Investigate The Effect And Mechanism Of Stem Cell Secretory Factors On The Improvement Of Ovary Function

Diminished ovarian reserve(DOR)refers to the decline of number or quality of oocytes in ovary.DOR can develop into premature ovarian failure(POF).In addition to old age,many factors can cause DOR,while the majority of the etiologies remain unclear.Hormone replacement therapy is used as a main clinical treatment for DOR,but the ovarian function of patients can not be recovered.At the same time,the success rate of assisted reproduction treatment is low.Stem cell therapy can improve and repair the function of damaged tissues,is a very promising treatment method.It has been employed in clinical applications in several fields,and many of pre-clinical researches and clinical trials are carrying out.Bone marrow transplant treatment indicates that stem cells have the potential to repair ovarian function,meanwhile animal experiments show that transplanted stem cells in the recipient can not differentiate into new oocytes.It will open up new windows for treatment of DOR/POF to investigate the specific mechanism of stem cells’ effectiveness on ovarian functionIn this study,different kinds of stem cell lines were established.Cell culture supernatants were collected and later injected to 32 weeks old female C57BL/6 mice(DOR model)via the tail vein.Then the mice ovary function was evaluated by analyzing the level of serum sex hormones,counting the number of follicles in the ovary and number of oocytes in vitro at different stages.Finally the expression levels of stem cell secretory factors were compared.This study accumulate laboratory data for investigate the effect and mechanism of stem cell secretory factors on the improvement of ovary function,and will provide a theoretical basis for the selection of seed cells for stem cell therapy of DOR/POF.Material and Methods(1)Establish different kinds of stem cell lines with different genders.Establish a new human embryonic stem cell(hESC)line by mechanical method.Establish new amniotic mesenchymal stem cell(AMSC)lines by enzymatic digestion and establish new umbilical cord mesenchymal stem cell(UMSC)lines by tissue culture.(2)Identification of cell lines.By immunofluorescence,embryoid body formation to confirm the pluripotency,by gene sequencing to clear the karyotype of hESC.By flow cytometry to detect MSC’s surface markers,and by induced differentiation into mesoderm lineages to evaluate MSC’s pluripotency.Genetic markers-STR was employed to detect MSC’s karyotype.(3)Evaluation of the quality of cell culture supernatant.Test the expression level of secrete factors in upernatants from above stem cells culture according to the ELISA Kit’s instruction manual.(4)DOR model selection and grouping.32-week-old female C57BL/6 was selected as DOR model.Total 81 mice were divided into nine groups.The saline,different stem cell culture media and different stem cell culture supernatants were injected through the tail vein respectively.(5)Statistics on the number of follicles in mice.Treated mice were sequentially intraperitoneally injected PMSG,.HCG(48h later)and thenwere killed(14-16h later injection).Ovulation from the oviduct ampulla was count under microscope.The number of follicles at different stages in ovary were count after paraffin sections and HE staining.(6)Detection of sex hormone levels in mice.According to the ELISA Kit’s instruction manual to test serum levels of estradiol,FSH and AMH in treated mice.(7)Deteceted the apoptosis of ovarian granulosa cells.According to the Tunel Kit’s instruction manual to detect the ovarian granulosa cells apoptosis.(8)Performed quantitive RT-PCR to analyze expression levels of secretory cytokines.Expression levels of proinflammatory factors,anti-inflammatory factor,growth factors,maxtix metalloprotein of MMP-1 and its inhibitorTIMP-1,Angiogein,LIF and BMP4 were analyzed..Results1 Establish different kinds of stem cell lines.1.1 The establishment and identification of hESC lineSuccessfully established a new hES cell line CCRM15.The cells appeared clonal growth with clear boundaries,and have a large,clear nucleoli,and high nuclear-cytoplasmic ratio.Cells were expressing pluripotent markers of Oct-4,Nanog,and ssea-4,in vitro culture could form embryoid bodies.Genome sequencing showed karyotype 46,XX.1.2 The establishment and identification of MSC linesSuccessfully established AMSC and UMSC lines,respectively named AMSC2.2(46,XX),AMSC1.2.2(46,XY)and UMSC2.2.2(46,XX),UMSC4.2.2(46,XY).The cells were spindle shape like fibroblasts,grew like radiation or spiral,had a strong capability of amplification in vitro.Expression levels of cell surface markers confirmed characteristics of established AMSC and UMSC lines are in line with typical MSC lines.All MSCs lines can be induced in vitro differentiation into adipogenic,osteogenic and cartilage.2 Stem cell culture supernatants improved mice ovarian function.2.1 Sex hormone levels were differently changed in mice serum after tail vein injection of different kinds of stem cell culture supernatantsSerum E2 levels in MSCs culture supermatant injected groups were higher than the control group,and the ascensional range was:male stem cell culture supermatant groups>female stem cell culture supernatant groups>PLT media group>saline group.There was significant difference between the MSCs culture supernatant groups(including AMSC2.2.2,AMSC 1.2.2,UMSC2.2.2 and UMSC4.2.2 group)with the the saline group(p<0.05、p<0.001、p<0.05、p<0.001),and at the same time there was significant difference between the AMSC 1.2.2 group with the PLT media group(p<0.01),the UMSC4.2.2 group with the PLT media group(p<0.001).However,there was no significant difference observed when serum E2 levels were compared between each hESCs group and the control group.After injection of stem cell culture supernatants,the FSH levels were all decreased,the descend range was:male stem cell culture supermatant groups>female stem cell culture supermatant groups>culture medium control group>saline control group.And there is significant difference between hESCs culture supernatant groups and hES medium group(p<0.05),and the MSCs culture supernatant groups with the the saline group(p<0.05).After injection of stem cell culture supernatants,serum AMH levels in mice in each group tended to increase,the ascensional range was:male groups>female groups>culture medium control group>saline control group.In addition,there was significant difference observed when compared CCRM22 cell culture supernatant group to hES medium control group(p<0.05).2.2 Number of follicles increased in mice after tail vein injection of different kinds of stem cell culture supernatantsAfter injection of stem cell culture supernatants,the number of ovulation were increased.And all had the trend:male stem cell culture supermatant groups>female stem cell culture supermatant groups>culture medium control groups>saline control group.There was significant difference observed when respectively compared the group of CCRM22,AMSC2.2.2,UMSC2.2.2 or UMSC4.2.2 to saline control group(p<0.05).And CCRM15,CCRM22 groups both showed significantly higher ovulation than hES culture medium control group(p<0.01).The number of ovarian follicles was count under the microscope after paraffin embedding,sectioning,HE staining.All groups have follicles of different stages,but number differences were showed in different groups.Follicles in all cell culture supernatants injected mice were increased.The ascensional range of primary and antral follicle was:male stem cell culture supernatant groups>female stem cell culture supermatant groups>stem cell culture medium control groups>saline control group.the ascensional range of primordial follicle in hESCs and UMSCs culture supernatant groups was:male stem cell culture supernatant groups>female stem cell culture supermatant groups>stem cell culture medium groups>saline group,but in AMSCs supernatant group was:female stem cell culture supernatant groups>male stem cell culture supermatant groups>stem cell culture medium groups>saline group.The ascensional range of secondary follicle in hESCs supernatant group was female stem cell culture supernatant groups>male stem cell culture supermatant groups>stem cell culture medium control groups>saline control group,and in MSCs supernatant groups were:male stem cell culture supernatant groups>female stem cell culture supermatant groups>stem cell culture medium control groups>saline control group.2.3 Comparison of apoptosis in mouse granulosa cells after tail vein injection of different kinds of stem cell culture supernatantsTunel detection found that the apoptosis rate of ovarian granulosa cells in the treatment group was lower than that in the control group.3 Expression levels of secretory cytokines in different kinds of stem cells were different by quantitive RT-PCR analysisAll kinds of stem cells expressed secretory cytokines.Female UMSCs expressed significantly high level of growth factors(SCF,VEGF,BDNF,HGF),Angiogein,LIF and IL-8(p<0.05).Female hESCs and male UMSCs expressed significantly high level of MMP-1(p<0.05).Conclusion:1.This study successfully established human embryonic stem cell line of CCRM15(46,XX),amniotic mesenchymal stem cell lines of AMSC2.2.2(46,XX)and AMSC(46,XY),and umbilical cord mesenchymal stem cell lines of UMSC2.2.2(46,XX)and UMSC4.2.2(46,XY).All cell lines have the normal cell morphology and normal cell properties,suggesting that they can be applied in later experimental research.2.In this study,mice with physiological ovarian dysfunction caused by age was selected as DOR model,and effects of different stem cell culture supermatants on DOR were explored via tail vein injection..And we found:in all supernatant-treated group,levels of serum sex hormones were beneficially changed,the number of ovarian follicles and ovulation increased,though not all of these parameters showed a significant difference when compared to control group,but the trend was clear.Our data indicates that stem cell culture superntant can improve ovarian function,suggesting that effectiveness of stem cell on tissue repairation can be achieved by the injection of stem cell-secreting cytokines.In addition,male MSCs culture supernatants showed stronger effects in improving ovary function,suggesting that the therapy effect of stem cell on ovarian function is cell type,cell sex dependent.The underlying function mechanism needs to be further studied.Our study is valuable to help to select appropriate type of stem cells as the seed cells for DOR/POF treatment.3.Different kinds of stem cells with different genders showed different ability in cytokines secreting,which might result in the differences in the effect of stem cell therapy.We predict female UMSCs show stronger promise in improving the blood supply,while female hESCs and male UMSCs may be more powerful in degradation of connective tissue.These data is helpful in selecting the suitable stem cells as the seed cells for specific diseases treatment.