Characterization of MSC Potential To Treat GVHD Using Molecular Markers Linked to MSC-Mediated Immunosuppression In Vitro.

Abstract Mesenchymal stem cells (MSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage and fat. The biological activities of MSCs suggest a number of potential clinical applications, where each particular application is related to a specific MSC activity mediated by a different mechanism. Osiris Therapeutics has developed a technology for isolation and expansion of hMSCs from adult bone marrow for clinical use. Data from pre-clinical and clinical studies suggest that the ability of MSCs to migrate to inflammatory sites, modulate immune response, down-regulate inflammation, and accelerate tissue repair in the local environment may have therapeutic effects. Therefore in developing therapeutic applications, the MSCs should be verified to display one or more of above-mentioned functions, calling for the need to develop predictive functional assays. Modulation of the immune response is an apparent in vivo therapeutic property of the MSC necessary for successful Graft versus Host Disease (GVHD) treatment. Based on previous knowledge regarding mechanisms underlying MSC-mediated immunosuppressive effects, several markers for developing an MSC potency assay have been proposed. In the present study a relationship between selected markers and hMSC-mediated immunosuppression was investigated in vitro. Results show that co-culture of hMSCs with anti-CD3/CD28-activated peripheral blood mononuclear cells (hPBMCs) caused inhibition of lymphocyte proliferation. The hMSC effect on lymphocyte proliferation is dose-dependent, causing > 50% inhibition at approximately 1:10–1:25 MSC: T-lymphocyte ratio. Supernatants of parallel co-cultures taken on days 1, 3, and 5 were analyzed for prostaglandin 2 (PGE2), tumor necrosis factor-α (TNF-α), and tryptophan. The results showed increased levels of PGE2, decreased levels of TNF-α and increased depletion of tryptophan related to indoleamine 2,3-dioxygenase (IDO) enzyme activity, associated with increasing number of MSCs in each well. The quantity of PGE2 on day 1 and the level of tryptophan on day 5 in the MSC-PBMC co-culture supernatants correlated to the level of inhibition of proliferation, with the PGE2 range from approximately 11,000 to 22,000 pg/mL and 50% tryptophan depletion resulting in a 50% inhibition of the lymphocyte proliferation point. Further studies demonstrated that the addition of TNF-α to MSCs induced PGE2 secretion at a level which was similar to that detected in the co-culture studies of MSCs-PBMC. Thus, a strong correlation between inducible PGE2 secretion/IDO enzyme activity and the inhibition of lymphocyte proliferation by hMSCs in vitro indicates key molecules responsible for hMSC functional activity related to the immunological responses involved with diseases such as GVHD, solid organ transplantation and autoimmune diseases.