| Part 1 Biological features of human umbilical cord mesenchymal stem cell-derived microvesiclesObjective:Here we set out to investigate the biological characteristics of human umbilical cord-derived MSC-MVs,including their morphology,size and immune-phenotype.Comparision the difference of miRNAs expression between umbilical cord MSC-MVs and adult BMSC-MVs,and finding the miRNAs expression characteristics of umbilical cord MSC-MVs.Methods:Human umbilical cord-derived MSCs were cultured and characterized by their immune-phenotype and multilineage differentiation capacity.MSC-MVs were isolated by differential centrifugation from the conditioned medium of MSCs and observed by a scanning electron microscope and a laser confocal microscopy.The size distribution characteristics were detected by qNano-TRPS,and the immune-phenotype of MSC-MVs were examined by flow cytometry.The expression of mRNAs and miRNAs enclosed in umbilical cord MSC-MVs and adult BMSC-MVs was determined by RNA and small RNA sequencing.Results:The immune-phenotype of umbilical cord-derived MSCs was in accordance with the classical immune-phenotype of MSCs,and the multilineage differentiation capacity of MSCs was confirmed in vitro.When observed by scanning electron microscopy and laser confocal microscopy,MSC-MVs showed a spheroidal morphology.The MSC-MVs extracted by differential centrifugation are those with diameters less than 1?m and their sizes are mainly distributed between 200-500nm.The immune-phenotype of MSC-MVs was similar to MSCs.Additionally,by using RNA sequencing,we discovered that umbilical cord MSC-MVs contained plenty of mRNAs,and these miRNAs were participate in cell proliferation and cell cycle.However,adult BMSC-MVs contained plenty of mRNAs,which participate in negative regulation of protein complex assembly and response to stimulus.miRNAs were the highest proportion of the RNA components in umbilical cord MSC-MVs,and the expression of miRNAs in umbilical cord MSC-MVs were higher than that in adult BMSC-MVs.Conclusions:Human umbilical cord MSCs could secret a large quantity of MVs,MSC-MVs are spherical bodies with diameters less than 1?m and having a membranous structure.These MVs share similar surface antigen with their parent cells.Umbilical cord MSC-MVs carry more "young" RNA molecular than adult BMSC-MVs,and carry a rich miRNAs component than that in adult BMSC-MVs.miRNAs is likely to be the key effector molecule of umbilical cord MSC-MVs.Part 2 Microvesicles identify the senescence in their parental MSCsObjective:Here,we aim to study the changes of the ability of secreting MSC-MVs and phenotypic characteristics of MSC-MVs in different passage MSCs.To investigate the expression pattern of miRNAs in different passage MSCs and MSC-MVs,and filter out specific miRNAs in MSC-MVs that reflect the senescence state of MSCs.Methods:MSC-MVs were derived from different passage umbilical cord MSCs.The concentration and size distribution of MSC-MVs was detected by qNano-TRPS.The surface antigen of MSC-MVs was examnied by flow cytometry.qRT-PCR and alizarin red staining were used to compare the pro-osteogenic differentiation ability of early and late passage MSC-MVs.The miRNAs expression in MSCs and MSC-MVs were measured by RNA sequencing.Identification of differentially expressed miRNAs in MSCs senescence,combined with the database of transcription factors and target genes in the GEO database,to construct the miRNA,transcription factors(TF)and genes co-regulatory networks of MSC senescence and to filter out key miRNAs in the network.Rely on the expression pattern of the key miRNAs in different passage MSCs and MSC-MVs,we screened out the specific miRNAs of MSC-MVs which could reflect the senescence status of MSCs.Results:We found that senescent late passage(LP)MSCs secreted higher levels of MSC-MVs with smaller size than did early passage(EP)MSCs,and the level of CD 105+MSC-MVs decreased with senescence in the parental MSCs.LP MSC-MVs exhibited a weaker pro-osteogenic differentiation ability than EP MSC-MVs.RNA sequencing results showed the same trend of decreasing number and expression level of miRNAs in senescent MSCs and MSC-MVs.The changes were more obvious in MSC-MVs than in MSCs.Furthermore,we confirmed that the expression of miR-146a-5p plays a key role in the senescence of MSCs and its expression is gradually up-regulated in MSCs and MSC-MVs.Conclusions:Our study has identified MVs,a component of SASP,as a novel source manifesting senescence in their parental cells in terms of concentration,size distribution,phenotypic markers,pro-osteogenic differentiation ability,and the expression of miR-146a-5p.Our findings provide evidence that MSC-MVs have considerable potential as secreted biomarkers to accurately identify the senescence of MSCs.Part 3 Umbilical cord mesenchymal stem cell-derived microvesicles reverse the senescence of mesenchymal stem cellsObjective:Here we set out to study the reversal effect of umbilical cord MSC-MVs on senescent adult BMSCs.To investigate the reversal effect of MSC-MVs on pro-angiogenesis and multilineage differentiation potential of senescent BMSCs.To explore the molecular mechanism of umbilical cord MSC-MVs reversing BMSCs senescence.Methods:The umbilical cord-derived MSC-MVs were co-cultured with the senescent adult BMSCs,and the proliferation capacity was detected by Edu staining.We detect the self-renewal ability by clone formation assay.P-galactosidase staining estimate the senescence of BMSCs.qRT-PCR and Western Blot detected the expression of P16,P21 and P53.The pro-angiogenesis ability of senescent BMSCs were examined by qRT-PCR,ELISA and tube formation assay.We detect the effect of MSC-MVs on the osteogenic differentiation capability of senescent BMSCs by alizarin red staining and subcutaneous heterotopic bone formation model in nude mice.To determined the effect of MSC-MVs on the adipogenic differentiation ability of senescent BMSCs by Oil red O staining and qRT-PCR.We compared the pro-osteogenic differentiation capability of umbilical cord MSC-MVs,adult BMSC-MVs,neonatal fibroblast derived MVs and Rnase treated umbilical cord MSC-MVs of senescent BMSCs by alizarin red staining.Comparision of the expression of miRNAs in umbilical cord MSC-MVs,adult BMSC-MVs and neonatal fibroblast derived MVs by high throughput sequencing.Results:Umbilical cord MSC-MVs can promote the proliferation and self-renewal ability of senescent BMSCs,and can decline the expression of aging-related P-gal,and inhibite the expression of P16,P21 and P53.ELISA,qRT-PCR and tube formation assay demonstrated that umbilical cord MSC-MVs can improve the pro-angiogenesis of senescent BMSCs.Umbilical cord MSC-MVs can enhance osteogenic differentiation capability,and inhibit the adipogenic differentiation capability of senescence BMSCs by qRT-PCR and cell chemical dyeing.Alizarin red staining showed that the ability of umbilical cord MSC-MVs to promote the osteogenic differentiation of aging BMSCs was stronger than that of BMSC-MVs and neonatal fibroblast derived MVs.Rnase digestion experiments and miRNA-seq analysis showed that the key effect of MSC-MVs on the osteogenic differentiation of senescent BMSCs was miRNAs.Conclusions:Umbilical cord MSC-MVs can reverse the senescence of adult BMSCs,enhance the pro-angiogenesis and osteogenic differentiation,and inhibit the adipogenic differentiation of aging BMSCs.The reversal effect of umbilical cord MSC-MVs on senescent BMSCs is likely to be mediated by horizol transfer of miRNAs.This study not only provides a new method for reversing the senescence of BMSCs in vitro,but also provides new strategies and ideas for the treatment of MSCs senescence-related diseases. |
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