Proteomic analysis of the human mesenchymal stem cells secretome using a label-free approach

BACKGROUND-AIM Mesenchymal stem cells (MSCs) have been shown to have a pivotal role in tissue repair and in local control of inflammation. In the last few years there has been a growing interest in using them to treat human inflammatory diseases, including severe steroid resistant acute graft versus host disease (aGVHD). 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 fact, it has been reported recently that 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 MSCs into the blood stream. METHODS Based on these evidences, we compared the secretome of MSCs, isolated from human bone marrow cells, before (unstimulated-MSCs) and after (stimulated-MSCs) treatment with pro-inflammatory cytokines (TNFs, IL1b and IL6). Secreted proteins were derivatized, digested and analyzed by LC-nanoESI-LTQ Orbitrap Velos, operated using a TOP20 data dependent method. Raw data files were processed using MaxQuant for protein identification and quantification (main search error 10 ppm, FDR 0.01% for peptide and protein identification). RESULTS Two biological replicates (each consisting of stimulated or unstimulated-MSCs secretome), were analyzed (five technical replicates of each sample) leading to the identification and quantification of 609 and 654 protein groups in the first and second biological replicate, respectively. According to the LFQ statistical analysis (using Perseus , t-test cut-off at 1% permutation-based False Discovery Rate) 231 proteins are differentially expressed in both replicates; in particular: 70 proteins are up-regulated or present only in stimulated MSCs secretome while 161 are down-regulated in stimulated-MSCs or present on in unstimulate-MSCs secretome. CONCLUSIONS These data explain the role of soluble factors responsible of the inflammatory effect of MSCs secretome in vivo. This project was founded in part by LINEA B “Piano di Sviluppo Unimi 2014”.