Effects Of Repsox On The Proliferation And Differentiation Potential Of Bovine Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells(BMSCs)are adult pluripotent stem cells that are widely present in animal bone tissues.They have strong ability of self-renewal and the potential of multi-differentiation,and great value in regenerative medicine and stem cell therapy.Since BMSCs has been discovered,a large number of studies have been carried out in human and mice.The in vitro isolation,purification,and culture systems have been established in human and mouse BMSCs.In large animals such as cattle,relatively few studies have been conducted on BMSCs.Currently,there is still controversy in the type identification of bovine BMSCs(bBMSCs).Additionally,during the in vitro culture process,along with the passage increase,the decline of the proliferation ability,cell senescence,and loss of pluripotency etc.usually occur.This study focused on the above issues.Firstly,bBMSCs were isolated and enriched from bovine fetus by whole bone marrow cell isolation,red blood cell lysis,and adherent purified culture.The cells were identified by immunofluorsent staining,flow cytometry,and multi-directional induction of differentiation.The results showed that the isolated cells were authentic bBMSCs,with strong capacity of clone and proliferate.Immunofluorescent staining showed that b BMSCs expressed typical mesenchymal stem cells(MSCs)positive markers CD73,CD90,and CD105,while not for the negative markers CD34 and CD45.Flow cytometry analysis futher showed that their purity was >90%.Additionally,induction differentiation tests showed that bBMSCs have strong potential of adipogenic,osteogenic and chondrogenic differentiation capacities.In order to optimize the in vitro culture conditions of bBMSCs,four fetal bovine serum(FBS)-knockout serum replacement(KSR)combined media were set up bychanging the concentration of FBS and KSR in the basical medium.The proliferation of bBMSCs in different concentrations of FBS-KSR showed that reducing the concentration of FBS and adding KSR were beneficial to the proliferation of bBMSCs in vitro.KSR could increase the expression of the proliferation-related gene.It could also promote the expression of anti-apoptosis gene Bcl2 and pluripotency-related gene Oct4,Sox2 and so on.Interestingly,we noticed that the proliferation of bBMSCs could be arrested when FBS was completely replaced by KSR.The above results indicate that KSR can increase the proliferation and pluripotent potential of bBMSCs in vitro.However,KBS can not be used to replace FBS completely because there may have some unclear components required for the proliferation of bBMSCs in FBS.Recently,studies have shown that the small molecule compound Repsox,an inhibitor of the TGF-? signaling pathway,plays an important role in the induction and stemness maintenance of pluripotent stem cells.However,for bBMSCs,whether Repsox could optimize their stemness and increase the proliferation and pluripotency has not been reported.In this study,the expression of proliferation and pluripotency-related gene in bBMSCs was significantly up-regulated after treatment with Repsox.Additionally,the osteogenic differentiation potential was significantly enhanced,while the chondrogenic differentiation potential was significantly reduced in bBMSCs.In this study,bBMSCs were isolated,identified and cultured in vitro,then the culture conditions were optimized to determine the effect of KSR on the biological characteristics of bBMSCs.The cells were further treated with Repsox to explore its effects on the proliferation and pluripotency of bBMSCs.Collectively,the present study was carried out to provide references for the optimization of the culture system,research of stemness characteristics and the investigation of reprogramming induction mechanism of bBMSCs,and also lay a foundation for their further application.