Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells Restore Ovarian Function Of Ovarian Insufficiency And The Exploration Of Therapeutic Mechanism

As the women’s chief sexual organ,the ovary is critical for regulating female reproductive health.The causes of ovarian insufficiency include natural reasons,such as ovarian aging;pathological reasons,such as premature ovarian insufficiency,and premature ovarian failure.The ovarian function is essential to maintain various physiological activities of women.The decline of ovarian function leads to perimenopausal symptoms in women,which affect multiple organs,including genitourinary symptoms,decreased bone density,and cardiovascular disease.However,there is currently no cure to fundamentally restore ovarian function.The increased apoptosis and decreased proliferation of ovarian granulosa cells are the critical causes of follicular atresia and ovaria function incapacitation.And they are the regulatory mechanisms for the treatment of ovarian insufficiency.Mesenchymal stem cells and their exosome-mediated therapeutic methods are currently the focus of research in the field of regenerative medicine.This study aims to investigate the therapeutic effect of umbilical cord mesenchymal stem cell-derived exosomes in the confrontation of ovarian insufficiency and to further explore the in-depth internal therapeutic mechanism.Part one Identification of umbilical cord mesenchymal stem cells,and isolation and identification of exosomesObjective:To complete the identification of human umbilical cord mesenchymal stem cells（hUCMSCs）.To extract and identify exosomes derived from hUCMSCs（hUCMSC-Exos）for subsequent cell and animal experiments.Methods:1.Third passage hUCMSCs were purchased from Qilu Cell Therapy Technology（Shandong,China）.We confirm their morphology by using a light microscope.Then,Alizarin Red staining was used for osteogenic identification;and Oil Red O staining was conducted for adipogenic differentiation.Finally,Flow cytometry was used to detect MSC-related surface markers（CD34,CD44,CD45,CD73,CD90,CD105,and HLA-DR）.2.Extract exosomes derived from hUCMSCs by ultracentrifugation.The hUCMSC-Exos were experimentally identified.First,we used Flow Nano Analyzer to measure exosomal size distribution and concentration.Then,we observed their morphologies by using transmission electron microscopy.Finally,surface markers of hUCMSC-Exos,including CD9,CD63,and CD81,were detected by Flow cytometry.Results:1.The hUCMSCs grow into long fusiform cells with fibroblast-like morphologies.Immunophenotypes of MSCs were characterized by flow cytometry using specific biomarkers.The results showed a high expression of CD73（98.8%）,CD105（98.1%）,CD44（99.9%）,and CD90（99.9%）,and a low expression of CD34（0.4%）,CD45（0.7%）,and HLA-DR（0.2%）in hUCMSCs.We induced hUCMSCs to develop into different cell types in a conditional culture system.The positive results of Alizarin Red and Oil Red O staining for induced hUCMSC demonstrated their multi-lineage differentiation potential.2.The hUCMSC-Exos were obtained by ultracentrifugation and were identified according to minimal experimental requirements for the definition of extracellular vesicles.The exosomes exhibited a cup-shaped morphology with their size distributions ranging from about 30 to 100 nm at a concentration of 8.69×10¹⁰particles/ml.Additionally,the surface marker proteins were positive in exosomes by conducting flow cytometry,including CD9,CD81,and CD63.These results confirmed that exosomes had been isolated from hUCMSCs.Conclusions:1.In this experiment,the international standard was used to identify hUCMSCs.Meanwhile,hUCMSC-Exos were successfully extracted by ultracentrifugation in this experiment,and their morphology,particle diameter,and exosomal marker were identified by using transmission electron microscopy,particle size analysis,and flow cytometry.2.Through the above experiments,the process of hUCMSCs culture and identification was established,and hUCMSC-Exos that met various standards were successfully extracted,which laid foundations for its application in subsequent experiments.Part two Exosomes derived from human umbilical cord mesenchymal stem cells promote granulosa cells proliferation by regulating the Hippo pathway in the treatment of premature ovarian insufficiencyObjective:The hUCMSC-Exos will be used to treat premature ovarian insufficiency（POI）mouse model and granulosa cell model in vitro to explore whether they can improve POI ovarian function and to investigate the intrinsic therapeutic mechanism.Methods:1.POI mice models were established through intraperitoneal injection of cyclophosphamide.Subsequently,transplantation of hUCMSC-Exos was conducted to administer POI mice.Ovaries and plasma of these mice models were harvested after two weeks of exosomal treatment.Ovarian morphology and follicle number were assessed by HE staining.Moreover,ELISA was used to detect hormone levels,which are related to ovarian function in serum.To assess the recovery of reproductive ability,we recorded the rate of pregnancy,the number of offspring,and the time of birth in different groups.The proliferation of ovarian cells in vivo was detected by immunohistochemistry and immunofluorescence staining.Western blot analysis was conducted to estimate the protein levels of Hippo-and proliferation-associated molecules in vivo.2.To explore the underlying mechanisms of exosome-mediated treatment,we conducted Ed U and CCK-8 assays to assess the proliferative ability of ovarian granulosa cells that were cultured in vitro with hUCMSC-Exos and YAP inhibitor.Western blot analysis was conducted to estimate the protein levels of Hippo-and proliferation-associated molecules in vitro.Results:1.After transplantation of hUCMSC-Exos,the ovarian function-related hormone levels and the number of ovarian follicles returned to nearly normal degrees.Meanwhile,there was a significant improvement in reproductive outcomes after exosomal treatment.Furthermore,the improvement of ovarian function and proliferation was associated with the regulation of Hippo pathway.2.In vitro,co-culture with exosomes significantly elevated the proliferation of ovarian granulosa cells by regulating Hippo pathway.However,the positive effects on the proliferation of granulosa cells were significantly depressed when the key Hippo pathway molecule was inhibited.Conclusions:1.This study completed the construction of the POI mouse model and cell model in vitro.2.The hUCMSC-Exos promoted ovarian functions and proliferation by regulating the Hippo pathway.3.Therefore,exosomal transplantation could be a promising and efficient clinical therapy for POI in the near future.Part three Exosomes derived from human umbilical cord mesenchymal stem cells improve ovarian function in natural ovarian aging by regulating the level of apoptosisObjective:This study was designed to investigate the therapeutic efficiency of hUCMSC-Exos on ovarian aging and then to analyze the regulation of phosphatase and tensin homologdeleted on chromosome ten（PTEN）and apoptosis levels in this exosomal treatment.Methods:1.The mice were fed for 14 months to build the natural ovarian aging（NOA）mouse model,and 10-week healthy female young mice were set as the Normal group.After the NOA mice were treated with hUCMSC-Exos,we used HE staining of ovary sections to detect the changes of ovarian tissue morphology and the number of follicles in different groups.We used RT-q PCR and western blotting analysis to detect the expression of PTEN and key apoptosis-regulation molecules of mice in the three groups.Furthermore,TUNEL analysis and Ed U test were examined on the ovarian sections to confirm the significant difference in cell apoptosis and cell proliferation between different groups.2.Human ovarian granulosa cell line was used to construct the cell apoptosis model.After the cell apoptosis model was co-incubated with hUCMSC-Exos,the expression levels of PTEN and apoptosis-related molecules were detected by Western Blot and PCR assay.Furthermore,TUNEL analysis was used to confirm the difference in apoptosis between different groups.3.High-throughput sequencing was used to detect the highly expressed miRNA in hUCMSC-Exos,and bioinformatics analysis was used to find miRNAs that could target and regulate PTEN.And then verify the regulation mechanism of the miRNAs on PTEN and apoptosis molecules.Results:1.In the ovarian tissue of NOA mice,the results of Western Blot and PCR showed that the expressions of PTEN and apoptosis-related molecules（Bax,Caspase-3,Caspase-9）were significantly up-regulated,while the expressions of Bcl-2 were significantly down-regulated.TUNEL staining showed that the apoptosis level of NOA ovarian tissue was higher than that of young mice.After using hUCMSC-Exos to treat NOA mice,Western Blot and PCR results showed that hUCMSC-Exos could regulate the expression of PTEN molecules and apoptosis-related molecules;TUNEL analysis and Ed U test showed hUCMSC-Exos could down-regulate the level of apoptosis and up-regulate the level of proliferation.2.Compared with the normal cells,TUNEL staining showed that the level of apoptosis was increased in the apoptosis cell model,and the results of Western Blot and PCR experiments showed that the expressions of PTEN and apoptosis-related molecules were up-regulated,which confirms the cell model was successfully constructed.After using hUCMSC-Exos to co-culture with the cell apoptosis model,Western Blot and PCR results showed that hUCMSC-Exos could regulate the expression of PTEN molecules and apoptosis-related molecules;TUNEL analysis could down-regulate the level of apoptosis of cell model.3.High-throughput sequencing of hUCMSC-Exos miRNA showed that miR-21-5p was the highest expressed molecule.PCR results showed that the expression level of miR-21-5p in hUCMSC-Exos was significantly higher than that of miR-26a-5p.After co-incubation with hUCMSC-Exos,human granulosa cells could uptake hUCMSC-Exos,and the expression level of intracellular miR-21-5p increased.When hUCMSC-Exos was co-cultured with the cell apoptosis model,the inhibitor of miR-21-5p was added.PCR and Western Blot results showed that after the cocultivation of hUCMSC-Exos,the m RNA and protein expression levels of PETN in the cell apoptosis model were significantly decreased than those in the untreated group.But the trend of decreased PETN expression was reversed after transfection with the miR-21-5p inhibitor.Conclusions:1.hUCMSC-Exos can inhibit the apoptosis of granulosa cells by regulating PTEN molecules,and finally improve the ovarian function of NOA mice.2.The miR-21-5p is the highest expressed miRNA molecule in hUCMSC-Exos,and it regulates the apoptosis of ovarian granulosa cells by targeting PTEN.