Study On The MiRNA Regulated Self-renewal Mechanism And Application Of Mesenchymal Stem Cells

As important seed cells for cell therapy,mesenchymal stem cells(MSCs)have shown prominent therapeutic potentials toward age induced functional degradation and other agerelated diseases such as cardiovascular disease,diabetes and neurodegenerative diseases.The primary problem for MSCs' usage is to satisfy the clinical amplication criteria of MSCs in both quality and quantity.On one aspect,senescence induced functional impairment during ex vivo expansion is considered the main obstacle for the culture of MSCs.Considering the vital role of mi RNAs in regulating the biological activities of stem cells,it is promising to solve this problem by modulating mi RNA expression.On the other aspect,the efficacy of components from MSCs in anti-aging therapy still need further investigation.In an effort to solving these problems,we performed series of studies and achieved the following progress:We investigated the mi RNA expression changes during senescence of MSCs.By a highthroughput sequencing method,we compared the mi RNA expression profiles of umbilical cord derived MSCs(UCMSCs)from early passage and late passage with obvious senescence phenotypes.By building a mi RNA-target genes interaction model,we analyzed the target genes expression regulated by mi RNAs.Functional cluster analysis of the differentially expressed target genes after UCMSCs senescence revealed the important regulatory roles of mi RNAs on senescence of MSCs.We revealed the mechanism of mi R-18 a on MSCs senescence.Through a continuous expression of mi R-18 a,we also developed a method to ameliorate senescence,maintain selfrenewal and biological functions of MSCs during ex vivo expansion.In this section,we first found that endogenous expression of mi R-18 a played a key role in resisting senescence of MSCs.Then,by a luciferase reporter assay and immune blot assay,we demonstrated that mi R-18 a inhibited the expression of CTDSPL by directly targeting its 3' un-translational region(3'UTR).Therefore,expression of mi R-18 a prevented CTDSPL induced senescence of MSCs.Finally,we used lentivirus transduction method to over-express mi R-18 a in MSCs and demonstrated that the continuous expression of mi R-18 a reduced the reactive oxygen level,ameliorated senescence,and promoted the self-renewal and biological functions of MSCs.We isolated and cultured MSCs from human cord blood(CBMSCs)and compared their function and therapeutic potentials with UCMSCs.The regulation of mi RNAs suggested different mechanism of senescence between them: the senescence of UCMSCs are mainly affected by dysregulation of differentiation related genes,while ubiquitin dependent proteolysis determines the senescence of CBMSCs.By a comparison in mi RNA and transcriptome profiles,we found UC-and CB-MSCs showed significant function differences in metabolism,adhesion,proliferation and immunoregulation.We also compared the secretome properties of both cells by detecting 55 cytokines they secreted.All of these results suggeste that CBMSCs have more prominent potentials in repairing neural,vascular and islet impairment,supporting hematopoietic stem cells function and immunoregulation than UCMSCs.We investigated the anti-aging effect of secretory compounds from CBMSCs.We first found that CBMSCs' secretory compounds significantly reduced the reactive oxygen levels induced by hydrogen peroxide and ameliorated cellular senescence.Then,by using 12 months old naturally aged mice as a model we evaluated the anti-aging effect of CBMSCs' secretory compounds.We found that administration of secretory compounds significantly improved the immune modulation functions of aged mice,as characterized by subpopulation changes of T cells,B cells,myeloid cells,NK cells,regulatory T cells and na?ve and memory T cells in peripheral blood.Further tissue section analysis also showed a significant improvement of skin and kidney tissues in aged mice after secretory compounds administration.These results suggest that CBMSCs' secretory compounds have the ability to suppress reactive oxygen induced cellular senescence,improve immune functions and finally ameliorate aging.