Proteomic analysis of equine amniotic mesenchymal stromal cells and their extracellular vesicles: comparing their regenerative properties.

Abstract

Aim: To compare specific functional features of equine amniotic mesenchymal cells (eAMCs) and their extracellular vesicles (EVs) through proteomic analysis. Methods: eAMCs were obtained by enzymatic digestion and their EVs were isolated by ultracentrifugation. Cells and EVs were characterized according to ISCT and MISEV guidelines. A proteomic analysis of both eAMCs and EVs was conducted. The raw data files were analyzed using FragPipe 22 and uniprotkb equus_caballus_reviewed database (20.02.2025) to obtain protein identifications (false discovery rate = 0.01) and their respective label-free quantification values using recommended parameters. Statistical analysis was performed based on the combined_protein.tsv file using FragPipe-Analyst. A cutoff of the adjusted P-value of 0.05 along with a |log2 fold change| of 1 has been applied to determine differentially expressed proteins in the comparison. Results: A total of 3,631 proteins were identified, of which 3,147 were identified with more than two peptides. Among these, 2,235 were exclusive to eAMCs, 697 were shared between eAMCs and EVs, and 71 were exclusive to EVs. eAMCs and EVs revealed distinguished proteomic profiles, differentially expressing proteins involved in biological processes related to tissue regeneration. Proteins promoting anti-inflammatory activity, oxidative stress resistance and angiogenesis exhibited increased expression levels in eAMCs, while extracellular matrix organization and deposition were predominantly upregulated in EVs. Conclusions: Equine eAMCs and EVs are characterized by a distinct proteomic profile showing the expression of different sets of proteins involved in regenerative processes and thus different therapeutic properties. This also highlights their coordinated activity on tissue homeostasis and regeneration mechanisms.