horse tendon cell analysis

The interfascicular matrix (IFM) binds tendon fascicles and contributes to the mechanics of energy storing tendons such as equine superficial digital flexor tendons (SDFT). IFM resident cells likely maintain IFM structure and tendon function, and deficits in IFM cell function with ageing may contribute to increased injury risk. The aim of this study was to use single cell RNA-sequencing (scRNAseq) and immunolabelling to profile IFM cells and establish how they are affected by ageing. Forelimb SDFTs were harvested from young (3-7y) and old horses (>17y; 4/group). Tendons were digested and 6,500 cells from each sample underwent scRNAseq. ~5x108 reads were generated across samples and aligned on the horse genome. Following quality control and data normalization, principal component analysis and integration was performed. Differentially expressed genes (DEGs) between age groups within each cluster were identified. SDFTs were also immunolabelled for cell markers. 11 cell clusters were identified; 3 tenocyte clusters of 2 tenocyte populations, 2 endothelial, 2 mural and 4 immune clusters. 1 tenocyte population, and the endothelial, mural and immune cells localised to the IFM, and 1 tenocyte population localised to the fascicles (FM). The IFM and FM tenocytes had distinct matrisomes. IFM tenocytes and mural cells also had the highest number of DEGs with ageing, with many DEGs associated with the matrisome, senescence, proteostasis loss and inflammation. We identified 9 cell types within the IFM, the largest being tenocytes that likely regulate IFM maintenance and repair and are disproportionately affected by ageing, indicating that senescence may result in loss of proteostasis and ability to regulate inflammation. This study uncovers the heterogeneity of tendon cells, with much of this complexity due to IFM cell populations. Loss of IFM cell phenotype and associated matrisome changes with ageing may contribute to increased injury risk.