Background
Mesenchymal stem cells (MSCs) are thought to be critical for successful regenerative medicine and immunosuppressive therapy. However, long term culturing of MSCs is known to impair cellular function and induces aging of MSCs.
Methods
To investigate the cellular mechanisms involved in the aging of MSC cultures, we analyzed human umbilical cord mesenchymal stem cells (hUMSCs) exposed to different culture conditions (glucose concentrations were 5.5 mM/25 mM/40mM) for 7/30 days using tandem mass tag (TMT) labeling quantitative proteomics, analyzing posttranslational protein modifications and applying bioinformatics methods. Differentially expressed proteins (DEPs) were clustered and functional annotated by Gene Ontology (GO) enrichment analysis. Pathway enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation tool. To identify the changes in N-glycans properties during culturing of hMSCs for either 7 or 30 days under three different glucose concentration, we analyzed the mesenchymal stem cell glycome using a mass spectrometry-based N-glycan profiling method.
Results
Here, we found that the morphology of hUMSCs during a long-term culturing period changed significantly compared with 7 days conditions. More specifically, we fund that a total of 66 proteins (fold change >1.50 and P-value <0.05 as criteria) were differentially expressed in long term culture hUMSCs, with 49 proteins upregulated and 17 proteins downregulated. Our GO analysis showed that aging exerts a side effect on
UMSCs by affecting variable molecular functions and biological processes, such as lysosome organization, exopeptidase activity, hydrolase activity, glycosaminoglycan binding and post-translational protein modification. KEGG analysis demonstrated that differential abundance proteins were significantly enriched in lysosomes, renin-angiotensin system, other glycan degradation and autophagy. The results of glycosylation analysis showed that cell N-glycans is associated with aging.
Conclusion
In conclusion, our comprehensive analysis revealed that the difference in differentially expressed proteins and post-translational protein modification levels between aging group vs control, which could provide a possible basis for aging and cell quality control study.