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Abstract
Tendon injuries in the horse are debilitating, costly, and require long periods of treatment and rehabilitation. Bone marrow derived mesenchymal stem/stromal cells (MSCs) have shown both experimental and clinical evidence that they have the capacity to improve tissue architecture and clinical reinjury rates; however, controlled studies cannot demonstrate their ability to enhance the biochemical composition or biomechanical strength of the tendon. Additionally, controlled and randomized human translational studies have been unable to prove the beneficial effect of MSCs in tendon injury. Therefore, discovering new techniques to enhance the reparative capacity of MSCs is crucial to improve treatment of both our veterinary patients and for translation into human tendon injury.
Benefits of MSCs therapy are attributed to their functional roles secreting bioactive molecules that stimulate angiogenesis, modulate immune cell function, curb cell apoptosis, and stimulate local stem/progenitor cell populations within the tissue microenvironment. These functions can be further modulated through MSC licensing (re: priming, preconditioning, stimulating) where naïve, unstimulated MSCs are exposed to cytokines, growth factors, small molecules, or hypoxia that enhances their secretory function. For this reason, our laboratory is interested in cytokine licensing as a method to enhance MSC secretory function in the context of tendon injury. Initially, we set out to determine if we could use the microenvironment of the injured tendon to enhance their function. Utilizing a surgically induced model of equine tendon injury, we implanted ultrafiltration probes in vivo and measured the temporal cytokine changes over 21 days. Using this data, we then moved into an in vitro setting and licensed MSCs with the two primary pro-inflammatory cytokines identified in tendon ultrafiltrate: IL-1b and IL-6.
Utilizing RNA sequencing, we discovered that IL-1b licensing, but not IL-6, greatly enhanced the reparative transcriptome of MSCs and had a variety of differentially expressed genes that could be relevant to the healing tendon. Additionally, when select downstream proteins were quantified by ELISA, we confirmed that a significant increase in protein production also resulted.
Next, a collaboration with a former PhD student of the laboratory examined the effects of TGF-b2 licensing of MSCs. While Dr. Berglund’s focus was on TGF-b2’s effect on MHC expression in equine MSCs, RNA sequencing also revealed that its effect also drastically enhanced extracellular matrix and growth factor production in MSCs and when co-cultured with tenocytes, enhanced their migration. This collaboration resulted in combining of our two cytokine licensing strategies: IL-1b + TGF-b dual licensed MSCs.
Our focus has now shifted to how IL-1b + TGF-b dual licensed MSCs could be translated to an equine preclinical model of tendon injury. First, we determined that IL-1b + TGF-b dual licensing significantly decreased surface expression of MHC class I. Next, RNA sequencing of the dual licensed cells showed many similar positive changes in gene expression as those cells licensed singly with IL-1b or TGF-b2, indicating that dual licensing did not compromise the benefits each cytokine had by itself. Finally, conditioned media generated from these dual licensed cells significantly enhanced tenocyte migration in vitro.
