The <i>N</i> ‐Glycome to Differentiate Mesenchymal Stem Cells Upon Chondrogenic Differentiation, Dedifferentiation, and Senescence

Mesenchymal stem cells (MSCs) are adult stem cells able to self-renewal or differentiation into different cell types, including chondrocytes. N-Glycans are post-translational modifications of glycoproteins that contribute to vital cell functions. In this work, we examined the cell surface N-glycome of human MSCs isolated from bone marrow to identify biomarkers for chondrogenic differentiation. In addition, we investigated for the first time the N-glycome of MSCs upon dedifferentiation and aging. Cell surface glycoproteins were released by tryptic digestion, then N-glycans were enzymatically cleaved, purified, permethylated, and analyzed by MALDI-TOF-MS combined with exoglycosidase digestions. We were able to detect 68 signals, comprising paucimannose, high-mannose, hybrid, and complex-type N-glycans, as well as structures containing polylactosamine motifs. A statistically significant decrease in antennarity and galactosylation, accompanied by an increase in sialylation, was observed during chondrogenic differentiation. Comparison of MSCs at passage 3 and passage 8 revealed increased levels of paucimannosylation, hybrid-type glycans, and sialylation, together with decreased biantennarity, bigalactosylation, and core-fucosylated N-glycans. Dedifferentiated MSCs exhibited a stem cell-like N-glycosylation profile, although statistically significant differences were still detected. These data show that the N-glycosylation profile of MSCs may serve as an indicator of the differentiation stage, dedifferentiation, and aging for the quality control of MSCs.