ISOLATION AND CHARACTERIZATION OF AMNION-DERIVED INHIBITORY FACTORS ON LYMPHOCYTE PROLIFERATION

Background Cells derived from the amniotic membranes of human term placenta are receiving particular attention because of their characteristics of stemness, multipotency and their immunologycal features, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously demonstrated that cells isolated from the mesenchymal region of the human amniotic membrane (human amniotic mesenchymal tissue cells, hAMTC) possess immunoregulatory properties, such as the ability to inhibit lymphocyte proliferation and cytokine production and to suppress generation and maturation of monocyte-derived dendritic cells, as also reported for MSC from other sources. All of the results described above were obtained when hAMTC were cultured either in direct cell contact or in a transwell system, thereby suggesting the involvement of soluble inhibitory factor(s) secreted by these cells. However, the precise factors and mechanisms responsible for the immunoregulatory roles of hAMTC remain unknown. Aims Therefore, in this study, we aimed to identify the soluble factors released by hAMTC which are responsible for the anti-proliferative effects of these cells on lymphocytes, and also to gain insight into the mechanisms underlying their actions through in vitro studies. Materials and Methods Conditioned medium (CM) was prepared by culture of isolated hAMTC (CM-hAMTC) and fragments of the whole human amniotic membrane (CMhAM) for 5 days in UltraCulture medium. Chemical and physical properties, such as the thermostability, chemical nature and molecular weight, of the factors associated with the anti-proliferative effects of hAMTC were evaluated. Blocking of specific synthetic pathways involved in the production of immunomodulatory factors, such as nitric oxide, kynurenine and prostaglandins, was achieved by adding specific inhibitors during CM production. Finally, the involvement of certain cytokines in the antiproliferative effects of the CM was evaluated by adding neutralizing antibodies to the CM during lymphocyte proliferation tests. Results In this study, we have demonstrated that: • the inhibitory factors are temperature-stable, have a small molecular weight, and are likely to be non-proteinaceous; • only inhibition of the cyclooxygenase pathway partially reversed the antiproliferative effects of hAMTC, suggesting that prostaglandins may be key effector molecules in this phenomenon; • factors which have been previously documented to play a role in the inhibitory effects of MSCs from other sources (HGF, TGF-β, NO and IDO) were not shown to be involved; • the anti-proliferative effects of the amniotic membrane are intrinsic to this tissue and its derived cells, since these effects are manifested in the absence of stimulating culture conditions, as opposed to MSC derived from the bone marrow, which possess anti-proliferative ability only when cultured in the presence of activating stimuli. Conclusions We have demonstrated that both isolated cells or the amniotic membrane in toto are capable of releasing factors that present with immunomodulatory capacity, thereby providing new insights toward the identification of some of these factors and toward the characterization of other factors which remain to be identified.