RESUMO
PURPOSE: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue. METHODS: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-ß1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFß1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFß1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFßRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFß1 secreted by L-MSC was able to suppress T cell proliferation. CONCLUSIONS: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.
