A mixed cell preparation and its use in the preparation of a medicament for the treatment of premature ovarian failure

Abstract

This invention discloses a mixed cell preparation and its application in the preparation of drugs for treating premature ovarian failure. The mixed cell preparation comprises granulosa cells and mesenchymal stem cells, mixed in a certain ratio (preferably 1:1). Granulosa cells are obtained by inducing differentiation from mesenchymal stem cells and express granulosa cell-specific markers (FOXL2, FSHR, etc.), participating in follicular structure reconstruction; mesenchymal stem cells can improve the ovarian microenvironment, inhibit inflammation, and resist fibrosis, providing a suitable "soil" for the survival of granulosa cells. In vitro experiments are expected to show that mixed culture can significantly increase estradiol secretion levels and cell survival rate. Animal models of premature ovarian failure are expected to show that mixed cell therapy can significantly restore the estrous cycle, improve hormone levels, increase the number of follicles at all stages, and improve pregnancy rates, with effects significantly superior to single-cell therapy. This invention utilizes the synergistic effect of the two cell types to achieve dual repair of the "seed" and the "soil," providing a novel treatment strategy for premature ovarian failure.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and regenerative medicine, specifically relating to a mixed cell preparation containing granulosa cells and mesenchymal stem cells, its preparation method, and its application in the preparation of drugs for treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) refers to the failure of ovarian function in women before the age of 40. Symptoms include amenorrhea, elevated gonadotropin levels, and decreased estrogen levels, leading to serious consequences such as infertility, osteoporosis, and increased risk of cardiovascular disease. Currently, the main treatment is hormone replacement therapy, but this only alleviates symptoms and cannot restore ovarian function and fertility.

[0003] Stem cell transplantation has brought new hope to the treatment of premature ovarian failure. Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potential, immunomodulatory functions, and tissue repair capabilities. They can inhibit ovarian granulosa cell apoptosis, promote angiogenesis, and improve the ovarian microenvironment through paracrine effects. However, MSC transplantation alone has the following limitations: Unable to directly rebuild follicular structure: MSCs mainly play the role of "soil improvement", improving the ovarian microenvironment, but they are difficult to directly transform into functional granulosa cells to participate in follicular reconstruction.

[0004] Limited duration of therapeutic effect: MSCs have a limited survival time in the body, and their paracrine effect gradually weakens with cell apoptosis.

[0005] Granulosa cell apoptosis is a key pathological step: one of the core pathological changes in premature ovarian failure is granulosa cell apoptosis leading to follicular atresia. Although MSC therapy alone can inhibit apoptosis, it cannot replenish the lost granulosa cells.

[0006] In recent years, research on inducing mesenchymal stem cells (MSCs) to differentiate into granulosa cells has made progress. Granulosa cells express granulosa cell-specific markers (FOXL2, FSHR, etc.) and have a certain estradiol secretion capacity, making them a potential "substitute" for granulosa cells in the construction of the follicular microenvironment. However, simple GLC transplantation also faces challenges: Unfavorable implantation environment: Inflammation and fibrosis in the damaged ovary are detrimental to the survival and integration of GLCs.

[0007] Insufficient functional support: GLCs require a suitable microenvironment to maintain their functionality and survival.

[0008] Therefore, there is an urgent need to develop a treatment strategy that can simultaneously play the dual role of "microenvironment repair" and "functional cell reconstruction" in order to more comprehensively restore ovarian structure and function. Summary of the Invention

[0009] Purpose of the invention The purpose of this invention is to provide a mixed cell preparation that combines granulosa cells and mesenchymal stem cells in a certain proportion for local ovarian injection to treat premature ovarian failure. This invention utilizes the synergistic effect of the two cell types: MSCs improve the ovarian microenvironment, inhibit inflammation, and resist fibrosis, creating a suitable "soil" for GLCs; GLCs participate in follicular structure reconstruction and replenish the granulosa cell pool, achieving synergistic repair of both the "seeds" and the "soil." Technical solution

[0010] The present invention provides a mixed cell preparation, characterized in that it comprises granulocytes and mesenchymal stem cells, wherein the granulocytes and mesenchymal stem cells are mixed in a certain proportion.

[0011] (a) Cellular components 1. Granulosa cells The granulocytes are obtained by induced differentiation of mesenchymal stem cells, characterized by expressing granulocyte-specific markers, including but not limited to: FOXL2: Forkhead box L2 transcription factor, a key biomarker for granulosa cells FSHR: Follicle-stimulating hormone receptor, a functional marker of granulosa cells AMH: Anti-Müllerian hormone, secreted by granulosa cells. Inhibin-α / β: Inhibin subunits, secreted by granulosa cells. The functional characteristics of granulocytes include: Responds to FSH stimulation It can secrete estradiol It can secrete AMH, inhibin, etc. 2. Mesenchymal stem cells The mesenchymal stem cells are derived from the umbilical cord, placenta, adipose tissue, or bone marrow, and are characterized by meeting the MSC criteria defined by the International Society for Cell Therapy: CD73⁺, CD90⁺, CD105⁺, CD34⁻, CD45⁻, HLA-DR⁻ It has the potential to differentiate into osteoblasts, lipoblasts, and chondrocytes. It has immunomodulatory function (ii) Mixing ratio The mixing ratio of granulocytes and mesenchymal stem cells is selected from one of the following ranges: 1:5 to 5:1 (GLCs : MSCs) Preferred ratios are 1:2, 1:1, and 2:1. The optimal ratio is 1:1 (equal proportion mixing). Basis for selecting the mixing ratio: Too high a proportion of GLCs: Insufficient microenvironmental support required from MSC An excessively high proportion of MSCs indicates insufficient granulocyte replenishment and limited structural reconstruction. The optimal ratio can be adjusted according to specific disease stages and patient conditions. (III) Cell preparation methods Includes the following steps: 1. Preparation of mesenchymal stem cells MSCs are isolated from human umbilical cord, placenta, adipose tissue or bone marrow, expanded and cultured to P3-P5 generation, and qualified by quality control.

[0012] 2. Induction of granulocyte differentiation Some MSCs were directed to differentiate into granulocyte-like cells. Induction methods included: Method A (Growth Factor Induction): Activin A, FSH, estrogen, etc., are added to the culture medium to induce MSCs to differentiate into granulosa cells. Method B (co-culture induction): Co-culture with ovarian granulosa cells or ovarian tissue to induce differentiation through cell contact and paracrine effects. Method C (Gene Modification Induction): Overexpression of key transcription factors such as FOXL2 drives differentiation into granulocytes. Identification of biomarker expression and functional characteristics of GLCs after induction.

[0013] 3. Cell mixing The expanded MSCs and induced GLCs were mixed in a predetermined ratio, resuspended in a drug carrier, and prepared into an injectable cell preparation.

[0014] 4. Quality Control For testing mixed preparations: Cell viability (>90%) Cell percentage (verified by flow cytometry) Sterility, mycoplasma, and endotoxin testing Functional testing (estradiol secretion capacity) (iv) Pharmaceutical carrier The cell preparation can be resuspended in the following pharmaceutical carriers: physiological saline Phosphate buffer Cell culture medium Medical liquids containing excipients such as hyaluronic acid Biodegradable hydrogel (extends local retention time) (v) Administration method The formulation of this invention is administered via local injection into the ovary, specifically comprising: Ultrasound-guided transvaginal ovarian injection Laparoscopic ovarian injection Ovarian injection under direct vision during open surgery The injection volume for one ovary is 50-200 μL, and the total number of cells is 1×10⁻⁶. 6 - 1×10 7 One ovary.

[0015] Mechanism of action The synergistic mechanism of action of the mixed cell preparation of this invention in treating premature ovarian failure includes: Mechanism 1: MSCs improve the microenvironment, paving the way for GLCs. Damaged ovaries exhibit inflammation, oxidative stress, and fibrosis, which are detrimental to cell survival. MSCs improve the ovarian microenvironment through the following pathways: Immune regulation: secretes TSG-6, IL-10, etc., inhibits M1 macrophages, promotes M2 macrophage polarization, and reduces inflammation. Anti-apoptosis: Secretes VEGF, HGF, IGF-1, etc., inhibiting granulocyte apoptosis. Anti-fibrotic: Secretes MMPs to degrade excessively deposited extracellular matrix. Promotes angiogenesis: secretes VEGF and bFGF, improving local blood supply. The “paving” effect of MSCs creates a microenvironment more suitable for the survival and integration of GLCs.

[0016] Mechanism 2: GLCs rebuild follicular structure and replenish "seeds" GLCs possess granulosa cell-like characteristics and can participate in follicular structure reconstruction: Integration into the ovarian stroma: migration to the perifollicular region Participating in follicle formation: Enveloping the oocyte to form the primordial follicle structure. Response to FSH: Expresses FSHR and responds to gonadotropin stimulation. Secretion of supportive factors: secretion of AMH, inhibin, etc., to regulate follicle development. The "seed" effect of GLCs directly replenishes the granule cell pool lost due to premature aging.

[0017] Mechanism 3: MSCs and GLCs interact to form a positive feedback loop. Factors secreted by MSCs promote the survival and functional maintenance of GLCs. Factors secreted by GLCs provide feedback regulation of MSC function The estradiol secretion level in the co-culture system was significantly higher than that in the monoculture system. Two types of cells work together to suppress inflammation and promote tissue repair. Mechanism 4: Restoring ovarian endocrine function Increased number of granulocytes → Restoration of estradiol secretion Elevated estradiol levels → negative feedback regulation of FSH → normalization of hormone levels Improved ovarian reserve function → AMH levels rise Mechanism 5: Restoring fertility Follicular structure reconstruction → Follicular development recovery Ovulation function restored → Natural conception possible Improved ovarian microenvironment → Increased oocyte quality Beneficial effects

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. Synergistic effect Effect dimensions: Individual MSCs, Individual GLCs, Synergistic effect of the mixed formulation of this invention Microenvironment improvement +++ + ++++ MSC-led Granulocyte supplementation +++++++ GLCs dominant Estradiol secretion synergistically enhanced. Follicle reconstruction + ++ ++++ Synergistic enhancement Anti-inflammatory effect +++ + ++++ Synergistic enhancement Long-term efficacy ++ + ++++ Synergistic enhancement 2. Overcoming the bottlenecks of single-cell therapy Technical bottlenecks: Single MSC therapy, single GLC therapy, this invention Direct supplementation of granulocytes cannot able The ability to improve the microenvironment is limited. able Cell survival requires support to support self-sufficiency. MSC provides support Paracrine function is mainly maintained and requires a suitable microenvironment. Microenvironment optimization 3. Adjustable ratio, personalized treatment The GLCs:MSCs ratio can be adjusted according to the patient's stage of illness. Early and mid-stage patients may benefit from an increased MSC ratio (focusing on microenvironment repair). For patients with advanced disease, the proportion of GLCs can be appropriately increased (with an emphasis on structural reconstruction). 4. The preparation process is feasible. Both cell types originate from the same MSC source, thus posing fewer ethical concerns. There is already a research foundation for induced differentiation technology. The quality control system is clearly defined. 5. Synergy with a series of patents It can be combined with the highly adhesive modified MSC of patent 2 (mixing GLCs with modified MSCs). It can be combined with the dynamic culture device of Patent 1 (for in vitro expansion and functional optimization). Detailed Implementation

[0019] (The following examples describe the intended implementation and expected results of the present invention. Specific experimental data will be provided in the form of a supplementary experimental report within 12 months from the priority date.) Example 1: Preparation of mesenchymal stem cells 1.1 Isolation and culture of MSCs from umbilical cord Aseptically collect umbilical cords from healthy full-term cesarean section fetuses (with informed consent), remove arteries and veins, cut Wharton's jelly into 1 mm³ pieces, and place them in α-MEM medium containing 10% FBS, incubate at 37°C and 5% CO2. After 7-10 days, cells will emerge and be passaged to P3 for future use.

[0020] 1.2 MSC Identification Flow cytometry detection of surface markers: Positive results: CD73, CD90, CD105 (>95%) Negative: CD34, CD45, HLA-DR (<2%) Verification of tridirectional differentiation ability: Osteogenesis induction: Alizarin Red staining positive Adipogenic induction: Positive for Oil Red O staining Chondrogenic induction: Alixin blue staining positive Example 2: Induction of differentiation of granulocytes 2.1 Growth Factor Induction Method P3 generation MSCs were inoculated into gelatin-coated culture dishes. The induction medium contained: DMEM / F12, 10% FBS, 10 ng / mL Activin A, 100 ng / mL FSH, and 1 μM estradiol. Induction culture was carried out for 14-21 days, with the medium changed every 3 days.

[0021] 2.2 GLCs Identification Marker detection: Immunofluorescence staining: FOXL2, FSHR, and AMH positive. qPCR: FOXL2, FSHR, AMH, and Inhibin-α / β mRNA expression were significantly upregulated. Functional testing: FSH stimulation experiment: cAMP levels increased after the addition of FSH. Estradiol secretion: E2 levels in culture supernatant detected by ELISA Expected results: After induction, GLCs express granulocyte markers, respond to FSH, and secrete estradiol.

[0022] Example 3: Preparation of Mixed Cell Preparations 3.1 Cell Expansion MSCs were expanded to passage P4, and the quantity met the demand. GLCs were induced and expanded to a sufficient number. 3.2 Cell Mixing Blend GLCs and MSCs in the following proportions: Groups, GLCs, Ratios, MSCs, Ratios, Uses The ratio of 1:1:2 indicates an MSC bias (emphasizing microenvironment remediation). A 2:1:1 ratio represents a balanced approach (expected optimal). The ratio 3:2:1 is biased towards GLC (emphasizing structural reconstruction). Comparison 1 1 0 pure GLCs Control 201 pure MSCs 3.3 Formulation resuspension Resuspend the mixed cells in physiological saline and adjust the concentration to 5 × 10⁻⁶. 7 / mL, dispensed into sterile cryovials, and stored at 4°C for later use (use within 24 hours).

[0023] 3.4 Quality Control Cell viability: Trypan blue staining, expected >90%. Flow cytometry analysis: confirmed that the ratio of GLCs to MSCs met the set parameters. Sterility testing: Negative cultures for aerobic bacteria, anaerobic bacteria, and fungi. Endotoxin: <0.5 EU / mL Example 4: Functional Validation of In Vitro Co-culture (Expected Plan) 4.1 Experimental Design Co-culture experiments were conducted in a 24-well Transwell plate system: Group 1: GLC cultured alone (lower chamber) Group 2: Individual MSC culture (lower chamber) Group 3: GLCs and MSCs co-cultured (lower chamber mixing) Group 4: GLCs (lower chamber) + MSCs (upper chamber) – non-contact co-culture 4.2 Detection Indicators Estradiol secretion: Supernatant was collected on days 3, 7, and 14, and E2 levels were detected by ELISA. Cell proliferation: CCK-8 assay Apoptosis: Annexin V / PI staining and flow cytometry detection Gene expression: qPCR detection of functional genes in granulocytes (FSHR, CYP19A1, etc.) 4.3 Expected Results The estradiol secretion level in the mixed culture group (group 3) was significantly higher than that in the single culture groups (groups 1 and 2). The apoptosis rate of GLCs in the mixed culture group was significantly lower than that in the single culture group. The effects of the non-contact co-culture group (group 4) were between those of group 3 and group 1, suggesting that both contact and paracrine secretion contributed. The optimal mixing ratio (1:1) yields the best results. Example 5: In vivo validation of a premature ovarian failure animal model (expected protocol) 5.1 Establishment of animal models A premature ovarian failure model was established using female SD rats (6-8 weeks old) through one of the following methods: Chemotherapy injury model: Intraperitoneal injection of cyclophosphamide (50 mg / kg / day) for 7 days + a single dose of busulfan (30 mg / kg) Autoimmune model: Induction of immune oophoritis by zona pellucida peptide injection Natural aging model: Rats over 12 months old Model success criteria: estrous cycle disorder, decreased serum E2, increased FSH, decreased AMH, and reduced number of follicles at all stages.

[0024] 5.2 Experimental Grouping Group processing sample size Group 1: Model Control (physiological saline) 15 Group 2 Pure MSCs transplanted 15 Group 3 Pure GLCs transplanted 15 Group 4 Mixed cell line (GLCs:MSCs = 1:2) 15 Group 5: Mixed cell line (GLCs:MSCs = 1:1) 15 Group 6: Mixed cell line (GLCs:MSCs = 2:1) 15 Group 7: Normal control (no treatment) 10 5.3 Cell Transplantation After anesthesia, the ovaries were exposed by laparotomy, and 50 μL of cell suspension (containing 2.5 × 10⁻⁶ cells) was injected into the parenchyma of each ovary using a microsyringe. 5 (One cell). Inject slowly, hold for 30 seconds, then withdraw the needle and apply pressure to stop bleeding. Suture the abdomen closed.

[0025] 5.4 Detection Indicators (1) Monitoring of the estrous cycle Starting from the second week after embryo transfer, vaginal smears were taken daily for two consecutive complete cycles to assess the recovery of the estrous cycle.

[0026] (2) Hormone level detection Blood samples were collected at weeks 4, 8, and 12 post-transplantation for ELISA testing. Estradiol (E2) Follicle-stimulating hormone (FSH) Anti-Müllerian hormone (AMH) (3) Ovarian histological analysis Five animals were sacrificed at each of the following three weeks post-transplantation: weeks 4, 8, and 12, and ovaries were harvested. HE staining: Counting follicles at each stage (primordial follicles, primary follicles, secondary follicles, antral follicles). Masson staining: assessing the area of ​​fibrosis TUNEL staining: Detection of granulocyte apoptosis Immunohistochemistry: Detection of FSHR and FOXL2 expression (4) Cell fate tracking The survival, distribution, and differentiation of transplanted cells were tracked using fluorescent labels (GFP-labeled MSCs and RFP-labeled GLCs).

[0027] (5) Fertility test Eight weeks after transplantation, the remaining female mice in each group were put in the same cage as male mice (2:1) to observe the conception rate and number of offspring.

[0028] 5.5 Expected Results (1) Recovery of the estrous cycle The estrous cycle recovery rate in the mixed cell therapy group (especially the 1:1 group) was significantly higher than that in the pure cell therapy group and was close to that in the normal control group.

[0029] (2) Improved hormone levels Group E2 (pg / mL) FSH (mIU / mL) AMH (ng / mL) Normal control group: 80-100, 5-8, 2.5-3.5 Model comparison: 20-30, 25-35, 0.3-0.6 Pure MSC group: 40-50, 18-22, 0.8-1.2 Pure GLCs group: 45-55, 16-20, 1.0-1.5 Mixed 1:1 ratio 70-85 8-12 2.0-2.8 The hormone levels in the 1:1 mixed group were significantly better than those in the pure cell group and were close to those in the normal control group.

[0030] (3) Follicle count Group: Primordial follicle, Primary follicle, Secondary follicle, Antral follicle Normal control 100% 100% 100% 100% Model comparison: 15-20% 10-15% 5-10% <5% Pure MSC group: 30-40%, 25-35%, 20-30%, 15-20% Pure GLCs group: 35-45%, 30-40%, 25-35%, 20-25% Mixed 1:1 ratio: 70-80%, 65-75%, 60-70%, 50-60% The number of follicles at each stage in the 1:1 mixed group was significantly higher than that in the pure cell group.

[0031] (4) Cell fate tracking At week 4 post-transfer, GFP-labeled MSCs and RFP-labeled GLCs were detected in ovarian tissue. Some GLCs integrated into the perifollicular region, while some MSCs were distributed in the ovarian stroma. The number of surviving GLCs in the mixed group was significantly higher than that in the pure GLCs group.

[0032] (5) Fertility Pregnancy rate and average number of babies born in each group Normal control 90-100% 10-12 Model control 0-10% 0-2 Pure MSC group 20-30% 3-5 Pure GLCs group 25-35% 4-6 Mix 1:1 ratio, 60-70%, 8-10 The pregnancy rate and number of litters in the mixed 1:1 group were significantly higher than those in the pure cell group.

[0033] Example 6: Optimal Ratio Screening (Expected Solution) 6.1 Dose gradient experiment Set a finer scaling gradient: GLCs:MSCs = 1:3, 1:2, 1:1, 2:1, 3:1, with three total cell doses (1×10⁻⁶) for each ratio: low, medium, and high. 5 5×10 5 1×10 6 / ovary).

[0034] 6.2 Overall Score Each group will be given a comprehensive score, with the following indicators: Hormone recovery (weight 30%) Follicle count (weight 30%) Pregnancy rate (weight 20%) Security (weight 20%) Expected results: GLCs:MSCs = 1:1, total cell dose 5 × 10⁻⁶ 5 / Ovarian is the best combination.

[0035] Example 7: Safety Evaluation (Expected Solution) 7.1 Acute toxicity High-dose mixed cells (1×10⁻⁶) were injected locally into the ovaries of rats. 7 (Ovary), observe for 7 days, with no expected death, no abnormal behavior, and no weight loss.

[0036] 7.2 Long-term toxicity Optimal dose of mixed cells injected locally into the ovaries of rats, observed for 3 months, expected results: Blood routine and liver and kidney function tests were normal. HE staining of major organs (heart, liver, spleen, lungs, kidneys) showed no abnormalities. The contralateral ovary is normal and has no tumor formation. 7.3 Tumorigenicity Nude mice were injected subcutaneously with mixed cells (1×10⁻⁶). 7 (each animal) was observed for 3 months, with no tumor formation expected.

[0037] 7.4 Biodistribution The distribution of the virus in vivo was tracked using fluorescently labeled cells, and it was expected to be mainly confined to the ovary on the injection side, with a small number migrating to the contralateral ovary. No abnormal aggregation was observed in distant organs such as the liver, spleen, lungs, and kidneys.

Claims

1. A mixed cell preparation, characterized in that, It comprises granulocytes and mesenchymal stem cells, wherein the granulocytes and mesenchymal stem cells are mixed in a certain proportion.

2. The mixed cell preparation according to claim 1, characterized in that, The granulocytes are obtained by induced differentiation of mesenchymal stem cells and express granulocyte-specific markers, which are selected from one or more of FOXL2, FSHR, AMH, Inhibin-α, and Inhibin-β.

3. The mixed cell preparation according to claim 1, characterized in that, The mesenchymal stem cells are derived from the umbilical cord, placenta, adipose tissue, or bone marrow, and meet the identification criteria for mesenchymal stem cells.

4. The mixed cell preparation according to claim 1, characterized in that, The mixing ratio of the granulocytes to mesenchymal stem cells is 1:5 to 5:1, preferably 1:2, 1:1 or 2:1, and most preferably 1:

1.

5. The mixed cell preparation according to claim 1, characterized in that, It also includes a pharmaceutically acceptable carrier selected from physiological saline, phosphate buffer, cell culture medium, medical liquid containing hyaluronic acid, or biodegradable hydrogel.

6. A method for preparing the mixed cell preparation according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Isolate and culture mesenchymal stem cells; (2) Induce some mesenchymal stem cells to differentiate into granulocytes; (3) Mix the mesenchymal stem cells from step (1) with the granulocytes from step (2) in a predetermined ratio; (4) Resuspend them in a pharmaceutical carrier to prepare an injectable cell preparation.

7. The method according to claim 6, characterized in that, The induction of differentiation in step (2) can be achieved by growth factor induction, co-culture induction, or gene modification induction.

8. The use of the mixed cell preparation according to any one of claims 1-5 in the preparation of a drug for treating premature ovarian failure.

9. The application according to claim 8, characterized in that, The mixed cell preparation is administered via local injection into the ovary.

10. The application according to claim 9, characterized in that, The total number of cells injected into each ovary was 1×102 5 Up to 1×10 7 indivual.

11. A method for treating premature ovarian failure, characterized in that, This includes administering the mixed cell preparation according to any one of claims 1-5 to an individual in need of treatment via local ovarian injection.

12. A system for treating premature ovarian failure, characterized in that, include: (1) The mixed cell preparation according to any one of claims 1-5; (2) An injection device for local injection into the ovary.