ANALYSIS OF MESENCHYMAL STEM CELLS (MSCS) SECRETOME FROM MOUSE MODELS AND HUMAN PATIENTS TO CHARACTERIZE THEIR IMMUNOMODULATORY PROPERTIES: A PROTEOMIC APPROACH.

Mesenchymal stem cells (MSC) are multipotent progenitor cells with self-renewable capacity and the potential to differentiate into various cell types, especially of the mesodermal lineages. They have immunomodulatory properties and, in particular, when exposed to pro-inflamatory cytokines, they acquire immunosuppressive and anti-inflammatory properties due in part to an array of soluble mediators. These cells can be used for a potential therapeutic application but, until now, a thourough characterization of pro-inflammatory primed MSC secretome is still lacking, because its characterization in vivo is very difficult. So, a commonly used approach is the analysis of media conditioned (CM) by cells in culture. The aim of this investigation is the proteomic characterization of bone marrow derived cultured MSC secretome following stimulation with pro-inflammatory cytokines, using two different models: mouse and human patients. This is a fundamental step in order to clarify the mechanism of MSC activity and to design strategies to modulate their properties for rational therapy design and improvement of existing therapies. The proteomic analysis of human MSC-CM and mouse MSC-CM confirms that exposure to pro-inflammatory cytokines results in significantly higher secretion of a number of immunomodulatory and angiogenesis-related proteins by MSC from both species. Notably, 62% of the proteins identified in st hMSC-CM were also identified in st mMSC-CM, clearly highlighting the existence of a common signature in the secretome of human and mouse MSC. However, although human and mouse MSC show a similar proteomic signature in response to stimulation by pro-inflammatory cytokines, our data indicate that they may induce different biological responses. In both species, several up-regulated proteins are associated with angiogenesis, a process that requires degradation of the vascular basement membrane and remodelling of the extracellular matrix to allow endothelial cells migration and invasion into the surrounding tissue. This is possible through the action of matrix metalloproteinases (MMPs) that degrade both matrix and non-matrix proteins and have central roles. So, the balance between MMPs and their natural inhibitors, the TIMPs, is critical for extracellular matrix remodelling and angiogenesis. Using both in vitro and in vivo assays, we identified the metalloproteinase inhibitor TIMP-1 as the molecule responsible for the anti-angiogenic effects of MSC. By identifying TIMP-1 as a critical effector of the anti-inflammatory properties of MSC, and by observing its anti-angiogenic role, both in mouse and in human, we can confirm the important role of TIMP-1 as key secreted molecule targeting endothelial cells. The identification of TIMP-1 as potential effector molecule responsible for the anti –angiogenic properties of MSC, both in mouse and in human, allow to confirm that MSC exert specific effect by secretion of a broad range of bioactive molecules and allow to design pre-clinical experiments and clinical trials, with the aim of exploit thei r potentially therapeutic role.