Donor‐derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes

Summary Reasons for performing study Recently, it has been shown that mesenchymal stem cells ( MSCs ) do not express the major histocompatibility complex ( MHC ) II antigen and are able to inhibit proliferation of MHC ‐mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo‐ MSCs , in vitro tests are required to confirm the safety of treatment protocols. Objectives To evaluate the immunosuppressive capabilities of equine bone‐marrow‐derived MSCs ( BM ‐ MSCs ) on MHC ‐mismatched lymphocytes. Study design In vitro experiment. Methods Phytohaemagglutinin‐stimulated peripheral blood mononuclear cells ( PBMCs ) from 3 T horoughbreds (recipients) were co‐cultured with mismatched BM ‐ MSCs from 3 C onnemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti‐ CD 4 and anti‐ CD 8 antibodies were performed. Connemara pony and T horoughbred BM ‐ MSCs were isolated, expanded and characterised by tri‐lineage differentiation. Finally, BM ‐ MSCs from both breeds were set up in co‐culture at different ratios with stimulated T horoughbred lymphocytes. Proliferation of CD4 + and CD 8 + cells was determined by flow cytometry. Results A high proportion of CD4/CD8 double‐positive lymphocytes were found in freshly isolated PBMCs , although this percentage decreased after 4 days of culture. Mismatched BM ‐ MSCs inhibited proliferation of stimulated lymphocytes in a dose‐dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM‐MSCs to PBMCs . Proliferation of CD4 + and CD8 + subpopulations decreased in 1:10 co‐culture, with statistical significance in the case of CD8 + cells, while that of the CD4/CD8 double‐positive population was similar to the phytohaemagglutinin control. Conclusions The results demonstrate dose‐dependent immunosuppression of stimulated lymphocytes by mismatched equine BM ‐ MSCs , supporting their future application in allo‐ MSC clinical treatments.