In vitro immunomodulatory activity of conditioned medium from horse amniotic derived multipotent progenitor cells

We recently demonstrated that heterologous transplantation of horse amniotic-derived cells (AMCs) can be useful for cell therapy applications in tendon diseases. We hypothesized that AMCs may promote tendon repair via paracrine-acting molecules and to test this hypothesis we examined the immuno-modulatory characteristics of AMCs and of its conditioned medium (AMCs-CM) in vitro. To produce AMCs-CM, fresh AMCs at passage three 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 lg/ml. To evaluate the effect of AMCs-CM, 50 or 100 ll/well of this supernatant or non-CM were added to activated PBMC. Effects of AMCs were studied either by cell-cell contact or by transwell system keeping a constant number of PBMC (2*105) and decreasing number of AMCs, to obtain ratios of PBMC : AMCs of 1 : 1, 1 : 0.5, 1 : 0.25, 1 : 0.125. Our 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 to the 1 : 1 ratio an inhibition of 90% (p < 0.05). This finding suggests that soluble factors are implicated, and this hypothesis is supported by PBMC inhibition with the AMCs-CM that could represent a novel therapeutic cell-free product in regenerative medicine.