Single-Cell Proteomic Analysis of Equine Adipose-derived Mesenchymal Stem Cells: a Pilot Study of Donor Variability

Background: Mesenchymal stem cells (MSCs) are used as an equine orthobiologic product in the treatment of musculoskeletal conditions. However, equine MSCs-based therapies demonstrate variable clinical outcomes, with donor-related factors potentially contributing to functional heterogeneity. Proteomics characterises protein expression and abundance, giving functional insight into MSCs' activities. Single-cell proteomics enables the identification of cellular heterogeneity masked by conventional bulk analyses. Objectives: To investigate the influence of donor age and sex on the protein profiles of equine adipose-derived MSCs using single-cell proteomics. It was hypothesised that significant donor-dependent heterogeneity would be present and that single-cell proteomic analysis would identify distinct proteomic profiles associated with age and sex. Study design: Pilot experimental study. Methods: Equine adipose-derived MSCs were isolated from eight donors, expanded to third passage and validated for trilineage differentiation using histological staining and quantitative PCR. Single- cell isolation was performed using the CellenONE platform. Individual cells were analysed by high-resolution nano-liquid chromatography coupled with tandem mass spectrometry. Protein abundance data were analysed using principal component analysis and linear modelling to evaluate the effects of donor age and sex. Results: Single-cell proteomics was performed on 784 AD-MSCs from seven horses aged 6–23 years (three mares and four geldings). Histological staining and qPCR confirmed trilineage differentiation. Principal component analysis showed more distinct separation by sex along PC1 (2.7% of variance) than by age along PC2 (1.6%). Eight proteins differed in abundance between mares and geldings (adjusted p-value < 0.05), whereas no proteins were significantly associated with age after correction for multiple testing. Conclusions: Single-cell proteomics identified donor-related heterogeneity in equine adipose-derived MSCs associated with age and sex. These preliminary findings provide a foundation for larger studies aimed at improving the understanding of equine donor variability.