCs mice was significantly higher than that in CTL mice (E). Figure 6 F). Immunofluorescence staining showed that the number of follicles expressing PCNA-positive (a marker of cell proliferation) and TUNEL-negative (a marker of apoptosis) in the ovaries of h-hADSCs / HEP14 mice was significantly higher than that in h-hADSCs mice and CTL mice. The expression of PCNA-positive and TUNEL-negative follicles in the ovaries of h-hADSCs mice was intermediate between that in h-hADSCs / HEP14 mice and CTL mice. Figure 5 G), significantly higher than CTL mice. Immunohistochemical staining results showed that, compared with h-hADSCs mice and CTL mice, h-hADSCs / HEP14 mice ovaries significantly overexpressed Stc1 and CYP26B1, while the expression of Stc1 and CYP26B1 in h-hADSCs mice ovaries was significantly higher than that in CTL mice ovaries. Figure 6 These results indicate that the activation of adipose-derived stem cells, alone or in combination with the HE P14 / PLGA microsphere composition, has a significant effect on promoting ovarian follicle development and regeneration in aged mice, and the combined effect is significantly better than the effect of activation of hADSCs alone.

[0126] III. Promoting the recovery of ovarian endocrine function in the elderly.

[0127] Furthermore, the recovery of serum hormone levels in the three groups of aged mice was assessed 4 weeks after treatment. Serum ovarian hormone levels showed that, compared with the aged control mice, h-hADSCs / HEP14 mice and h-hADSCs mice exhibited significantly elevated levels of E2 (estradiol), AMH (anti-Müllerian hormone), lNHA, and lNHB. Figure 6 J, 6, K, 6L and 6M) and decreased FSH (follicle-stimulating hormone) and T (testosterone) levels ( Figure 6 N and 6O). The improvement in serum levels in h-hADSCs / HEP14 mice was superior to that in h-hADSCs mice. These results indicate that both h-hADSCs alone and h-hADSCs-enhanced combinations significantly promote the regeneration and endocrine function of aged ovarian tissue.

[0128] In summary, both treatment with energized h-hADSCs alone and treatment with a combination of energized h-hADSCs (energized h-hADSCs combined with HEP14 / PLGA microspheres) have the effects of resisting ovarian tissue fibrosis in aged mice, promoting ovarian follicle development and regeneration, and restoring endocrine function. The effect of the h-hADSCs / HEP14 combination treatment is better than that of energized adipose stem cells alone.

[0129] Example 10: Mechanism study of adipose-empowered stem cells and a combination of adipose-empowered stem cell-based treatments for premature ovarian failure in women of reproductive age.

[0130] I. Empowering adipose-derived stem cells: Alone or in combination with HEP14 / PLGA microspheres, this combination reduces ovarian fibrosis and promotes ovarian angiogenesis by enhancing the secretion of MMP1 and PDGFD.

[0131] Follicular development is characterized by angiogenesis and extracellular matrix remodeling. Therefore, we assessed the degree of fibrosis and angiogenesis in the ovarian tissue of four groups of mice 4 weeks after treatment. Collagen content was determined by Sirius Red (PSR) staining, and collagen I (red) and collagen III (green) were distinguished using polarized light microscopy. The results showed that collagen fibers in normal ovaries were thinner and more uniformly distributed, while collagen fibers in the ovaries of POI mice were significantly increased, relatively thicker, and clustered. The collagen fibers in the ovaries of POI mice were significantly more abundant than those in h-hADSCs / HEP14 mice and HEP14-hADSC mice. Figure 7 A and 7B). The collagen content in the ovaries of h-hADSCs mice was between that in h-hADSCs / HEP14 mice and POI mice. Figure 7 A and 7B). Further immunohistochemical and immunofluorescence (IHC and IF) staining results showed that the myofibroblast (MFB) marker protein α-SMA (A and 7B) was present in the ovaries of h-hADSCs / HEP14 mice. Figure 7 C) significantly decreased, and the signal of the endothelial cell-specific marker protein CD31 increased ( Figure 7 D), with expression levels similar to the normal control group, but in stark contrast to the significant increase in α-SMA expression and the significant decrease in CD31 expression in the ovaries of POI mice. Figure 7 D). The expression of α-SMA and CD31 in the ovaries of h-hADSCs mice is between that in the ovaries of h-hADSCs / HEP14 mice and POI mice. Figure 7 D). The above results demonstrate that empowering adipose-derived stem cells, alone or in combination with the HEP14 / PLGA microsphere composition, can reduce fibrosis in ovarian tissue and promote angiogenesis in ovarian tissue.

[0132] To gain a deeper understanding of the molecular mechanisms underlying the anti-ovarian fibrosis and pro-angiogenic effects of hADSCs alone or in combination with HEP14 / PLGA microspheres, we examined the expression of three key differentially expressed genes, MMP1, PDGFD, and PDGFR, in the ovarian tissues of mice in each group. MMP1 is the most abundant matrix metalloproteinase, capable of degrading type I, II, and III collagen. PDGFD is a potent angiogenic growth factor, reported to promote angiogenesis by binding to its homologous receptor PDGFRβ. Western blot confirmed that, compared to c-hADSCs, HEP14 significantly increased the expression of MMP1, PDGFD, and PDGFRβ in h-hADSCs. Figure 7 E). IHC and ICC staining of ovarian tissue sections showed that the expression of MMP1, PDGFD, and PDGFRβ in the ovaries of h-hADSCs / HEP14 mice was significantly higher than that in the ovarian tissues of h-hADSCs mice and POI mice. Figure 7 F-7G). The expression levels of MMP1, PDGFD, and PDGFRβ in the ovaries of h-hADSCs mice were significantly higher than those in POI mice (F-7G). Figure 7 F-7G). To confirm the enhanced anti-fibrotic and angiogenic capabilities observed above, conditioned media including c-hADSCs-CM and h-hADSCs / HEP14-CM were collected. Results were consistent with those obtained from real-time qPCR and Western blot analyses. Figure 2 F and Figure 7 E), enzyme-linked immunosorbent assay (ELISA) results confirmed that HEP14 treatment did indeed increase the secretion level of MMP1 in hADSCs. Figure 7 H). Simultaneously, human umbilical vein endothelial cells (HUVECs) were cultured using the aforementioned conditioned medium and a known PDGFD neutralizing antibody (PDGFDNab) for tube formation experiments. Tube formation results based on total length and number of branch points indicated that h-hADSCs / HEP14-CM medium significantly increased the tube formation ability of HUVECs compared to c-hADSCs-CM conditioned medium. Figure 7 I). IF staining of CD31 confirmed the formation of these tubes ( Figure 7 These results strongly support the claim that HEP14 enhances the angiogenic capacity of hADSCs. In summary, these findings indicate that HEP14-enhanced hADSCs significantly enhance the anti-fibrotic and angiogenic effects of h-hADSCs.

[0133] The above results indicate that adipose-empowered stem cells, alone or in combination with HEP14 / PLGA microspheres, stimulate the secretion of MMP1, PDGFD, and PDGFRβ, thus reducing ovarian fibrosis and promoting ovarian angiogenesis. The anti-fibrotic and angiogenesis-promoting effects of the combination of adipose-empowered stem cells and HEP14 / PLGA microspheres are stronger than those of adipose-empowered stem cell therapy alone.

[0134] II. HEP14 stimulates hADSCs to secrete STC1, which specifically promotes ovarian follicle development and functional recovery.

[0135] Anti-Vimentin antibody (Ab) was confirmed to be a human cell-specific antibody and did not react with mouse tissue cells (analysis certificate, ab8069, Abcam). Immunohistochemical staining results showed that Vimentin expression signaling overlapped with CM-DIL signaling for labeling and empowering adipose-derived stem cells, therefore Vimentin-positive adipose-derived stem cells (hADSCs) Vimentin+ ) represents the energized h-hADSCs transplanted into the ovary ( Figure 7 K). Four weeks after h-hADSCs transplantation, empowered h-hADSCs were detected in the ovaries of h-hADSCs / HEP14 mice. Vimentin+ Significantly more h-hADSCs were found in the ovaries of mice that received h-hADSCs-enhanced transplantation than in mice that received h-hADSCs-enhanced transplantation alone. Vimentin+ Eight weeks post-transplantation, empowered hADSCs remained in the ovaries of h-hADSCs / HEP14 mice. Vimentin+ Significantly higher levels of empowered hADSCs in the ovaries of mice compared to h-hADSCs Vimentin+ ( Figure 7 K). Furthermore, these cells were found to be involved in the granulosa cell layer or theca cell layer of follicles in the ovaries of h-hADSCs / HEP14 mice (K). Figure 7 K). hADSCs were detected in the ovaries of h-hADSCs mice. Vimentin+ They are mainly distributed in the ovarian stromal tissue or around growing follicles. Figure 7 These results indicate that HEP14 promotes the survival and proliferation of h-hADSCs in the ovary. More importantly, these hADSCs Vimentin+ Vimentin signaling overlaps with FSHR or CYP19A1 expression signals in the ovarian granulosa layer. Figure 7 L); overlaps with CYP17A1 expression signals in the theca cell layer ( Figure 7FSHR and CYP19A1 are markers of granulosa cells in ovarian tissue, while CYP17A1 is a marker of follicular cells in ovarian tissue. These results indicate that the combined application of HEP14 / PLGA microspheres and empowered hADSCs promotes the survival and retention of transplanted empowered hADSCs and induces their transdifferentiation into functional granulosa-like cells or follicular-like cells. Therefore, the combination of HEP14 / PLGA microspheres and empowered hADSCs has a stronger effect on promoting follicular proliferation and development and restoring ovarian function.

[0136] Consistent with RNA-seq results ( Figure 2 E), Western blot results showed that HEP14 significantly promoted the secretion of cytokines such as STC1 and CYP9A by activated adipose-derived stem cells (EDS). Figure 7 Immunofluorescence staining revealed that STC1 expression in the ovaries of h-hADSCs / HEP14 mice was significantly higher than that in the ovaries of h-hADSCs mice and POI mice. The expression level in h-hADSCs mice was significantly higher than that in POI mice (N). Figure 7 Further western blot results showed that knocking down STC1 expression in ovarian granulosa cell lines with siRNA led to a decrease in CYP19A1 expression, indicating that STC1 promotes CYP19A1 expression in granulosa cells in ovarian tissue. Figure 7 The main function of CYP19A1 is to stimulate granulosa cells to produce estradiol. Granulosa cells are also the target site for estradiol in ovarian tissue, thus promoting the regeneration and development of ovarian follicles.

[0137] In summary, this invention has found that HE P14 can stimulate the secretion of multiple secretory factors, particularly STC1, CYP19A1, MMP1, and PDGFD. These factors work synergistically, on the one hand, by enhancing the anti-ovarian fibrosis and promoting ovarian angiogenesis of transplanted energized mesenchymal stem cells through MMP1 and PDGFD, thus improving the ovarian tissue microenvironment; on the other hand, STC1 and CYP19A1 synergistically stimulate the growth of ovarian follicular granulosa cells, thereby promoting follicular development and improving ovarian hormone secretion and endocrine function. In conclusion, these results demonstrate that the energized mesenchymal stem cells and the energized mesenchymal stem cell composition of this invention have excellent effects in promoting the regeneration and functional recovery of damaged ovarian tissue.

[0138] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0139] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for empowering mesenchymal stem cells, characterized in that, The enhanced mesenchymal stem cells were obtained by treating human mesenchymal stem cells with HEP14, and the human mesenchymal stem cells were mesenchymal stem cells derived from adipose tissue; HEP14 is a compound with the following molecular structure: ； The treatment of human mesenchymal stem cells with HEP14 includes the following steps: (1) Cultivating and expanding human mesenchymal stem cells; (2) Add HEP14 at a concentration of 1.25~20μM to the culture medium and treat human mesenchymal stem cells for 12 hours to 48 hours; (3) Human mesenchymal stem cells were digested and made into a single-cell suspension; (4) The human mesenchymal single cells were eluted with physiological saline to obtain the energized mesenchymal stem cells suspended in physiological saline.

2. The empowered mesenchymal stem cells according to claim 1, characterized in that, The step (2) of treating human mesenchymal stem cells with HEP14 involves adding 2.5 μM HEP14 to the culture medium and treating the human mesenchymal stem cells for 32 hours.

3. A composition for empowering mesenchymal stem cells, characterized in that, The composition comprises: Empowering mesenchymal stem cells, wherein the empowering mesenchymal stem cells are the empowering mesenchymal stem cells as described in claims 1-2; HEP14 / PLGA microspheres, wherein the HEP14 / PLGA microspheres are prepared using polylactic acid-glycolic acid copolymer (PLGA) as a carrier, and are HEP14 / PLGA microspheres with high HEP14 loading and long-lasting HEP14 sustained release.

4. The mesenchymal stem cell-enhancing composition according to claim 3, characterized in that, The HEP14 / PLGA microspheres with high HEP14 loading and long-lasting HEP14 sustained release were prepared by the following method: In a dark environment, polylactic acid-glycolic acid copolymer (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5-10:1, and stirred with a magnetic stirrer at a speed of 400-500 rpm. Then, the resulting solution was added to an aqueous solution containing 1-2% (w / v) polyvinyl alcohol, and stirred with a magnetic stirrer at a speed of 300-500 rpm at 25°C to allow the dichloromethane to evaporate for at least 12 hours. Then, the microspheres were collected by centrifugation and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol. Finally, the microspheres were placed in a freeze dryer for freeze drying to obtain HEP14 / PLGA microspheres.

5. The mesenchymal stem cell-enhancing composition according to claim 4, characterized in that, The polylactic acid-hydroxyacetic acid copolymer and HEP14 were dissolved in dichloromethane at a mass ratio of 5:

1.

6. The use of the empowered mesenchymal stem cells according to any one of claims 1 to 2 in the preparation of a medicament for treating organ dysfunction, wherein the organ dysfunction is caused by damage to the regenerative function of an organ or aging, and the organ is the ovary; wherein the organ dysfunction is premature ovarian failure or ovarian dysfunction due to aging.

7. The application of the empowered mesenchymal stem cells according to claim 6 in the preparation of drugs for treating organ dysfunction, characterized in that, The drug is administered via local injection into the ovary of the recipient, using a saline solution containing the said energized mesenchymal stem cells; the drug has at least one of the following functions: (1) Anti-ovarian tissue fibrosis; (2) Promotes angiogenesis in ovarian tissue; (3) Promotes ovarian tissue regeneration and functional recovery; (4) Restoring ovarian estradiol, anti-Müllerian hormone, and follicle-stimulating hormone levels. The restoration of ovarian estradiol, anti-Müllerian hormone, and follicle-stimulating hormone levels is as follows: Compared with the premature ovarian failure group of reproductive age, the empowered mesenchymal stem cell treatment group showed higher estradiol and anti-Müllerian hormone levels, but lower follicle-stimulating hormone levels; Compared with the elderly ovarian function decline group, the empowered mesenchymal stem cell treatment group showed significantly increased estradiol and anti-Müllerian hormone levels and decreased follicle-stimulating hormone levels.

8. The use of the mesenchymal stem cell-enhancing composition according to any one of claims 3 to 5 in the preparation of a medicament for treating organ dysfunction, wherein the organ dysfunction is caused by damage to the regenerative function of an organ or aging, and the organ is the ovary; wherein the organ dysfunction is premature ovarian failure or ovarian dysfunction due to aging.

9. The use of the mesenchymal stem cell-enhancing composition according to claim 8 in the preparation of a medicament for treating organ dysfunction, characterized in that, The drug is administered via local injection of a saline solution containing the described energized mesenchymal stem cells into the ovary of the recipient, combined with an injection of a saline solution containing the described HEP14 / PLGA microspheres; the drug has at least one of the following functions: (1) Anti-ovarian tissue fibrosis; (2) Promotes angiogenesis in ovarian tissue; (3) Promotes ovarian tissue regeneration and functional recovery; (4) Restoring ovarian estradiol, anti-Müllerian hormone, and follicle-stimulating hormone levels, wherein the restoration of ovarian estradiol, anti-Müllerian hormone, and follicle-stimulating hormone levels is as follows: compared with the premature ovarian failure group of reproductive age, the group treated with the empowered mesenchymal stem cell composition showed higher estradiol and anti-Müllerian hormone levels, but lower follicle-stimulating hormone levels; compared with the elderly ovarian function decline group, the group treated with the empowered mesenchymal stem cell composition showed significantly increased estradiol and anti-Müllerian hormone levels and decreased follicle-stimulating hormone levels.

10. The use of the mesenchymal stem cell-enhancing composition according to claim 9 in the preparation of a drug for treating organ dysfunction, characterized in that, The injection routes for the physiological saline solution containing the HEP14 / PLGA microspheres include any one of intraperitoneal injection, subcutaneous injection, and intramuscular injection.

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