Application of Human Umbilical Cord Highly Active Mesenchymal Stem Cells

Abstract

The present invention discloses an application of highly active human umbilical cord mesenchymal stem cells. HA-MSCs were isolated from human umbilical cord tissue using TeSR-E8 containing 5% CloneR and LN521-Coated culture flasks, and a technical system for HA-MSC isolation, expansion, and cryopreservation was established. Compared with conventionally cultured MSCs, the HA-MSCs prepared by the present invention have higher proliferation activity, smaller size, a higher nuclear-cytoplasmic ratio, and highly express embryonic stem cell-associated marker antigens SOX2, Nanog, and OCT4. They have the potential to differentiate into neural, myocardial, and hepato-intestinal progenitor cells derived from the three germ layers and possess excellent anti-aging activity.

Description

Technical Field

[0001] The present invention relates to the technical field of human umbilical cord stem cells, and in particular to an anti-aging application of highly active human umbilical cord MSCs. Background Art

[0002] Mesenchymal stem cells (MSCs) are a group of adult pluripotent stem cells with the ability to self-renew and differentiate. MSCs can be obtained from virtually any human tissue, such as adipose tissue, dental pulp, menstrual blood, placenta, amniotic fluid, umbilical cord, and synovial fluid. MSCs possess immunomodulatory properties, secreting cytokines and immune receptors to modulate the host tissue microenvironment. They have demonstrated significant therapeutic potential in cardiovascular disease, autoimmune disorders, vascular ischemia, tissue damage, and various degenerative diseases. Therefore, MSCs hold broad promise for both disease prevention and treatment.

[0003] Because MSC morphological and functional heterogeneity weakens their therapeutic effects, it introduces differences in clinical efficacy assessments and mechanistic studies, which may partly explain why data from MSC-based clinical trials are largely inconsistent. To address the differences in efficacy caused by heterogeneity, reports have been published on the isolation of more potent and primitive MSC subpopulations from MSCs derived from different tissues. Although these subpopulations have high proliferative and differentiation potential, whether they can maintain multi-lineage differentiation capacity, similar to the plasticity of embryonic stem cells, remains to be determined.

[0004] The reported methods for isolating high-potential MSC subpopulations include physical separation, which separates trypsin-resistant, multidirectionally differentiated, and pressure-resistant cells based on their reaction to trypsin; separation based on immunophenotype, which separates cell subtypes with pluripotency surface marker molecules by flow cytometry, or introduces exogenous genes for cell re-editing. These separation methods are relatively cumbersome, with low cell activity and low yield, making it difficult to obtain cell therapy quantities for subsequent efficacy studies, and safety is difficult to guarantee. In addition, most culture systems contain animal-derived serum, making them unsuitable for clinical translation. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of human umbilical cord highly active MSCs, especially for anti-aging effects.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions:

[0007] A method for anti-aging using highly active human umbilical cord MSCs.

[0008] Use of highly active human umbilical cord MSCs in the preparation of anti-aging preparations.

[0009] Further preferably, the method for preparing the above-mentioned human umbilical cord highly active MSCs comprises the following steps:

[0010] The following steps are involved:

[0011] S1. Reagent preparation;

[0012] S2, primary culture;

[0013] S3, subculture;

[0014] Preferably, it also includes S4 and high-activity MSC cryopreservation.

[0015] Further preferably, S1 includes:

[0016] S11. Prepare high-activity MSC complete medium: Add TeSR-E8 Supplement to TeSR-E8 Basal Medium, mix thoroughly by inversion, and then add CloneR. Adjust the pH to 7.2-7.4 with 6.6% NaHCO3 and store at 4°C until ready for use.

[0017] S12. Preparation of high-activity MSC culture flask: slowly thaw the laminin LN521 stock solution at 2-8°C, and add 1× DPBS (Ca 2+ / Mg 2+ ) Dilute the laminin LN521 stock solution to prepare the coating solution. Add the coating solution to the culture flask to evenly cover the entire bottom of the flask and incubate at 2-8°C until ready for use.

[0018] Further preferably, S2 includes:

[0019] S21. Cleaning the collected human umbilical cord to remove residual blood;

[0020] S22, disaggregate the umbilical cord into small tissue pieces and transfer them into a high-activity MSC culture flask;

[0021] S23. Add high-activity MSC complete medium to the high-activity MSC culture bottle, shake the bottle slowly, and place it in an incubator at 37°C and 5% CO2 for primary culture;

[0022] S24, on the 3rd day, supplement with high-activity MSC complete culture medium, and then change the medium every 3-4 days

[0023] S25. During passage, the detached tissue pieces are collected and can be cultured for a second time according to the above method;

[0024] Further preferably, S3 includes:

[0025] S31. Remove cells from the incubator, discard old culture medium, and gently rinse once with normal saline.

[0026] S32, primary high-activity MSCs were added with Recombinant Trypsin-EDTA Solution and placed in a 37°C, 5% CO2 incubator for 3-5 minutes;

[0027] S33. After the cells were completely detached, an equal amount of high-activity MSC complete medium was added to terminate the digestion, and the cells were collected, added with normal saline, and centrifuged at 1500 rpm for 4 mins.

[0028] S34. After centrifugation, discard the supernatant, add high-activity MSC complete medium, gently pipette to mix the cells, and count the number of viable cells using trypan blue staining;

[0029] S35, according to 5000 cells / cm 2 The cells were seeded into new high-activity MSC culture flasks at a seeding density of

[0030] S36. Place in an incubator at 37°C and 5% CO2 for expansion culture;

[0031] Further preferably, S4 includes:

[0032] Select highly active MSCs in the logarithmic growth phase and digest them with Recombinant Trypsin-EDTA Solution. After the cells shrink and become round, add highly active MSC complete medium to terminate the digestion. After centrifugation at 1500 rpm for 4 minutes, discard the supernatant, gently pipette to make the cells uniform, and then divide them into sterile cryopreservation tubes. Place the cryopreservation tubes containing cells in a programmed cooling box in a -80°C refrigerator. After 4 hours, place them in a -196°C liquid nitrogen tank for storage until use.

[0033] Further preferably, in step S11, the ratio of TeSR-E8 Basal Medium, TeSR-E8 Supplement, and CloneR is 24:1:1.

[0034] More preferably, in step S12, the laminin LN521 stock solution is diluted to 5 μg / ml.

[0035] Further preferably, in step S21, the human umbilical cord is repeatedly rinsed with physiological saline containing 100u / ml of the dual antibody.

[0036] Further preferably, the above-mentioned anti-aging preparation is a preparation for preventing, improving and / or treating ovarian aging; more preferably, the preparation is a pharmaceutical agent.

[0037] A method for preparing highly active human umbilical cord MSCs comprises the preparation steps defined in any one of the above items.

[0038] Human umbilical cord highly active MSCs are prepared by the above preparation steps.

[0039] More preferably, the human umbilical cord highly active MSCs have the functions of wound repair, cell repair, promotion of tissue / cell regeneration and differentiation, immune regulation, anti-aging, and the like.

[0040] A use of the human umbilical cord highly active MSCs in the preparation of cosmetics, health products and medicines.

[0041] More preferably, the human umbilical cord highly active MSCs are in the form of cell culture or lyophilized powder.

[0042] More preferably, the dosage form of the above-mentioned health care products and medicines is selected from injections, oral preparations, tablets, capsules or patches.

[0043] More preferably, the cosmetics further include common adjuvants such as antioxidants, stabilizers, emulsifiers, solubilizers, pigments and fragrances.

[0044] A use of the above-mentioned human umbilical cord highly active MSCs in preparing a preparation for preventing, improving or treating aging.

[0045] Further preferably, the above-mentioned human umbilical cord highly active MSCs are used to prepare a medicament for preventing, improving or treating physiological and / or pathological aging / functional impairment of the skin, liver, heart, ovary, uterus, testicles, muscle, nerves, kidneys, immunity, metabolism, or systemic aging / functional impairment.

[0046] Further preferably, the above-mentioned human umbilical cord highly active MSCs are used to prepare a preparation for preventing, improving and / or treating ovarian aging.

[0047] More preferably, the above preparation is a pharmaceutical preparation.

[0048] More preferably, the human umbilical cord highly active MSCs are used to prepare drugs for preventing, improving and / or treating aging.

[0049] More preferably, the human umbilical cord highly active MSCs are used to prepare anti-aging drugs.

[0050] More preferably, the human umbilical cord highly active MSCs are used to prepare anti-aging drugs.

[0051] More preferably, the human umbilical cord highly active MSCs are used to prepare drugs for preventing, improving and / or treating ovarian aging.

[0052] More preferably, the human umbilical cord highly active MSCs are used to prepare anti-ovarian aging drugs.

[0053] More preferably, the human umbilical cord highly active MSCs are used to prepare drugs for reversing ovarian aging.

[0054] More preferably, the above-mentioned medicine further comprises one or more pharmaceutically acceptable excipients, diluents or carriers.

[0055] A preparation comprising any one of the above-mentioned highly active human umbilical cord MSCs.

[0056] A medicament comprising any one of the above-mentioned highly active human umbilical cord MSCs.

[0057] A pharmaceutical composition comprising any one of the above-mentioned highly active human umbilical cord MSCs.

[0058] A pharmaceutical composition, characterized in that it comprises highly active MSCs prepared by the preparation method according to any one of claims 1 to 7.

[0059] A pharmaceutical composition, the active ingredient of which is prepared by any of the above-mentioned methods for preparing highly active human umbilical cord MSCs; preferably, further comprising one or more pharmaceutically acceptable excipients, diluents or carriers.

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

[0061] First, improve the culture medium and supporting culture conditions, including TeSR-E8 culture medium, ClonR, cell density and recombinant laminin coating, in order to create an overall in vivo ecological microenvironment suitable for primitive cells. In this culture condition, TeSR-E8 culture medium does not contain animal origin and has a low protein content. Only one growth factor, ClonR, is added to the entire culture condition, and the use of recombinant laminin coating maintains the high pluripotency of the cells. Under these conditions, the primary culture is carried out by whole-tissue adhesion. Compared with other separation methods, this method is simple and easy, and the time from tissue removal to entering the incubator is shorter, which maximizes and maintains very high cell activity.

[0062] Secondly, the primary culture method allows the original cells or highly pluripotent cells in the tissue to be sorted out from the primary generation, and can be stably passaged and preserved, providing a prerequisite for subsequent expansion.

[0063] Third, compared with conventionally cultured MSCs, highly active MSCs have higher proliferation activity, smaller size, higher nuclear-cytoplasmic ratio, and high expression of embryonic stem cell-related marker antigens SOX2, Nanog, and OCT4. They have the potential to differentiate into neural, myocardial, and hepato-intestinal progenitor cells derived from the three germ layers, indicating that this cell is a sub-totipotent stem cell among highly active MSCs.

[0064] Fourth, highly active MSCs were more effective than conventionally prepared MSCs in treating ovarian aging in macaques, improving ovarian tissue structure and secretory function, regenerating follicles, and restoring ovulation and menstruation, but failing to restore reproductive function. The study found that highly active MSCs reversed ovarian aging through multiple mechanisms, including GC differentiation, reversing GC aging, resisting oxidative stress, inhibiting ovarian cell apoptosis, increasing vascular density, promoting ovarian GC division and proliferation, and promoting the expression of specific marker molecules. These results provide a new technical approach and scientific basis for the clinical treatment of ovarian aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The morphology of umbilical cord HA-MSC and MSC under an inverted phase contrast microscope (×50); 1A: On the 8th day, HA-MSC cells can be seen growing scattered around the tissue mass, showing short spindle shapes (red circles); 1B: On the 12th day, HA-MSC formed large colonies, and the colonies were arranged like fish schools; 1C: On the 10th day, MSC cells grew scattered around the tissue mass, and the cells were long spindle-shaped and fibroblast-like; 1D: On the 15th day, MSC colonies were arranged in a whirlpool shape.

[0066] Figure 2 Figure 2 shows the cell morphology of HA-MSC and MSC at three passages after passage under an inverted phase contrast microscope (×50); 2A, 2B, and 2C are P3, P6, and P9 of HA-MSC after passage, respectively. Each passage maintains a uniform short cell morphology; 2D, 2E, and 2F are P3, P6, and P9 of MSC after passage, respectively. With the increase of passage, the cell body becomes larger, the edges become irregular, some cells are polygonal, and the number of granules in the cytoplasm increases.

[0067] Figure 3 The P3 generation HA-MSC and MSC were dynamically observed under a holographic living cell microscope for 48 hours.

[0068] Figure 4 Figure 4 is the growth curve of HA-MSC and MSC; 4A: The growth curve of P3 generation HA-MSC and MSC showed a typical "S" shape; 4B: The growth curve of P9 generation HA-MSC remained unchanged, while the growth curve of P9 generation MSC tended to be flat, and the cell proliferation ability decreased.

[0069] Figure 5 is the population doubling time of HA-MSC and MSC.

[0070] Figure 6 This is a transmission electron micrograph of HA-MSC; 6A: Bar = 5 μm; 6B: Bar = 2 μm.

[0071] Figure 7 MSC transmission electron microscopy; 7A: Bar = 5 μm; 7B: Bar = 2 μm.

[0072] Figure 8 Figure 8A shows the nuclear-cytoplasmic ratio of HA-MSCs and MSCs. Figures 8A, 8B, and 8C show HA-MSCs; Figures 8D, 8E, and 8F show MSCs. Figure 8G shows the statistical results. Bar = 5 μm. n = 3. **P < 0.01.

[0073] Figure 9 9A and 9B are the cell cycle of HA-MSC and MSC, respectively.

[0074] Figure 10 10A and 10B are the results of HA-MSC and MSC, respectively.

[0075] Figure 11 11A is the flow cytometric graph of the expression of HA-MSC and MSC surface markers (SSEA-3, SSEA-4, TRA-1-60); 11A and 11B are the results of HA-MSC and MSC, respectively.

[0076] Figure 12 Figure 12A and 12B show the electrophoresis images and expression statistics of HA-MSC and MSC pluripotency transcription factors detected by Western blotting.

[0077] Figure 13 It is the identification of the tri-lineage differentiation ability of HA-MSC and MSC.

[0078] Figure 14 Expression of ectoderm differentiation markers in HA-MSCs and MSCs. DAPI localizes cell nuclei, and the secondary antibody is labeled with Cy3, revealing red fluorescence. Bar = 20 μm. n = 3, ***P < 0.001, **P < 0.01. Figures 14A and 14B show the fluorescence images and statistical results of differentiation marker expression, respectively.

[0079] Figure 15 Identification of mesodermal differentiation markers in HA-MSCs and MSCs; DAPI localizes cell nuclei, and the secondary antibody is labeled with Cy3, revealing red fluorescence. Bar = 20 μm. n = 3, ***P < 0.001, **P < 0.01. Figures 15A and 15B show the fluorescence imaging and statistical results of differentiation marker expression, respectively.

[0080] Figure 16 Expression of endoderm differentiation markers in HA-MSCs and MSCs. DAPI localizes cell nuclei, and the secondary antibody is labeled with Cy3, revealing red fluorescence. Bar = 20 μm. n = 3, ***P < 0.001. Figures 16A and 16B show the fluorescence imaging and statistical results of differentiation marker expression, respectively.

[0081] Figure 17 This is a graph showing changes in serum AMH.

[0082] Figure 18 This is the abdominal ultrasound of the HA-MSC treatment group 5 months after transplantation.

[0083] Figure 19 19A and 19B are the tissue staining images and statistical results, respectively.

[0084] Figure 20 This is the colonization of dual-mode labeled cells in ovarian tissue (40×).

[0085] Figure 21 Detection of ROS levels in ovarian tissue (40×). 21A and 21B are fluorescence imaging and statistical results, respectively.

[0086] Figure 22 The ovarian tissue vascular density is detected (40×). 22A and 22B are the fluorescence imaging and statistical results, respectively.

[0087] Figure 23 is ovarian cell proliferation and apoptosis detection (40×); Figure 23 A and 23B are the fluorescence imaging and statistical results of HA-MSC, respectively. Figure 23 C and 23D are the fluorescence imaging and statistical results of MSCs, respectively.

[0088] Figure 24 is the expression of ovarian granulosa-specific markers (40×); Figure 24 A and 24B are the fluorescence imaging and statistical results of HA-MSC, respectively. Figure 24 C and 24D are the fluorescence imaging and statistical results of MSCs, respectively.

[0089] Figure 25 Telomere length in macaques of different ages and changes after HA-MSC treatment.

[0090] Figure 26 β-galactosidase staining of human ovarian granulosa cells (50×). Figure 26 A and 26B are the cell staining images and statistical results, respectively.

[0091] Figure 27 3D holographic live cell dynamic observation of aged hGC and HA-MSC (600×); Figure 27 A, Figure 27 B shows the results before and after the addition of HA-MSC.

[0092] Figure 28The morphological changes of MSCs and HA-MSCs induced into GCs in vitro (50×). Figure 28 A is the morphological change diagram of the in vitro induction process. Figure 28 B is the morphology of hGC cells.

[0093] Figure 29 It is a specific marker molecule for identification of stem cells induced into GC in vitro (40×); Figure 29 A and 29B are the fluorescence imaging and statistical results of HA-MSC. Figure 29 C and 29D are the fluorescence imaging and statistical results of MSCs.

[0094] Figure 30 These are the changes in AMH secretion induced by HA-MSCs into GCs in vitro. DETAILED DESCRIPTION

[0095] The present invention will be described in detail below with reference to examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0096] the term

[0097] “HA-MSC”, “highly active MSC” and “highly active mesenchymal stem cells” are used interchangeably throughout the text.

[0098] "Anti-aging" refers to reversing, preventing, alleviating or inhibiting the progression of aging and a disease, disorder or condition associated with aging, or one or more symptoms of a disease, disorder or condition.

[0099] "Treat" means to slow, alleviate, ameliorate or alleviate at least one symptom of a disease, or to reverse its onset after onset.

[0100] "Prevention" refers to the process of taking action before the onset of a disease to prevent the development of the disease or to reduce the severity of the disease or slow its development.

[0101] "Agent" refers to a substance that produces or is capable of producing an effect and may include, but is not limited to, chemicals, pharmaceuticals, drugs, biologics, small molecules, antibodies, nucleic acids, peptides, and proteins.

[0102] "Pharmaceutical composition" refers to a composition at any stage of the manufacturing process, including the final pharmaceutically acceptable product and any process intermediates thereof.

[0103] Excipients are substances added to pharmaceutical preparations besides the main drug, also known as auxiliary materials. Examples include preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0104] The various terms and phrases used in the present invention have general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases herein. If the mentioned terms and phrases are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail.

[0105] Example 1: Preparation of Human Umbilical Cord Highly Active MSCs (HA-MSCs)

[0106] 1. Materials

[0107] 1.1 Source of human umbilical cord

[0108] Umbilical cords for this experiment were obtained from the Department of Obstetrics and Gynecology of the 920th Hospital of the Joint Logistics Support Force of the People's Liberation Army. Eligible mothers were required to have no infectious diseases, no tumors, no family history of genetic diseases, and no congenital diseases in their full-term fetuses. Umbilical cords were collected at the time of delivery, with the consent of the mother and her family, who signed the "Informed Consent Form." Umbilical cord collection was approved by the Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the People's Liberation Army, approval number: 2019-036(Section)-01.

[0109] 1.2 Main instruments

[0110]

[0111] 1.3 Main Reagents

[0112]

[0113]

[0114] 2. Experimental Methods

[0115] The following steps are involved:

[0116] S1. Reagent Preparation

[0117] S1-1. Screening and preparation of culture medium:

[0118] S1-1-1. Based on DMEM / F12, the culture medium components are gradually enriched, such as replacing bovine serum with human platelet lysate, replacing bovine serum albumin with human serum albumin, and gradually replacing animal-derived components with human-derived components, thereby reducing the immune rejection reaction caused by differences in species origin; there is also a practice of completely abandoning animal-derived components based on DMEM / F12, that is, completely free of animal-derived components (including those from human sources), and adding trace elements such as selenium, vitamins, transferrin, insulin, basic fibroblast growth factor, as well as buffers to maintain the pH stability of the solution, antioxidants and other substances. We tried using DMEM / F12 as the base medium, supplemented with human platelet lysate instead of FBS. We observed that cell proliferation remained strong, but in vitro osteogenic and adipogenic abilities decreased. We then added translocase, insulin, and basic fibroblast growth factor, and observed that cell morphology more closely resembled native stem cells: small, translucent cells with clear outlines and a high nuclear-cytoplasmic ratio. In vitro trilineage differentiation capacity remained strong, suggesting that these additives play a key role in maintaining stem cell stemness. To control the cloning efficiency of single stem cells, we also added CloneR, and observed that cells maintained strong proliferation even at low seeding densities. We ultimately settled on the complete HA-MSC medium described in S1-1-2.

[0119] S1-1-2. Preparation of HA-MSC complete culture medium: Add 20 ml TeSR-E8 Supplement to 480 ml TeSR-E8 Basal Medium, mix thoroughly by inversion, and then add 20 ml CloneR. Adjust the pH to 7.2-7.4 with 6.6% NaHCO3 and store in a refrigerator at 4°C until use.

[0120] S1-2. Optimization of the culture environment

[0121] S1-2-1. Coating Fluid Optimization and Screening: Focusing on creating a pristine microenvironment for stem cell growth, we considered optimizing the two-dimensional space. We switched from pre-treating the culture flasks with serum and / or mouse embryonic fibroblast feeder layers to using gelatin, Matrigel, and LN521. After repeated experimentation, we discovered that gelatin coating rarely resulted in growth when primary cells were isolated. After eliminating gelatin coating, we investigated Matrigel and LN521 extracellular matrices, finding that both maintained a relatively pristine cell state for our isolated cells. Ultimately, we settled on the coating fluid and culture flask treatment methods described in S1-2-2.

[0122] S1-2-2. Coating solution for HA-MSC culture flask: slowly thaw the laminin LN 521 stock solution at 2-8°C and use 1× DPBS (Ca 2+ / Mg2+ ) Dilute the laminin LN521 stock solution to 5μg / ml to prepare the STSC coating solution. 2 Add 5 ml of coating solution to evenly cover the entire bottom of the bottle, incubate at 2-8 ° C overnight, and mark the bottle as LN521-CoatedT75cm 2 .

[0123] S2. Primary culture of human umbilical cord HA-MSCs

[0124] S2-1. In a clean bench, repeatedly rinse the collected human umbilical cord with physiological saline containing 100u / ml of double-antibody, remove residual blood, and place in a 10cm culture dish.

[0125] S2-2, first use tissue scissors to cut the umbilical cord into 1mm pieces 3 Each small tissue block was transferred to a 1.5ml EP tube. The tissue in the EP tube was separated with iris scissors until it became a paste and then transferred to LN521-Coated T75cm 2 In culture flask.

[0126] S2-3. After adding 3 ml of HA-MSC complete culture medium to each bottle, shake the bottle slowly to evenly disperse the microtissue blocks. Mark the culture time, cell name and passage number on the bottle and place it in an incubator at 37°C and 5% CO2 for primary culture.

[0127] On day 3 of S2-4, add 3 ml of complete culture medium. Change the medium every 3-4 days thereafter and observe cell growth under an inverted phase-contrast microscope. When changing the medium, gently remove the old culture medium with a pipette and discard it. Do this gently, taking care not to dislodge any adherent tissue fragments.

[0128] S2-5. During subculturing, the detached tissue pieces are collected and secondary adherent culture can be performed according to the above method.

[0129] Subculture of S3 and HA-MSCs

[0130] When the primary cells grow to about 80% confluence, they need to be subcultured. The specific steps are as follows:

[0131] S3-1. Remove the cells from the incubator, discard the old culture medium in the clean bench, and gently rinse once with saline.

[0132] S3-2. Add 4 ml of Recombinant Trypsin-EDTA Solution to each bottle of primary HA-MSCs and place them in an incubator at 37°C and 5% CO2 for 3-5 minutes.

[0133] S3-3. After complete cell detachment under a microscope, add an equal amount of HA-MSC complete medium to terminate digestion. Collect the cells into a 50 ml centrifuge tube, add physiological saline, and centrifuge at 1500 rpm for 4 minutes.

[0134] S3-4. After centrifugation, discard the supernatant, add 2 ml of complete culture medium, gently pipette to mix the cells, and count the number of living cells using trypan blue staining.

[0135] S3-5, according to 5000 cells / cm 2 The cells were seeded into new LN521-coated T75 culture flasks at a seeding density of 1:1.

[0136] S3-6. Mark the culture time, cell name, and passage number on the bottle and place it in a 37°C, 5% CO2 incubator for expansion. Take photos of each passage.

[0137] S4. Highly active MSC cryopreservation

[0138] HA-MSCs in the logarithmic growth phase were selected and digested with Recombinant Trypsin-EDTA Solution. After the cells shrank and became round, the corresponding complete culture medium was added to terminate the process. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. TM Gently pipette D10 Cryopreservation Medium to evenly distribute the cells. Then aliquot into sterile cryopreservation tubes, adding 1.8 ml of liquid to each tube. Label the cryopreservation tubes with the cell name, batch number, and freezing time, and keep records. Place the cryopreservation tubes containing cells in a programmed cooling box at -80°C. After 4 hours, store in a liquid nitrogen tank at -196°C until use.

[0139] Example 2: Preparation method of conventional MSCs

[0140] The conventional MSC preparation method includes the following steps:

[0141] S1. Reagent Preparation

[0142] S1-1. Preparation of MSC complete culture medium: Add 55 ml of fetal bovine serum to 500 ml of DMEM / F12 culture medium, adjust the pH value to 7.2-7.4 with 6.6% NaHCO3, and store in a refrigerator at 4°C until use.

[0143] S1-2, MSC uses ordinary T75cm 2 Culture flask.

[0144] S2. Primary culture of human umbilical cord MSCs

[0145] S2-1. In a clean bench, repeatedly rinse the collected human umbilical cord with physiological saline containing 100u / ml of double-antibody, remove residual blood, and place in a 10cm culture dish.

[0146] S2-2, first use tissue scissors to cut the umbilical cord into 1mm pieces 3 Each small tissue block was transferred to a 1.5ml EP tube. The tissue in the EP tube was separated with iris scissors until it became a paste and then transferred to a regular T75cm 2 In culture flask.

[0147] S2-3. After adding 3 ml of MSC complete culture medium to each bottle, shake the bottle slowly to evenly disperse the microtissue blocks. Mark the culture time, cell name and passage number on the bottle and place it in an incubator at 37°C and 5% CO2 for primary culture.

[0148] On day 3 of S2-4, add 3 ml of complete culture medium. Change the medium every 3-4 days thereafter and observe cell growth under an inverted phase-contrast microscope. When changing the medium, gently remove the old culture medium with a pipette and discard it. Do this gently, taking care not to dislodge any adherent tissue fragments.

[0149] S2-5. During subculturing, the detached tissue pieces are collected and secondary adherent culture can be performed according to the above method.

[0150] S3. Subculture of MSCs

[0151] When the primary cells grow to about 80% confluence, they need to be subcultured. The specific steps are as follows:

[0152] S3-1. Remove the cells from the incubator, discard the old culture medium in the clean bench, and gently rinse once with saline.

[0153] S3-2. Add 4 ml of 0.25% trypsin-EDTA solution to each bottle of primary MSCs and place them in an incubator at 37°C and 5% CO2 for 3-5 minutes.

[0154] S3-3. After the cells have completely detached under a microscope, add an equal amount of MSC complete medium to terminate digestion. Collect the cells into a 50ml centrifuge tube, add physiological saline, and centrifuge at 1500 rpm for 4 minutes.

[0155] S3-4. After centrifugation, discard the supernatant, add 2 ml of complete culture medium, gently pipette to mix the cells, and count the number of living cells using trypan blue staining.

[0156] S3-5, according to 5000 cells / cm 2 The cells were seeded into new T75 culture flasks at a seeding density of 1:1.

[0157] S3-6. Mark the culture time, cell name, and passage number on the bottle and place it in a 37°C, 5% CO2 incubator for expansion. Take photos of each passage.

[0158] S4. Conventional MSC cryopreservation

[0159] Select MSCs in the logarithmic growth phase and digest them with Recombinant Trypsin-EDTA Solution. After the cells shrink and become round, add the corresponding complete medium to terminate the process. Centrifuge at 1500 rpm for 4 minutes and discard the supernatant. Add the prepared freezing solution (containing 10% DMSO and 5% glycerol) to the MSCs and adjust the cell density to 4×10 6 Gently pipette to evenly distribute the cells. Then aliquot into sterile cryovials, adding 1.8 ml of liquid to each tube. Indicate the cell name, batch number, and freezing time on the cryovials and keep records. Place the cryovials containing cells in a programmed cooling box at -80°C. After 4 hours, store in a -196°C liquid nitrogen tank until ready for use.

[0160] Example 3: Comparison of Highly Active MSCs and Conventionally Cultured MSCs

[0161] In the following text and the accompanying drawings, HA-MSC refers to highly active MSC, and MSC refers to conventionally cultured MSC.

[0162] 1. Growth Morphology of Primary Cultures of Human Umbilical Cord HA-MSCs and MSCs

[0163] Using the whole-tissue adherence method, which does not strip away blood vessels or the capsule, two types of stem cells were cultured. Umbilical cord HA-MSCs were cultured in TeSR-E8 medium containing 5% ClonR. On day 8, cells were observed to be scattered around the tissue mass in a culture flask coated with laminin LN 521. Umbilical cord HA-MSCs were small, short, and spindle-shaped, with a high nuclear-to-cytoplasmic ratio. On day 12 after the medium was changed, a large number of cells were observed to migrate from the periphery of the tissue mass. Figure 1 A, 1B). On the 10th day, umbilical cord MSC cells were observed to grow sporadically around the tissue mass in an elongated spindle shape. On the 15th day, they grew in a swirling fusion pattern ( Figure 1 C, 1D). HA-MSCs are smaller in size, have a higher nuclear-cytoplasmic ratio, and contain fewer foreign cells in primary cells compared to MSCs.

[0164] 2. Subculture of Human Umbilical Cord HA-MSCs and MSCs and 3D Holographic Live Cell Dynamic Observation

[0165] 2.1 Subculture

[0166] When the primary cells grow to about 80% confluence, they are subcultured. The two stem cells that are subcultured to P3 maintain their morphology and grow well ( Figure 2 A, 2D). Compared with cells derived from the same umbilical cord, HA-MSCs maintained a short rod-shaped morphology and good activity until P9. Figure 2 B, 2C); P9 MSC cells increased in size, became wider and flatter, and some cells became polygonal. The number of granules in the cytoplasm increased, indicating an aging state ( Figure 2 E, 2F).

[0167] 2.23D holographic dynamic observation of living cells

[0168] Under a 3D holographic live cell microscope, HA-MSC and MSC of the P3 generation were dynamically observed for 48 hours. One cell field of view was selected for dynamic observation. HA-MSC was small in size, with regular cell body edges, little cytoplasm, a distinct nucleus and obvious nucleolus, and dense genetic material with strong refractive index in the nucleus, indicating that it was in the early stage of division. Before the end of the 48-hour observation, HA-MSC was about to enter the division phase, and MSC was larger in size, with irregular cell bodies and a small number of particles visible in the cytoplasm. The cell nucleus was small and the nucleolus was obvious. Before the end of the 48-hour observation, the MSC did not divide ( Figure 3 ). Figure 3 The HA-MSC cells showed larger nuclei, less cytoplasm, and a gradual increase in nuclear staining, indicating they were about to enter the division phase. The MSC cells were irregular in size, with abundant cytoplasm, large nuclei, prominent nucleoli, and a constantly changing and wandering cell body (×400).

[0169] 3. Growth Curve and Population Doubling Time of Human Umbilical Cord HA-MSCs and MSCs

[0170] 3.1 Growth curve

[0171] The growth curves of HA-MSC and MSC were measured by MTS method. The two P3 cells were in the retention period 2 days after inoculation, with no obvious proliferation. They entered the logarithmic growth period 3-7 days later, with vigorous cell growth and good activity ( Figure 4 A); on the 8th day, it entered the plateau phase, and the growth curve showed a typical "S" shape. Compared with the same generation HA-MSC, the logarithmic growth curve of P9 MSC was flat, and it entered the plateau phase earlier, indicating that the cell proliferation ability was reduced ( Figure 4 B).

[0172] 3.2 Population doubling time

[0173] The population doubling time is the time required for the cell number to double under culture conditions, reflecting the proliferation ability of the cells. The population doubling time (PDT) of the two cells was calculated by taking the OD value of the logarithmic growth phase on the growth curve. The PDT of P3-HA-MSC was lower than that of P3-MSC (P < 0.01); the PDT of P9-HA-MSC was significantly lower than that of P9-MSC (P < 0.001); This shows that the proliferation ability of HA-MSC of P3 and P9 is higher than that of MSC ( Figure 5 ).

[0174] 4. Transmission Electron Microscopy Observation and Nuclear-Cytoplasmic Ratio of Human Umbilical Cord HA-MSCs and MSCs

[0175] 4.1 Transmission electron microscopy observation

[0176] HA-MSC cells under transmission electron microscopy showed slight edema. The cell membrane was intact, with abundant pseudopodia and protrusions on the surface. The cell shape was slender, the intracellular matrix was uniform, the organelles were evenly distributed, and the cells were slightly swollen. The cell nucleus (N) was irregular in shape, with an intact nuclear membrane and slightly marginalized heterochromatin ( Figure 6 A); Mitochondria (M) are abundant, most with clear structures, intact membranes, and parallel cristae. Some membranes have a lighter matrix and fewer cristae. The rough endoplasmic reticulum (RER) is significantly expanded, with a large number of flocculent aggregates and endoplasmic reticulum retention in the cisternae. The Golgi apparatus (Go) is enlarged and the vesicle membrane is expanded. A small number of autolysosomes (ASS) are present, and two ( Figure 6 B).

[0177] Under the transmission electron microscope, the MSC cells were slightly edematous, with some organelles slightly swollen and the cytoplasm evenly distributed. The cell membrane was intact, with abundant pseudopodia (PS) around it in a slender shape, and a small number of organelles in the membrane were vacuolated. The nucleus (N) was large and irregular in shape, with an intact nuclear membrane, a normal perinuclear space, uniform chromatin, a large nucleolus (Nu), and a dense structure ( Figure 7 A); Mitochondria (M) are abundant, mostly slightly swollen, with pale intramembrane matrix, broken and reduced cristae, and in some severe cases, cristae disappear and become vacuolated; the rough endoplasmic reticulum (RER) is partially dilated, and a small amount of flocculent aggregates can be seen in the endoplasmic reticulum cisternae. Lysosomes (Ly) are occasionally present. Glycogen granules (GL) are locally aggregated. There are a large number of autophagolysosomes (ASS), with 5 ( Figure 7 B).

[0178] 4.2 Nuclear-cytoplasmic ratio

[0179] The nuclear-cytoplasmic ratio was calculated by measuring 3 cells in each group. The nuclear-cytoplasmic ratio of HA-MSC was significantly higher than that of MSC (P < 0.01). Figure 8 .

[0180] 5. Cell Cycle

[0181] By flow cytometry, 79.48% of HA-MSCs and 63% of MSCs at P3 were in the G0 / G1 phase, which is consistent with the characteristics of MSCs cultured in vitro. HA-MSCs have a high proportion of G0 / G1 phase, proving that they have a high differentiation potential ( Figure 9 ).

[0182] 6. Expression of surface markers

[0183] HA-MSCs and MSCs highly expressed MSC-related surface markers CD73, CD105, and CD90, and low or no expression of CD34 and CD45 (Table 1, Figure 10 ), which is consistent with the phenotypic characteristics of MSC; HA-MSC partially expressed ESC-related surface markers SSEA-3, SSEA-4 and TRA-1-60, while MSC was affected by the serum in the culture environment, with SSEA-4 expression of 25.8% and no expression of SSEA-3 and TRA-1-60 (Table 1, Figure 11 ).

[0184] Table 1 Expression percentage of surface markers of HA-MSC and MSC

[0185]

[0186] 7. Western blot (WB) detection of pluripotency transcription factor expression

[0187] Western blot was used to detect the expression of two stem cell pluripotency transcription factors. It was found that HA-MSC expressed pluripotency transcription factors SOX2, OCT4, and Nanog. Analysis of the grayscale value of the target band showed that the expression levels of SOX2 and Nanog were significantly higher than those of MSC (P < 0.001); while MSC only expressed OCT4, and the expression level was no different from that of HA-MSC (P > 0.05) ( Figure 12 ), indicating that HA-MSCs are in an earlier stage of development than MSCs.

[0188] 8. Identification of Trilineage Differentiation Ability

[0189] Adipogenic differentiation: After 14 days of adipogenic differentiation induction, red lipid droplets can be seen in the cytoplasm after Oil Red O staining; osteogenic differentiation: After 21 days of induction, calcium nodules can be stained red by Alizarin Red; chondrogenic differentiation: After 14 days of induction, cartilage collagen matrix can be stained blue by Alpha-Cyanine Blue. From the staining degree analysis, HA-MSC has stronger ability to differentiate into three lineages than MSC ( Figure 13 ).

[0190] 9. Expression levels of marker molecules after differentiation of the three germ layers

[0191] After the two stem cells were induced to differentiate into three germ layers, two specific markers were selected for each germ layer (ectoderm markers: Nestin, PAX6; mesoderm markers: Meso-CXCR4, Brachyury (T); endoderm markers: Endo-CXCR4, SOX-17) and the expression of markers was detected by immunofluorescence. The results showed that HA-MSCs could differentiate into the ectoderm, with the cell morphology showing an elongated neural progenitor cell-like shape, and expressed ectoderm-specific markers by immunofluorescence identification; at the same time, they could also differentiate into the mesoderm and endoderm and express specific markers of the corresponding germ layers, while MSCs could only differentiate into the mesoderm ( Figure 14-16 ).

[0192] Example 4: Ovarian aging reversal test

[0193] 1. Animal Origin

[0194] Macaques for this experiment were purchased from the Experimental Animal Center of the Kunming Institute of Zoology, Chinese Academy of Sciences, under the Laboratory Animal Production License No. SCXK(Yunnan)K2017-0003. Young male macaques were 8 years old and weighed 2 kg; female macaques were 22-28 years old and weighed 3.5-6.5 kg. They were housed at the Experimental Animal Center of the Basic Medical Laboratory of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army, under the Laboratory Animal Use License No. SYXK(Military)2017-0051. They were of normal size and maintained in a clean, hygienic, and temperature-controlled environment. All animal studies were approved by the Laboratory Animal Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army, under the approval number 2020-034(Ke)-01.

[0195] 2. Screening of Ovarian Aging Macaques

[0196] Nine elderly female macaques meeting the inclusion criteria were screened and exhibited a distinct aging phenotype, with dull or partially lost fur, sparse teeth, and poor mobility. Abdominal ultrasound revealed ovarian atrophy or non-detectable ovaries, with fewer than two follicles or no follicles. Peripheral blood sex hormone monitoring for two consecutive months revealed significantly lower levels of E2, P, and AMH in elderly macaques compared to reproductive-aged macaques. Inhibin B was below the limit of detection, while FSH and LH levels were undetectable, as they showed no cross-reactivity with human reagents.

[0197] 3. Animal Grouping and HA-MSC Transplantation Treatment

[0198] The 9 elderly female macaques were randomly divided into HA-MSC treatment group (n=3), MSC treatment group (n=3), and elderly model group (n=3). 7Double-labeled HA-MSCs and MSCs were transplanted into the hind limb vein at a dose of 10 cells / kg / time. The elderly model group was infused with an equal volume of normal saline as a control. The infusion was continued for three consecutive days, and no adverse reactions occurred after the infusion.

[0199] 4. Evaluation of the efficacy of HA-MSC transplantation therapy

[0200] 4.1 Mating and menstrual observation

[0201] After cell transplantation, the monkeys were housed with young male macaques. Two macaques in the HA-MSC group were observed to resume menstruation 7 and 10 days after cell transplantation, respectively. One macaque resumed menstruation on day 29, while the rest showed no signs of menstruation. On day 90 after transplantation, the macaques in the HA-MSC group that had resumed menstruation twice were observed mating with young male macaques.

[0202] 4.2 Serum AMH monitoring

[0203] Compared with the levels before transplantation, the AMH levels of the HA-MSC and MSC treatment groups increased at 1 month after transplantation. The increase in the HA-MSC treatment group continued until the 2nd month, when it began to decline and was slightly higher than the level before transplantation at 5 months. The AMH level of the MSC treatment group decreased to the level before transplantation after the increase at 1 month. The AMH level of the elderly model group did not increase during the observation period ( Figure 17 ).

[0204] 4.3 Abdominal ultrasound of macaques

[0205] HA-MSC treatment group: 1 / 3 (94346) left ovary: 0.95 cm × 0.57 cm × 0.57 cm, OV = 0.16 cm 3 , 2 follicles in the left ovary: 1, 0.23cm×0.22cm, 2, 0.49cm×0.45cm; right ovary: 0.9cm×0.5cm×0.49cm, follicle: 0.18cm×0.17cm, see ( Figure 18 ).

[0206] MSC treatment group: 2 / 3 (99324, 98318) 99324 left ovary: 1.16cm×0.55cm×0.48cm, OV=0.16cm 3 , follicle: 0.64cm×0.53cm, right ovary not shown. 98318 left ovary: 0.98cm×0.63cm×0.53cm, OV=0.17cm 3 , follicle: 0.66cm×0.25cm, right ovary: 0.79cm×0.31cm×0.27cm, OV=0.04cm 3 , no follicles. Elderly model group: bilateral ovaries were not shown.

[0207] 4.4 Ovarian gross anatomy and histological changes

[0208] At 5 months of age, macaques were euthanized and specimens were obtained. Gross specimens revealed that the ovaries of the HA-MSC-treated group were clearly visible, with a grayish-white color. These ovaries were larger than those in the aged control and MSC-treated groups. HE staining revealed that follicles of all levels were visible in the HA-MSC-treated group, with neatly arranged medullary stroma and distinct boundaries. In the MSC-treated group, no mature follicles were observed, but a small number of primary and primordial follicles were visible. In the aged control group, occasional atretic follicles were observed, along with abundant fibrous connective tissue.

[0209] 4.5 Changes in ovarian tissue fibrosis

[0210] Masson staining was used to reflect the fibrosis of ovarian tissue. Collagen fibers in the tissue appeared blue, while muscle fibers, cellulose, and red blood cells appeared red. The staining results showed that the elderly control group had a large amount of collagen fiber deposition in the medulla and interstitium, with a large fiber area and obvious fibrosis. The HA-MSC treatment group had a small amount of collagen fiber deposition under the cortex layer, with a smaller area, and the degree of fibrosis was significantly reduced compared with the elderly control group (P < 0.001). The MSC treatment group had some collagen fiber deposition in the medulla, and the degree of fibrosis was significantly reduced compared with the elderly control group (P < 0.001). Figure 19 ).

[0211] 5. Mechanism of HA-MSCs in reversing ovarian aging

[0212] 5.1 Dual-labeled HA-MSC colonization

[0213] Under the fluorescence microscope, the HA-MSC treatment group showed green fluorescence expression of eGFP-labeled cells (red circle), and blue was the cell nucleus. No green fluorescence was found in the MSC treatment group and the elderly model group. In addition, Prussian blue staining solution showed that the HA-MSC treatment group had SPION-labeled cells colonized in the subcortex, medulla, and around the follicles of the ovary. SPIONs appeared blue (red circle) and the cell nuclei were red (red circle). Figure 20 ).

[0214] 5.2 Changes in Reactive Oxygen Species Content in Ovarian Tissue

[0215] The DHE method was used to detect the level of reactive oxygen species (ROS) in ovarian tissue. DHE will be oxidized into red fluorescence in the presence of ROS in the tissue. The intensity of red fluorescence is proportional to the level of ROS in the tissue. The results showed that the ROS level in the elderly model group was higher, and the ROS level decreased after cell therapy. The ROS level in the HA-MSC treatment group was lower than that in the MSC treatment group (P < 0.05), indicating that HA-MSC inhibited the production of reactive oxygen species in ovarian tissue, suggesting an anti-oxidative stress effect ( Figure 21 ).

[0216] 5.3 Changes in vascular density in ovarian tissue

[0217] Immunofluorescence technology was used to detect the vascular endothelial cell marker CD34 to reflect the density of blood vessels in ovarian tissue. Red fluorescence indicates CD34-positive cells, and blue indicates cell nuclei. The results showed that CD34 expression was lowest in the elderly model group. After cell therapy, CD34 expression increased, indicating increased blood vessel density and angiogenesis. The CD34 expression level in the HA-MSC treatment group was higher than that in the MSC treatment group (P < 0.05), indicating that HA-MSC has a stronger ability to promote angiogenesis than MSC ( Figure 22 ).

[0218] 5.4 Changes in ovarian cell proliferation and apoptosis

[0219] Immunofluorescence technology was used to detect ovarian cell proliferation and apoptosis using Ki67 antibody and Tunel staining. In the proliferation assay, red fluorescence indicates Ki67-positive proliferating cells, and blue indicates cell nuclei; in the apoptosis assay, red fluorescence indicates positive apoptotic cells, and blue indicates cell nuclei. The results showed that compared with the elderly model group, the ovarian cell proliferation rate increased and the apoptosis rate decreased after cell transplantation. Among them, the ability of HA-MSC to promote proliferation and inhibit apoptosis was greater than that of MSC (P < 0.01) ( Figure 23 ).

[0220] 5.5 Expression of ovarian granulosa cell-specific markers

[0221] Immunofluorescence technology was used to detect the expression of AMH and FSHR, the specific marker molecules of granulosa cells. Red fluorescence indicates AMH and FSHR positive cells, and blue indicates cell nuclei. The results showed that compared with the elderly model group, the expression of AMH and FSHR increased after cell therapy, and the expression level in the HA-MSC treatment group was greater than that in the MSC treatment group (P < 0.01) ( Figure 24 ).

[0222] 5.6 Measurement of relative telomere length in peripheral blood mononuclear cells

[0223] The Q-PCR method was used to detect the relative length of telomeres in peripheral blood mononuclear cells in 6 age groups and 1 month (T1) and 3 months (T3) after HA-MSC treatment. The results of the relative telomere length measurements in the 6 age groups showed that telomere length gradually shortened with age. After 1 month of HA-MSC treatment, the telomeres of the 22-year-old group were extended to between 13 and 17 years old. After 3 months of treatment, the telomere length was measured again and returned to the pre-treatment level ( Figure 25 ).

[0224] 5.7 In vitro co-culture of human ovarian granulosa cells and HA-MSCs

[0225] 5.7.1 Transwell Co-culture of Human Ovarian Granulosa Cells and HA-MSCs

[0226] H2O2-induced senescent hGC were co-cultured with HA-MSC and MSC, respectively, and their ability to reverse senescent hGC was assessed using β-galactosidase staining. Senescent cells are stained blue by β-galactosidase. The results showed that both HA-MSC and MSC could reverse senescent hGC after 72 hours of co-culture. Compared with the elderly model group, the number of blue-stained senescent cells decreased (P < 0.001), and there was no difference with the normal control group. There was no difference in the ability of the two cells to reverse senescent hGC ( Figure 26 ).

[0227] 5.7.2 Dynamic Changes of 3D Holographic Live Cells Co-cultured with Human Ovarian Granulosa Cells and HA-MSCs

[0228] After hGC was treated with 273mM H2O2 for 24 hours, the results of 3D holographic live cell microscopy for 36 hours showed that the number of granular autophagosomes in the four hGC cells photographed under the field of view increased, the cell activity was poor, there was no proliferation within 36 hours, and the cells were in a senescent state ( Figure 27 A). After the addition of HA-MSCs, two HA-MSCs entered the original observation field, one of which began to divide into two STSCs after attaching to the wall. Six hours later, the other HA-MSC also began to divide. After the addition of HA-MSCs, the autophagolysosomes in the original four senescent hGCs gradually decreased, and the hGCs began to divide one by one. By the end of the 36-hour observation period, all four hGCs had divided and proliferated ( Figure 27 B).

[0229] 5.8 Detection of the ability of HA-MSC to induce granulosa cells in vitro

[0230] After 20 days of induction of HA-MSC and MSC by the prepared granulosa cell complete induction solution, the morphology of some cells gradually changed from fibroblast-like to round and oval-like, similar to granulosa cells ( Figure 28 Immunofluorescence technique was used to identify the induced cells. Both cells expressed AMH and FSHR, markers specific for granulosa cells. HA-MSCs were more effective than MSCs in inducing granulosa cells. Figure 29 To identify whether the cells were induced to be functional granulosa cells, the collected culture supernatant was tested for AMH. The results showed that a small amount of AMH secretion began at D4 of induction, and the amount of AMH secretion gradually increased with the extension of induction time ( Figure 30 ).

[0231] In summary, compared with conventionally cultured MSCs, HA-MSCs have higher proliferation activity, smaller size, higher nuclear-cytoplasmic ratio, and highly express embryonic stem cell-related marker antigens SOX2, Nanog, and OCT4. They have the potential to differentiate into neural, myocardial, and hepato-intestinal progenitor cells derived from the three germ layers, indicating that these cells are a sub-totipotent stem cell among UC-MSCs.

[0232] Furthermore, HA-MSCs were shown to be more effective than conventionally prepared MSCs in treating ovarian aging in macaques, improving ovarian tissue structure and secretory function, regenerating follicles, and restoring ovulation and menstruation, but failing to restore reproductive function. HA-MSCs were found to reverse ovarian aging through multiple mechanisms, including differentiation into GCs, reversing GC aging, resisting oxidative stress, inhibiting ovarian cell apoptosis, increasing vascular density, promoting ovarian GC division and proliferation, and promoting the expression of specific marker molecules. These results provide a new technical approach and scientific basis for the clinical treatment of ovarian aging.

[0233] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A use of highly active human umbilical cord mesenchymal stem cells (MSCs) in the preparation of an anti-aging preparation, characterized in that: The method for preparing the highly active MSCs comprises the following steps: S1. Reagent Preparation S11. Prepare high-activity MSC complete medium: Add TeSR-E8 Supplement to TeSR-E8 Basal Medium, mix thoroughly by inversion, and then add CloneR. Adjust the pH to 7.2-7.4 with 6.6% NaHCO3 and store at 4°C until ready for use. S12. Preparation of high-activity MSC culture flasks: Slowly thaw the laminin LN521 stock solution at 2-8°C and use a solution containing Ca 2+ / Mg 2+ Dilute the laminin LN521 stock solution with 1× DPBS to make the coating solution. Add the coating solution to the culture flask to evenly cover the entire bottom of the flask and incubate at 2-8°C for later use. S2. Primary Culture Stem cells are cultured using the whole-tissue adherence method without stripping off blood vessels and capsule; S21. Cleaning the collected human umbilical cord to remove residual blood; S22, disaggregate the umbilical cord into small tissue pieces and transfer them into a high-activity MSC culture flask; S23. Add high-activity MSC complete medium to the high-activity MSC culture bottle, shake the bottle slowly, and place it in an incubator at 37°C and 5% CO2 for primary culture; S24: On day 3, high-activity MSC complete culture medium was added, and the medium was changed every 3-4 days thereafter; S25. During passage, the detached tissue fragments were collected and cultured for a second time according to the above method; S3. Subculture When the primary cells grow to about 80% confluence, subculture them; S31. Remove cells from the incubator, discard old culture medium, and gently rinse once with normal saline. S32, primary high-activity MSCs were added with Recombinant Trypsin-EDTA Solution and placed in a 37°C, 5% CO2 incubator for 3-5 minutes; S33. After the cells were completely detached, an equal amount of high-activity MSC complete medium was added to terminate the digestion, and the cells were collected, added with normal saline, and centrifuged at 1500 rpm for 4 mins. S34. After centrifugation, discard the supernatant, add high-activity MSC complete medium, gently pipette to mix the cells, and count the number of viable cells using trypan blue staining; S35, according to 5000 cells / cm 2 The cells were seeded into new high-activity MSC culture flasks at a seeding density of S36. Place in an incubator at 37°C and 5% CO2 for expansion culture; The anti-aging preparation is a medicine for treating ovarian aging.

2. The use according to claim 1, characterized in that The method for preparing highly active MSCs further comprises S4, cryopreservation of highly active MSCs, Select highly active MSCs in the logarithmic growth phase and digest them with Recombinant Trypsin-EDTA Solution. After the cells shrink and become round, add highly active MSC complete medium to terminate the digestion. After centrifugation at 1500 rpm for 4 minutes, discard the supernatant, gently pipette to make the cells uniform, and then divide them into sterile cryopreservation tubes. Place the cryopreservation tubes containing cells in a programmed cooling box in a -80°C refrigerator. After 4 hours, place them in a -196°C liquid nitrogen tank for storage until use.

3. The use according to any one of claims 1 to 2, characterized in that In step S11, the ratio of TeSR-E8 Basal Medium, TeSR-E8 Supplement, and CloneR is 24:1:

1.

4. The use according to claim 3, characterized in that In step S12, the laminin LN521 stock solution is diluted to 5 μg / ml.

5. The use according to claim 4, characterized in that In step S21, the human umbilical cord is repeatedly rinsed with physiological saline containing 100u / ml of double antibody.

6. The use according to claim 5, characterized in that The anti-aging preparation further comprises one or more pharmaceutically acceptable excipients, diluents or carriers.

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