Human Amniotic Mesenchymal Stem Cells Promote Spermatogonia Cells Proliferation And Its Mechanisms

Objective: Spermatogenesis is accomplished by spermatogonial cells(SCs)in seminiferous tubules of testis.This is a highly coordinated and orderly process.As a type of germline stem cell,SCs,like embryonic stem cell cells,have the potential to proliferate and differentiate,some of these cells maintaining a relatively constant number of cells through self renewal,the other continuously differentiating into various transitional spermatogonia cells,finally produce sperm.For adult males,SCs are the only cells capable of transmitting genetic information to their offspring.As precursor cells for sperm production,it has broad clinical application prospects such as the treatment of non-obstructive azoospermia,the fertility preservation of prepubertal male cancer patients,and cell replacement therapy by transdifferentiation into other cell types.The limited number of SCs in testis tissue is the main obstacle to the study and clinical application of them.Feeder layer provide many necessary cytokines and transmitters for the proliferation of SCs,but feeder cells make the culture conditions of SCs complex and uncontrollable.Except GDNF(glial cell line-derived neurotrophic factor)and FGF2(fibroblast growth factor 2),few growth factors are known to promote the proliferation of SCs in vitro.Thus,discovery of new cytokines and their interactions is critical for full understanding of the developmental microenvironment of SCs.In addition,it is of great significance to discover new cytokines to promote SCs proliferation,further achieve feeder-free culture and to continuously understand the biological characteristics of SCs.A variety of autocrine/paracrine factors secreted from mesenchymal stem cell(MSC)are important for the therapeutic effects of these cells.Human amniotic mesenchymal stem cell not only has various characteristics of MSCs,it also has advantages such as rich sources,low immunogenicity and stable proliferation.Therefore,hAMSCs has broad prospect for repair and regeneration of autologous cells.It has not been reported whether the cytokines secreted by hAMSCs are beneficial for the proliferation of SCs.Based on the previous findings of our group,there are proteins related to cell proliferation in the secretory proteome of hAMSCs.The purpose of this study was to explore whether human amniotic mesenchymal stem cell conditioned medium(hAMSCs-CM)could promote the proliferation of SCs in vitro and whether unknown cytokines in hAMSCs-CM could be used to enhance the proliferation ability of SCs in vitro.The aim of this study is: 1)To study the effect of hAMSCs-CM on the proliferation of SCs in vitro.2)Through bioinformatic analysis,finding candidate proteins related to cell proliferation secreted by hAMSCs.3)To investigate the effect of target protein POSTN on the proliferation of SCs and its mechanisms.4)The effect of POSTN on the proliferation and differentiation of SCs were studied.Method:The effects of hAMSCs-CM on the proliferation of GC-1 spg cells in vitro1.HAMSCs were isolated by enzyme digestion,the cell surface markers of hAMSCs were identified by flow cytometry,and their differentiation potential was also identified under the osteoblasts,adipocytes and chondrocytes condition medium.2.HAMSCs were cultured in serum-free medium for 24 hours,and the supernatants were collected as the conditioned medium for the follow-up experiments.3.Human SCs were isolated by two-step enzymatic digestion.Different concentrations(0,25%,75% and 100%)of hAMSCs-CM were co-cultured with SCs for different time(0,24,48,72,96 hours).MTS assay was used to evaluated the proliferation of SCs in vitro.4.The proliferation of GC-1 spg cells was detected by MTS when co-cultured with different concentrations(0,25%,75% and 100%)of hAMSCs-CM for different time(0,24,48,72,96 hours).EdU assay was used to evaluate the proliferation of GC-1spg cells when co-cultured with different concentrations(0,25%,75%,and 100%)of hAMSCs-CM for 24 hours.5.The exocrine proteins of hAMSCs-CM were analyzed by bioinformatics analysis;proteins related to cell proliferation were selected.6.The expression of POSTN and THBS1 in hAMSCs-CM was confirmed by ELISA.The effect of POSTN on the proliferation of GC-1 spg cells and its mechanism1.POSTN and THBS1 were cultured with GC-1 spg cells at different concentrations(0,10,50,100,200 ng/m L)for different time(0,24,48,72,96 hours).MTS assay was used to evaluate the effect of POSTN and THBS1 on the proliferation of GC-1 spg cells.2.POSTN siRNA was used to silence POSTN in hAMSCs for 24 hours,then POSTN expression was detected by Real-time PCR.After that hAMSCs conditioned medium was collected and the POSTN level was detected by ELISA.3.After POSTN silence,the proliferation of GC-1 spg cells was examined by MTS when co-cultured with hAMSCs-CM for 0,24,48,72 and 96 hours.4.GC-1 spg cells were pretreated with Wnt inhibitor XAV939,and then co-cultured with POSTN for different time(0,24,48,72,96 hours),the proliferation of GC-1spg cells was investigated by MTS.Meanwhile,GC-1 spg cells were co-cultured with POSTN for 24 hours,the effects of POSTN on the proliferation of GC-1 spg cells was detected by EdU assay.5.GC-1 spg cells were pretreated with Wnt inhibitor XAV939,and then co-cultured with POSTN.Western Blot was used to detect the expression of downstream proteins GSK3?,?-catenin and Cyclin-D1 in the Wnt pathway.6.Flow cytometry was used to examine the effect of POSTN on the cell cycle of GC-1spg cells.The effect of POSTN on the proliferation of human SCs in vitro1.Human SCs were isolated by enzyme digestion,and identified by immunofluorescence and RT-PCR.2.The effect of POSTN on the expression of OCT4 was evaluated by immunofluorescence and the proliferation of human SCs was detected by Ki67 staining.In addition,MTS and Real-time PCR were used to detect the proliferation of human SCs when treated with POSTN.3.POSTN was co-cultured with human SCs,and Western Blot was used to detect the expression of GSK3?,?-catenin and Cyclin-D1 in the downstream of Wnt pathway.4.Human SCs were differentiated in vitro by three-dimensional culture.After differentiation,the expression of haploid molecular markers was detected by Real-time PCR,Western Blot and immunofluorescence.Results:HAMSCs-CM promotes the proliferation of SCs and GC-1spg cells1.HAMSCs were isolated by enzyme digestion,then cultured to the second generation,they could differentiate into osteogenic,adipogenic and chondroblast.Flow cytometry results indicated that cell surface markers CD73,CD44,CD105 were positive and HLA-DR,CD31,CD45 were negative,all of these characteristics are consistent with hAMSCs.2.MTS results showed that with increase of the concentration of hAMSCs-CM,the proliferation ability of SCs increased.When 100% conditioned medium and 75%conditioned medium added,the cell proliferation increased significantly compared with the control group(p < 0.05).3.MTS results showed that with increase of the concentration of hAMSCs-CM,the proliferation of GC-1 spg cells increased.From 48 hours,the cell proliferation of GC-1 spg cells in 100% conditioned medium and 75% conditioned medium was significantly enhanced compared with that in control group,the difference was statistically significant(p < 0.05).EdU staining also indicated that the proportion of EdU positive cells in 100% and 75% conditioned medium groups was significantly higher than that in control group(p < 0.05).4.Bioinformatics analysis screened out POSTN and THBS1 protein were highly expressed in hAMSCs-CM,which may be related to cell proliferation.5.ELISA results confirmed that POSTN and THBS1 were highly expressed in hAMSCs-CM,in consistent with the mass spectrometric analysis results.POSTN in hAMSCs-CM promotes the proliferation of GC-1 spg cells1.MTS results showed that 100ng/m L and 200ng/m L POSTN significantly promote the proliferation of GC-1 spg cells from 48 hours.GC-1 spg cells co-cultured with different concentrations of THBS1 showed no significantly difference in cell proliferation compared with the control group.In addition,EdU staining results demonstrated that the proportion of positive cells treated with 100ng/m L and200ng/m L POSTN were significantly higher than the control group(p < 0.05).We choose 100ng/m L POSTN in the follow-up experiment.2.Both siRNA sequences effectively reduced the expression of POSTN in hAMSCs.Furthermore,POSTN secretion in hAMSCs-CM was significantly lower after POSTN silence(p < 0.05).3.MTS results showed that the proliferation ability of GC-1 spg cells co-cultured with CM treated with si-POSTN was lower than that of the control group.The percentage of EdU positive cells also decreased.MTS results indicated that when pretreated with Wnt inhibitor XAV939,cell proliferation was significantly decreased compared with the POSTN group(p < 0.05).EdU assay results were consistent with the MTS results.4.Western Blot was used to detect the expression of GSK3?,?-catenin and Cyclin-D1 in GC-1 spg cells after POSTN treatment.When treated with POSTN,the expression of GSK3? was decreased;?-catenin and Cyclin-D1 were increased.5.Cell cycle analysis indicated that the proportion of S phase cells was increased when treated with POSTN.6.POSTN could significantly inhibit the apoptosis of SCs.POSTN promotes the proliferation of human SCs1.Human SCs were isolated by enzyme digestion;immunofluorescence results showed that OCT4 and PLZF were positive in SCs.RT-PCR results indicated that OCT4,PLZF,SALL4 and VASA were positive.2.Ki67 immunofluorescence staining results showed that POSTN could promote the proliferation of human SCs.MTS results and Real time-PCR results also showed that POSTN could promote the proliferation of human SCs.3.Western Blot was used to detect the expression of GSK3?,?-catenin and Cyclin-D1 downstream of Wnt pathway in SCs.When treated with POSTN,the expression of GSK3? was decreased and the expression of ?-catenin and Cyclin-D1 were increased.4.Three dimentional culture methods were used for cell differentiation.Real time-PCR results showed that the levels of Crem-1,LDH,Boule and PROTAMINE,which are molecular markers of haploid cells,were increased in the POSTN group compared with the control group.Western Blot and immunofluorescence results also showed that the expression levels of Acrosin and TNP1 were higher in POSTN group than in the control group.Conclusion: HAMSCs-CM promotes the proliferation of SCs cells.The results of bioinformatics analysis screened out the candidate protein POSTN in hAMSCs-CM.POSTN promotes proliferation of SCs cells in vitro by activating Wnt signaling pathway.Furthermore,the expression of molecular markers of haploid cells increased after POSTN treatment.