| Background:Chemotherapy can significantly improve the survival rate of cancer patients,but it can also cause serious damage to female fertility,which can lead to a decrease in the number of follicles in the ovaries,which in turn can lead to endocrine disruption and reproductive dysfunction or primary ovarian insufficiency(POI)or,in more severe cases,to premature ovarian failure(POF).ovarian failure(POF).Chemotherapy in female cancer patients is one of the most common causes of POI.To date,there is no specific drug to treat POI,and it is difficult to reverse POI once it occurs.Therefore,it is of great concern how to protect against chemotherapy-induced reproductive toxicity,especially in young and fertile female patients.Several studies have found that extracellular vesicles(EVs)derived from mesenchymal stem cells(MSCs)can play an important role in the repair of injured tissues.Induced pluripotent stem cell derived mesenchymal stem cells(iPSC-MSCs)can be induced to differentiate from a wide variety of cells,are derived from a wide variety of sources,and can expand indefinitely.Laboratory-induced differentiation of iPSC-MSCs also has the advantage of low heterogeneity and avoidance of disease transmission.Thus EVs of iPSC-MSCs are very promising therapeutic options in regenerative medicine.However,to date,no studies have reported the use of iPSC-MSC-EVs in chemotherapy-induced ovarian damage.Objective:To investigate whether iPSC-MSC-EVs can have a palliative effect on chemotherapy-induced ovarian injury and to elucidate their potential mechanisms in tissue repair.Methods and Results:We differentiated iPSCs into iPSC-MSCs using the MSC Progenitor Cell Kit and used fluorescence-activated cell sorting for cells that successfully expressed the MSCs positive markers CD73,CD90,CD 105 while not expressing the MSCs negative markers(CD45/CD34/CD11b/CD19/HLA-DR).The cells were stably passaged and the supernatants of the third to seventh generation iPSC-MSCs were collected and the EVs were separated by ultrafiltration and centrifugation.iPSC-MSC-EVs were identified by protein immunoblotting,transmission electron microscopy,nanoparticle size analysis and RNA distribution analysis.iPSC-MSC-EVs were injected into adult mice from the tail vein at different times while using chemotherapy-induced ovarian injury.The protective effects of iPSC-MSC-EVs on chemotherapy-induced ovarian injury in mice were evaluated using H&E staining,follicle counting,and immunohistochemical staining.The ovaries of mice born 2-3 were cultured in vitro using chemotherapy-induced ovarian injury,while co-cultured with iPSC-MSC-EVs.The effect of iPSC-MSC-EVs on chemotherapy-induced apoptosis of primordial follicles in the ovaries was assessed using H&E staining,follicle counting,and immunohistochemical staining.Mouse primary granulosa cells were extracted for in vitro culture and chemotherapy was used to induce granulosa cell injury,while iPSC-MSC-EVs were used for co-culture.The effect of iPSC-MSC-EVs on cell viability was detected using the MTS kit,and proliferation apoptosis in granulosa cells was detected using western blot.RNA was extracted from primary mouse granulosa cells of control group,chemotherapy group,and chemotherapy and iPSC-MSC-EVs co-treatment group in vitro and then transcriptome sequencing was performed,and differentially expressed genes were analyzed and screened by bioinformatics analysis,and the relationship between samples was observed by PCA.iPSC-MSC-EVs were screened by KEGG enrichment analysis and signaling pathway analysis to attenuate the The signaling pathways of iPSC-MSC-EVs to mitigate the effects of chemotherapy damage were screened by KEGG enrichment analysis and signaling pathway analysis.Overexpression of ILK in granulocytes by lentiviral transfection and inhibition of ILK function in granulocytes using ILK inhibitor ILK-IN3.The effect of ILK on the cell viability of different treatment groups was examined by MTS.The effect of ILK expression in granulocytes on downstream pathways was examined using western blot to explore the role played by ILK in chemotherapy-induced granulocyte injury.miRNAs(microRNAs)in iPSC-MSC-EVs were sequenced,and the top 50 expressed miRNAs were analyzed and targets were predicted by bioinformatics analysis such as GO enrichment analysis,DIANA miRPath,miRWalk and miRTarBase.The analysis results were validated using qPCR.RNA in iPSC-MSC-EVs was degraded using RNase to verify the mechanism by which iPSC-MSC-EVs exert their functions.whether it is achieved by the RNA they carry.Results:Cell morphological observations showed that iPSCs were gradually transformed from round to typical MSCs long shuttle cells,and purified iPSC-MSCs were obtained by fluorescence-activated cell sorting and grew well after passaging.Protein immunoblotting showed that iPSC-MSC-EVs isolated by ultrafiltration were positive for EVs positive markers CD 63,CD 9,CD 81 and Hsp 70,but negative for EVs negative marker Calnexin.Under transmission electron microscopy,iPSC-MSC-EVs showed a typical circular cup-shaped EVs structure.Particle size analysis showed that the diameters of iPSC-MSC-EVs ranged from approximately 50-150 nm.RNA distribution analysis showed that the peak RNA extracted from iPSC-MSC-EVs was concentrated in the range of 20-200 nt.All experimental results indicate that we successfully extracted iPSC-MSC-EVs.The results of follicle counts in adult mouse ovaries and in vitro cultured ovaries showed that transplantation of iPSC-MSC-EVs significantly improved the chemotherapy-induced decrease in follicle numrbers.Immunohistochemical staining showed that transplantation of iPSC-MSC-EVs promoted cell proliferation and inhibited chemotherapy-induced apoptosis in adult mouse ovaries and cultured ovaries in vitro.In granulosa cell in vitro culture experiments,MTS and western blot results showed that iPSC-MSC-EVs could effectively inhibit chemotherapy-induced granulosa cell apoptosis and had a protective effect on mouse granulosa cells.The results of granulocyte transcriptome sequencing data,qPCR,western blot,and immunohistochemical staining experiments showed that iPSC-MSC-EVs could reverse chemotherapy-induced downregulation of ILK-PI3K/AKT pathway,inhibit apoptosis,accelerate cell cycle,and promote cell proliferation.RNase degradation experiments indicated that the iPSC-MSC-EVs function through the RNAs they carry.miRNA sequencing and bioinformatics analysis of iPSC-MSC-EVs suggest that iPSC-MSC-EVs may function by transferring functional miRNAs that target ILK pathway genes.Conclusions:1.iPSC-MSC-EVs transplantation significantly attenuated chemotherapy-induced ovarian injury,inhibited chemotherapy-induced apoptosis of mouse follicles and granulosa cells,and promoted granulosa cell proliferation.2.iPSC-MSC-EVs attenuated chemotherapy-induced ovarian injury by reversing chemotherapy-induced downregulation of the ILK-PI3K/AKT pathway.3.In granulosa cells,ILK accelerates the cell cycle and promotes cell proliferation by phosphorylating downstream AKT molecules.4.iPSC-MSC-EVs may protect against chemotherapy-induced granulosa cell apoptosis by delivering regulatory miRNAs targeting the ILK pathway.Significance:It provides a new idea for therapeutic approaches to improve ovarian injury in female chemotherapy patients and enhance ovarian function in POI patients,and preliminarily elucidates the underlying molecular mechanisms by which iPSC-MSC-EVs attenuate chemotherapy-induced ovarian injury. |
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