In vitro immunomodulatory activity of conditioned medium obtained from amnion-derived horse progenitor cells and its first clinical application in horse tendon injuries

We recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) is useful for cell therapy applications in tendon diseases (Lange-Consiglio et al., 2012, 2013). Whether MSCs differentiate into tenocytes, supply immunomodulatory and trophic factors or if a combination of the two mechanisms occurs, is still debated. To test this hypothesis, we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMCs-CM) in vitro, and studied the therapeutic effect of AMCs-CM in horse tendon injuries in vivo. To produce AMCs-CM, AMCs at passage 3 were cultured for 5 days. Supernatants were lyophilized and stored at 4°C until use. Control (non-CM) was generated in the same way but without cells culturing. Lymphocyte proliferation was induced by stimulating peripheral blood mononuclear cells (PBMC) by phytohemagglutinin at the concentration of 2 μg/ml. To evaluate the effect of AMCs-CM, 50 or 100 μl/well of this supernatant or no-CM were added to activated PBMC. Effects of AMCs were studied either by cell-cell contact or by transwell system maintaining constant the number of PBMC (2*105) and decreasing the number of AMCs, to obtain ratios of PBMC:AMCs of 1:1, 1:0.5, 1:0.25, 1:0.125. Sterile CM was intralesionally injected under ultrasonographic guidance in spontaneously damaged tendons of 13 private sport horses. Patients were clinically and ultrasonographically monitored monthly. Success criteria was return to former athletic function and absence of relapses. Results demonstrated that AMCs are capable of inhibiting PBMC proliferation in a dose-dependent manner, either in cell-cell contact or in transwell system reaching a 90% (P≤0.05) decrease of PBMC proliferation at a ratio of 1:1. The same effect was also observed for the AMCs-CM but not for the control media. In vivo, intralesional procedures were well tolerated and a marked reduction in swelling and tendon cross sectional area were noticed. A treatment related neovascularization was Power Doppler imaged, in the affected area, in the early healing phase but not at later recovery stage. An obvious improvement in lesional ecogenicity and architecture was clearly noticeable just after 30 days. Two years after CM injection 84% of horses showed no relapses. Our findings suggest that soluble factors are implicated in inhibiting PBMC proliferation and in tendon regenerative process that may initiate an anti-inflammatory and angiogenic response leading to tendon regenerative process. CM should be considered a safe, novel biologic cell-free therapeutic agent in regenerative medicine. 1. Lange Consiglio et al. Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane. J Tis Eng Reg Med 2012; 6: 622. 2. Lange Consiglio et al. Investigating the efficacy of amnion-derived compared with bone marrow-derived mesenchymal stromal cells in equine tendon and ligament injuries. Cytotherapy epub 2013.