Changes And Verification Of Gene Expression In Human Umbilical Cord Mesenchymal Stem Cells After Continuous Passage

Background: Stem cell-based therapy is one of the most widely used treatments nowadays,particularly in malignant tumor treatment.However,the side effects of classic stem cell transplantation on the human body are also cannot be avoided.Basic research has found that Mesenchymal stromal cells(MSCs)derived from various human tissues;not only have the basic characteristics of stem cells,but also have a strong secretome,as well as their unique multipotency,low immunogenicity and other characteristics.In recent years,more successful applications of MSCs-based cell therapy have made MSCs quickly become one of the most promising candidate cells than stem cells.More studies have shown that,the use of MSCs for allogeneic treatment has fewer side effects and strong tissue recovery capabilities,which further promotes the process of MSCs to clinical application.Although MSCs have many advantages,the difference between batches and the difficulty of mass production are important reasons hindering their clinical application,especially when continuously culture in-vitro;MSCs will quickly undergo morphological changes,and their functions will also decline to various degrees,resulting in a decrease in the efficiency of MSCs,and the reasons behind these changes are still unclear.Human umbilical cord(hUC)is rich in MSCs,which-Despite of the ethical issues-could have a wide range of applications.Studies have shown that human umbilical cord-derived MSCs(hUC-MSCs)have more passage potential in-vitro than those derived from other tissues.Moreover,their functions are relatively more stable.Accordingly,in this study,we aimed to clarify the difference between hUC-MSCs and MSCs from other tissue sources through the transcriptomic analysis of hUC-MSCs after continuous passage,and to identify the key factors that could affect hUC-MSCs stable passage and verification.Methods: 1.Primary hUC-MSCs were isolated from human umbilical cord Wharton’s jelly by explant method,and their surface markers were identified by flow cytometry to insure meeting the ISCT identification criteria for MSCs in-vitro,and their in-vitro differentiation potential was identified by directional induction;2.The successfully identified hUC-MSCs were continuously sub-cultured until no-proliferation stage then the cells subjected to flow cytometry,plate clone formation assay,Poly-A RNA FISH,Western Blot and SA-β-Gal and other experimental techniques to identify cell morphology and function of the early,middle and late generations;3.Transcriptomics analysis was performed on the early and late passaged cells,and the enrichment analysis of differential genes expression was carried out using bioinformatics techniques to screen the key genes that could affect the functional changes of hUC-MSCs after passage;4.RT-PCR was used for verification to further clarify the impact these key genes on the performance of MSCs after continuous passage.Results: 1.First,we optimized the hUC-MSCs isolation system based on the explant method,and successfully isolated primary hUC-MSCs from five newborn umbilical cords,which were labeled as hUC-MSCs-n-P0(n stands for umbilical cord number),and confirmed that it has osteogenic differentiation potential when cultured in-vitro,high expression of CD105/CD73/CD90,low or no expression of CD45/CD34/HLA-DR,which meets the ISCT identification standard for MSCs cultured in-vitro;2.Next,we performed continuous passages on these five cell lines.The first three cell lines were passaged 15 times(P15),and the cells almost completely lost their proliferation capability and fibroblast morphology.These phenomena occurred when the 4th and 5th hUC-MSCs were subcultured to P18 and P11,respectively.Accordingly,we selected the first three cell lines for the next downstream experiments.We identified the surface markers of the three strains of P2,P8,and P15 cells,and found that the change of hUC-MSCs from different passages was insignificant,at the same time colony formation experiments showed that the colonies of hUC-MSCs were significantly reduced after passage;3.Subsequently,We used senescence-associated β-galactosidase(SA-β-Gal)on the P2,P8,and P15 generation cells and found that the higher the number of cell passages,the more serious the senescence of the cells.Along with SA-β-Gal staining,we also detected the cell cycle,and the results showed that hUC-MSCs had obvious G0/G1 phase arrest after continuous culture;4.We performed total RNA extraction followed by RNA-Seq analysis for P2,P8,and P15 generation cells.GO analysis showed that compared with early cells,the up-regulated differential genes in late-passage cells were mainly enriched in the stimulation response of cells to various inflammatory factors.Moreover,Reactome enrichment analysis showed that the up-regulated differential genes were significantly enriched in the cell senescence-associated secretory phenotypes(SASP);5.Then we verified the RNA-Seq results at RNA level;RT-PCR results showed that after serial passage,the mRNA levels of the key factors of SASP(IL6 and IL8)were significantly up-regulated,indicating that after serial passage hUC-MSCs had obvious cellular senescence accompanied by SASP secretion phenotype;6.We detected the level of endogenous mRNA(PolyA RNA-FISH)on the cells of P2,P8,and P15,and found that the higher the cell generation,the more obvious the nuclear retention of mRNA.Hence more,we detected the key protein expression levels of mRNA transcription-export(TREX)components,and found that the expression of UAP56/TAP/Thoc2/ALYREF was consistent with the nuclear and cytoplasmic distribution of mRNA indicating that after continuous passage of hUC-MSCs could cause nuclear retention of endogenous mRNAConclusion: After detecting the surface markers of the serially passaged hUC-MSCs,we found that there was no significant change.However,the cell morphology changed significantly,the cells became larger and more flat,in addition to the slow cell proliferation;through the detection of the senescence level of hUC-MSCs of different generations,we found that hUC-MSCs had obvious cell senescence after continuous passage,and there were obvious SASP-related secretion phenotypes;The detection of endogenous mRNA levels reveals that with the increase of generations,the mRNA level in the nucleus increased significantly,indicating that aging hUC-MSCs had obvious mRNA nuclear retention.