Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles

Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and eAMC-EVs were spotted on microarray slides, and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. Signal intensity was detected using a microarray scanner. Both eAMC and eAMC-EV microarrays interacted with all the lectins, indicating the presence of N- and O-linked glycans. With respect to eAMCs, eAMC-EVs, were found to be (1) enriched in Gal beta 1,3GalNAc terminating O-glycans, alpha 2,3-linked sialoglycans, and high-mannose N-glycans (Con A); (2) diminished in N-acetyllactosamine, GalNAc, Gal, GlcNAc, and fucose terminating glycans; and (3) unchanged in alpha 2,6 linked sialoglycans content. These results suggest that eAMC-EVs emerge from a specific eAMC microdomain, and that the high simultaneous presence of Gal beta 1,3GalNAc, alpha 2,3 sialic acid, and high-mannose N-linked glycans may constitute markers of the eAMC-EVs. The role of these sugars in equine regenerative medicine requires further investigation.