Complete analysis of the mwsenchymal stem cells secretome using A label-free approach

Mesenchymal stem cell (MSCs) have recently been shown to have a pivotal role in tissue repair and in local control of inflammation. Therefore, in the last few years there has been a growing interest in using them to treat human inflammatory diseases, including severe (grade III-IV) steroid resistant acute graft versus host disease (aGVHD). In vitro studies indicated that MSCs display immunomodulatory activities: they inhibit T and NK cell activation, B-cell terminal differentiation and dendritic cell maturation and functions. Although there is evidence from in vivo studies that MSCs are able to reduce inflammatory damage, it is not clear whether their immunomodulatory effects rely on soluble factors or cell-cell contacts. In a recent study, by comparing the immunomodulatory potential of mouse MSC either injected intravenously, or subcutaneously entrapped through the use of microcapsules, it has been reported that the encapsulated MSCs (E-MSCs) are extremely efficient in down-modulating in vivo immune responses, such as antigen-specific T cell responses and acute GVHD, and that this activity is based on soluble factors released by MSC into the blood stream [1]. Based on these evidences, in collaboration with Humanitas Clinical and Research Center, we compared the secretome of MSC, isolated from murine bone marrow cells, before & after treatment with pro-inflammatory cytokines (TNFs, IL1b, and IL6). To achieve this goal, we employed a label-free quantitative approach that allows us to examine differences in global protein expression between different samples. Samples have been derivatized, digested and analyzed by LC-nanoESI-LTQ Orbitrap Velos equipped with a RPC18 column for peptides separation prior to MS/MS analysis. For protein identification and quantification, raw data files were processed and analyzed using MaxQuant 1.3.0.5.