The Key Role Of Stemness Of Mesenchymal Stem Cells In Regulation Of Multiple Cells

Mesenchymal stem cells(MSCs),derived from mesoderm,are a kind of cells characterized by sternness that have the ability to proliferation while retaining the pluripotency to differentiate into multiple lineages.This unique property of MSCs makes themselves attractive candidates for tissue engineering and clinical application.However,loss of proliferation and sternness due to culture senescence in vitro is a major factor limiting their application.It is therefore a big task to identify the the optical culture medium for sternness maintaineance of MSCs and explore the molecular mechanism that regulate this process.On the other hand,mesenchymal stem cells co-cultured with endothelial cells(ECs)have attracted most attention in strategies that seek to achieve vascularized bone in tissue engineering.However,the interactions between osteogenesis of MSCs and angiogenesis of ECs remain elusive as well as the underlying mechanisms coupling these processes.Here,we identified an optimal culture medium for the sternness maintainance of hMSCs through comparison of three culture media and exploit the critical role of KLF2 in sternness maintained hMSCs.Based on these analysises,we further demonstrated the key role of sternness in directing the interaction between the angiogenesis of HUVECs and osteogenesis of hMSCs.This work therefore provides new insights into understanding the dominant role of sternness in MSCs and will have important directive to design of new strategies in tissue engineeringPart ⅠSystem construction for the stemness maintainence of mesenchymal stem cellsMesenchymal stem cells(MSCs)are a kind of cells characterized by sternness that can differentiate into multiple lineages,thus possessing great potential in tissue engineering and clinical therapy.However,loss of proliferation and sternness due to culture senescence in vitro is a major factor limiting their application.Identifying the optimal culture medium for sternness maintainence of MSCs and understanding the molecular mechanism that regulate this process,are critical to future advances in tissue and organ regeneration.Here we compared the cell morphology,proliferation,cell typical surface markers and differentiation related gene expression in hMSCs(human mesenchymal stem cells)under three media and identified the best culture medium for its sternness maintainance.After that,we screened out the critical role of KLF2(Kruppel like factor 2)in stemness maintained hMSCs,as indicated by gene expression in three culture media.To investigate the role of KLF2 in detail,a knockdown experiment in hBMSCs was performed.After KLF2 knockdown,cell proliferation,colony forming ability and multi-lineage differentiation ability were down-regulated.In addition,quantitative RT-PCR indicated an decreased expression of pluripotency associated genes and increased expression of differentiation related genes in KLF2 knockdown hMSCs as compared to control cells,which futher confirmed that KLF2 contributes to the sternness maintainance of hMSCs.Our results identify an optimized culture medium for the sternness maintainance of hMSCs and the key role of KLF2 that underlying this process,which would lay down the foundation for the future application of hMSCs on tissue engineering and clinical therapy.Part ⅡStemness directs the smart synergistic strategy betweenmesenchymal stem cells and endothelial cellsMesenchymal stem cells(MSCs)co-cultured with endothelial cells(ECs)have attracted most attention in strategies that seek to achieve vascularized bone.However,the interactions between osteogenesis of MSCs and angiogenesis of ECs remain elusive as well as the underlying mechanisms coupling these processes.Here,we demonstrated there was a smart synergistic strategy between human mesenchymal stem cells(hMSCs)and umbilical vein endothelial cells(HUVECs)for better ossification that sternness maintained hMSCs initiated and promoted the angiogenesis of HUVECs,and in turn stimulating the osteogenic differentiation of hMSCs.The sternness maintainence of hMSCs was controlled by the critical transcription factor(Kruppel-like factor 2,KLF2)using three typical culture media as we reported previously.KLF2 knockdown also downregulated the sternness of hMSCs as well as the angiogenesis of HUVECs and obtained poor ossification finally.Mechanistic dissection studies showed that sternness maintained hMSCs might induce basic fibroblast growth factor(bFGF)secretion through activation of core NANOG-OCT4-SOX2 pluripotency network and result in vascular endothelial growth factor(VEGF)up-regulation to mediate higher angiogenesis of HUVECs.Meanwhile,up-regulation of VEGF would in turn stimulate hMSCs osteogenesis through targeting of HUVECs to regulate the communication between MSCs and ECs.The three-dimensional culture model within hydrogel matrices that mimic in vivo environment further confirmed the sternness maintained hMSCs facilitated the formation of vascular-like network.This work therefore sheds light on the fundamental understanding of stemness directed smart synergistic strategy between hMSCs and HUVECs,and will have a profound impact on further strategies designing in bone tissue engineering.