The Isolation Of Microvesicles From Human Umbilical Cord Mesenchymal Stem Cells And Detection Of Specific MicroRNAs

BackgroundMicrovesicles (MVs) are circular fragments of membrane, which can be released from cells. Recently many evidences indicate they play a pivotal role in cell-to-cell communication. MVs can transfer various bioactive molecules.ObjectiveTo isolate MVs from human umbilical cord mesenchymal stem cells(MSCs) and detect specific microRNAs expression, for exploring the potential cell-to-cell communication mode of MSCs, which aimed to provide the scientific basis for the research of liver failure protection treatment mechanism by MSCs transplant.Methods1.MSCs were isolated from human umbilical cord by tissue mass culture methods and amplificated by subcultures. MVs were isolated from MSCs conditon medium.2.The third generation cells were labelled by CD29-PE, CD44-APC, CD34-FITC, CD45-PerCP and analysised by flow cytometric.3.Total RNA was extracted from MVs and cells using TRIzol reagent. The expression levels of miR-100and miR-21were detected by real-time PCR, with U6as the internal control respectively.Results1.Cells formed a long spindle. Cells were90%confluent and arranged in a spiral shape at the time of12d primary culture or4d subculture.2.Human umbilical cord MSCs did not express hematopoietic cells mark CD34(0.87%), CD45(1.28%); strongly expressed β2-integrin CD29(96.68%), substrate receptors CD44(92.78%).3.MiR-100was detected in the MVs and cells. The level of miR-100expression in cryopreserved cells was obviously lower than that in normal cells(P<0.05). Meanwhile, no changes were found in MVs and normal cells(P>0.05).4. MiR-21was detected in the MVs and cells. The level of miR-21expression in MVs was significantly higher than that of normal cells(P<0.05). While the level of miR-21expression in cryopreserved cells was obviously lower than of that in normal cells(P<0.05).Conclusions1.We can harvest a large number of identified MSCs from human umbilical cord.2.MVs can be isolated from MSC-CM. MiR-100and miR-21can be detected in MSCs and MVs.3.The enrichment of microRNAs in MVs is specific. The content of microRNAs in cryopreserved MSCs is obvious lower than that in normal cells.