The Senescence Of Human Mesenchymal Stem Cells During Culture And Expansion In Vitro

Mesenchymal stem cells(MSCs)are multipotent stromal cells that can differentiate into a variety of cell types.In vitro cell expansion,or cell culture,is the most practical way to obtain large numbers of stem cells in basic research.However,there are several problems of maintaining stemness in long-term culture.Cell senescence is induced by changing cell microenvironments,increasing the pressure of replication and altering intercellular information.Thus,to investigate whether there is senescence of stem cells during the culture would pave the way for application.In this study we investigated the senescence of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs),which were characterized by low immunogenicity and widely application.hUC-MSCs of passaging 5,10,15,20,25 and 30 were cultured to observe cell changes.Firstly,we found that although hUC-MSCs maintained the shape of fibroblastlike cell,hUC-MSCs became larger,flatter and had more vesicles with passaging.The expression of stem cell marker CD105 was decreased gradully.But colony forming unit assay showed no difference in different passage.Then our result indicated that ?-galactosidase expressed after passage 10.Additionally,the proliferation ability of P10,P15,P20,P25 and P30 hUC-MSCs was significantly lower than that of P5 hUC-MSCs.Secondly,we used RNA sequencing to compare the difference of transcripts between passage 5 and 25.The GO enrichment analysis showed that there were significance differences in DNA duplication,cell cycle phase regulation,chromatin condensation and nuclear division,and the KEGG enrichment analysis showed that p53-signaling pathway,ECM-receptor interaction,DNA replication,cellular senescence and cell cycle also had significance differences.It was verified that the cyclin-dependent kinase inhibitor p16,p21,p53 gradually increased with passaging by western blot,while cell cycle had no significant difference.Finaly,we detected H4K20 methylation modification levels and found that the level of H4K20 me,H4K20me2,H4K20me3 decreased.Thus,we conclude that there is senescence of hUC-MSCs after passage 10.These results will shed light on the Basic research and clinical application of hUC-MSCs.future.