Characterization of inflammatory factor-induced changes in mesenchymal stem cell exosomes and sequencing analysis of exosomal microRNAs

BACKGROUND Treatments utilizing stems cells often require stem cells to be exposed to inflammatory environments,but the effects of such environments are unknown.AIM To examine the effects of inflammatory cytokines on the morphology and quantity of mesenchymal stem cell exosomes(MSCs-exo)as well as the differential expression of microRNAs(miRNAs)in the exosomes.METHODS MSCs were isolated from human umbilical tissue by enzymatic digestion.Exosomes were then collected after a 48-h incubation period in a serum-free medium with one of the following the inflammatory cytokines:None(control),vascular cell adhesion molecule-1(VCAM-1),tumor necrosis factor(TNF)α,and interleukin(IL)6.The morphology and quantity of each group of MSC exosomes were observed and measured.The miRNAs in MSCs-exo were sequenced.We compared the sequenced data with the miRBase and other non-coding databases in order to detect differentially expressed miRNAs and explore their target genes and regulatory mechanisms.In vitro tube formation assays and Western blot were performed in endothelial cells which were used to assess the angiogenic potential of MSCs-exo after inflammatory cytokine stimulation.RESULTS MSCs-exo were numerous,small,and regularly shaped in the VCAM-1 group.TNFαstimulated MSCs to secrete larger and irregular exosomes.IL6 led to a reduced quantity of MSCs-exo.Compared to the control group,the TNFαand IL6 groups had more downregulated differentially expressed miRNAs,particularly angiogenesis-related miRNAs.The angiogenic potential of MSCs-exo declined after IL6 stimulation.CONCLUSION TNFαand IL6 may influence the expression of miRNAs that down-regulate the PI3K-AKT,MAPK,and VEGF signaling pathways;particularly,IL6 significantly down-regulates the PI3K-AKT signaling pathway.Overall,inflammatory cytokines may lead to changes in exosomal miRNAs that abnormally impact cellular components,molecular function,and biological processes.

Development of a Handheld Nano-centrifugal Device for Visual Virus Detection

Gold nanoparticles(AuNPs)colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis.However,this assay is not effective for visually detecting low-concentration targets due to the faint color change.Here,we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs.Results showed that the handheld nano-centrifugal device could easily reach more than 6000 r/min within 10 s simply by stretching and tightening the coiled rope in an appropriate rhythm.Further,combined with the CRISPR/Cas12a nucleic acids recognition system,a field-deployable colorimetric platform termed handheld nano-centrifugal device assisted CRISPR/Cas12a(Hand-CRISPR)has been validated.Moreover,clinical diagnostics applications for Epstein-Barr virus(EBV)and severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)detection with high sensitivity and accuracy(100%consistency with reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)test results)have been demonstrated.Overall,the Hand-CRISPR platform showed great promise in point-of-care-test(POCT)application,expected to become a powerful supplement to the standard nucleic acid testing method in remote or poverty-stricken areas.