Abstract
Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of EYA2 than GPRIN3, and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified EYA2 and GPRIN3 as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.
Highlights
Tendinopathy is one of the most frequently diagnosed sports-related injury both in human and equine athletes [1,2,3], predominantly affecting energy-storing tendons such as the superficial digital flexor tendon (SDFT) in horses [3] and the Achilles tendon in humans [4]
Cartilage, ligament and tendon tissue were harvested from young adult Warmblood horses and in the case of tendon, tissue quality was confirmed by macroscopic and histological evaluation [34]
We identified 12 genes that were selectively expressed in either tendon (THBS4, TENM4, SCX, ENPEP), ligament (TNMD), bone (BTLN9, CD36, MASP2, SNCG), or cartilage (CHODL, ACAN, THBS3), and two genes that were exclusively expressed in tendon (EYA2 and G-protein regulated inducer of neurite outgrowth 3 (GPRIN3)) (Figure 3)
Summary
Tendinopathy is one of the most frequently diagnosed sports-related injury both in human and equine athletes [1,2,3], predominantly affecting energy-storing tendons such as the superficial digital flexor tendon (SDFT) in horses [3] and the Achilles tendon in humans [4]. The failure to achieve regeneration of injured tendon tissue is thought to be the result of an inadequate intrinsic cellular healing response [5,6]. The regenerative approach to tendon healing aims to regenerate tendon tissue through the application of growth factors or implantation of stem cells [5,7,8,9,10,11]. The clinical use of mesenchymal stem cells (MSCs) to treat horses suffering from tendon injury was introduced. Several in vitro and in vivo studies have since been conducted to evaluate various aspects of stem cell therapy of tendon injury in the horse [13,14,15,16]. Since it is known that the pathophysiology of tendon injury and the healing response are very similar between horses and humans, the horse serves as an important model in tendon research [4]
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