Human Umbilical Cord Matrix Stem Cells Isolation,Identification And Research On Differentiation Potential To Germ Cells

Background and Purpose:Traditional theory holds that human oocytes are non-renewable cells.Immune,environmental and genetic factors and the disease progression,treatment process of the gynecological tumor can damage the oocytes,lead to ovarian function decrease,even failure,loss of reproductive function.And the aspects of nerve,bone,endocrine affect women's physical and mental health.The patients don't have access to,even by assisted reproductive technology,have the function eggs.Therefore,oocytes are the important way to help such patients.In recent years,the study found that stem cells have the ability to differentiate into all types of cells in the body at home or abroad.The series of researches found that the embryonic stem cells which cultured in vitro and then transplanted in vivo can produce functional gametes and obtain healthy offspring.The study of embryonic stem cell therapy is restricted because of the ethical controversy,the inconvenience of drawing materials and tumorigenic potentiality.Therefore,the researchers have turned to adult stem cells.etc.Umbilical cord mesenchymal stem cells?UCMSCs?come from the relatively pure tissue of neonatal umbilical cord tissue,with the advantages of low cost,free from ethical issues and no damage to the provider,which offers a source of the seed cells.Research found that UCMSCs as a potential alternative for oocytes.It can express the germ cell markers and survive in ovarian.But until now we didn't get the functional gametes.The reason maybe the microenvironment in vitro can't completely simulate the microenvironment in the body.Studies have shown that the somatic cells from gonads which can induce germ cells to differentiate into oocytes,and the synchronization of ovarian cells can promote the primitive germ cell meiosis.Thus,we hold that meiotic prophase embryos co-culture with stem cells,which is expected to better simulate the microenvironment of the ovarian eggs occurred in the body.At the same time make full use of ovarian cells secreted cytokines to environmental inducion of stem cells,which help to observe the ovarian function of microenvironment in oogenesis.To explore the possible mechanisms how the adult stem cells become a germ cells.We isolate,culture and identify UCMSCs,and then co-culture with primordial germ cells?PGCs?,and induced the cells to differentiate into oocyte like cells.To explore its possible mechanisms of induction of germ cells in vitro,provide the new thought of the research about the completely oocytes regeneration.1 Experimental methods1.1 Isolation,culture and identification of UCMSCsThe wharton jelly of umbilical cords were isolated in vitro and cut up,then UCMSCs were seeded and adhered to the culture dishes.Test the immune phenotype of UCMSCs by the flow cytometry phenotype,and take the P3 generation UCMSCs as seeded cells.1.2 Isolation,culture and identification of PGCsThe 12.5d.p.c mouse embryonic gonads were isolated in vitro and cut up,then primordial germ cells were seeded and adhered to the culture dishes.Identify the P3 generation PGCs by the alkaline phosphatase staining and the flow cytometry phenotype.1.3 Study on biological characteristics of PGCsStudy on biological characteristics of the P3 generation PGCs by the transcription-polymerase chain reaction?RT-PCR?and immunofluorescence.1.4 Flow cytometry observed the differentiation of UCMSCs into Primordial germ cell-like cells?PGCLCs?The third passsge UCMSCs were cultured in different medium,control group and the experimental group were set.The experimental group cultured in PGCLCs differential medium,and control group cultured in UCMSCs medium,and cultured in incubator of 37 ? and 5%CO₂ condition.The inverted microscope was used to observe cellular morphology.Flow cytometry was used to identify the stage specific embryonic antigens of cultured cells.1.5 Differentiation of UCMSCs by co-cultureControl group and the experimental group were set.Group 1 is PGCLCs and PGCs.Group 2 is UCMSCs and PGCs.Control group is UCMSCs and UCMSCs.Cells were co-culture in 0.4?m transwell,and cultured in incubator of 37? and 5%CO₂ condition.1.6 Detection of SCP3 protein expression of UCMSCs by Western blotUCMSCs which co-cultured 14 days later were collected and then detect the expression of SCP3 protein by Western blot method.1.7 Statistical analysisUsing SPSS18.0 statistical software,measurement data used `x±s,that three groups were compared by chi-square test.Results2.1 Isolation,culture and identification of UCMSCsMicroscope observation showed that primary UCMSCs isolated grow adherently and swirlingly had few miscellaneous,which could fuse up to 90% when were cultured 7 days.After underground passage,the cells were more uniform,regular shape,and fast proliferation.And could fuse up when subculture 2 days.Flow cytometry detection the expression of UCMSCs showed that the positive rate of mesodermal origin molecule CD73 and CD29,was 62.9% and 95.5%,expressing endothelial cell charateristics molecule CD31,the positive tate was 0.221%,the expressing of hematopoietic stem cells charactized molecul CD45,the positive tate was 0.186%,the expressing of stage special embryo antigen-1,the positive tate was 0.946%,in line with the UCMSCs feature.2.2 Isolation,culture and identification of PGCsMicroscope observation showed that primary PGCs isolated grow adherently and swirlingly had some miscellaneous,which could fuse up to 70%-80% when were cultured 5-7 days.After underground passage,the cells were more uniform,regular shape,and fast proliferation.And could fuse up when subculture 3 days.The alkaline phosphatase staining is positive.Flow cytometry detection the expression of PGCs showed that the positive rate of the expressing of stage special embryo antigen-1,the positive tate was 92.5%,the expressing of stage special embryo antigen-4,the positive tate was 0.71%,in line with the UCMSCs feature.2.3 Study on biological characteristics of PGCsTranscription-polymerase chain reaction?RT-PCR?and immunofluorescence detection the expression of PGCs showed that Oct4,Stra8,Vasa,Scp3,Zp3 were positive.2.4 Flow cytometry observed the differentiation of UCMSCs into Primordial germ cell-like cells?PGCLCs?Microscope observation showed that experimental group began to appear Primordial germ cell-like cells when were cultured 7-9 days.Control group didn't like that.Flow cytometry detection the expression of experimental group showed that the positive rate of the expressing of stage special embryo antigen-1,the positive tate was 73.83%,the expressing of stage special embryo antigen-3,the positive tate was 66.6%.The expression of control group showed that the positive rate of the expressing of stage special embryo antigen-1,the positive tate was 0.69%,the expressing of stage special embryo antigen-3,the positive tate was 0.45%.These findings suggest that UCMSCs have the potential to differentiate into PGCLCs.2.5 Detection of SCP3 protein expression of UCMSCs by Western blotAfter 14 days,the expression of SCP3 protein of group 1 was the highest,And the group 2 higher than control group,differences were statistically significant?P<0.05?.Conclusion1.Isolation high purity of UCMSCs.2.Isolation high purity of PGCs.3.Using the expression methods can culture the high purity of PGCs with the excellent stem cell properties and extremely strong proliferative ability,and the cells can reach the meiosis stage.4.UCMSCs have the potential to differentiate into PGCLCs.5.UCMSCs have the potential to differentiate into oocyte like cells in vitro.