Investigating the immunoregulatory potential of human Adipose-derived Stromal Cell secretome: proteomic analysis of their Conditioned Medium in comparison to human Dermal Fibroblasts’ one

Abstract

OBJECTIVE: Human Adipose-derived Stromal Cells (hASCs) possess a well-recognized immunoregulatory potential. Nowadays it is widely accepted that this beneficial action is mainly mediated by released bioactive factors and extracellular vesicles (EVs) rather than depending on cell-cell contact. In this perspective, we recently demonstrated the efficient performance of hASC secretome (Conditioned Medium, CM) in contrasting different inflammatory pathologies, both on an in vitro model of TNF\u3b1-induced osteoarthritis and in vivo on diabetic neuropathy. Interestingly, CM deriving from human Dermal Fibroblasts (hDFs) never exerted any effect. Through a proteomic approach we analysed the presence of several soluble molecules in the CM of hASCs and hDFs, focusing on the factors differentially expressed between the two populations that may be involved in hASC immunoregulatory properties. MATERIALS AND METHODS: Primary cultures were isolated from the subcutaneous fat or the dermal connective tissue of 5 female donors subject to plastic surgery, following the procedure approved by IRCCS Galeazzi Orthopaedic Institute (PQ 7.5.125, version 4). CM was collected from confluent hASCs or hDFs cultured for 72 hours in starving conditions (absence of FBS). Samples were then concentrated through Amicon Ultra-15 Centrifugal Filter Devices with 3 kDa cut-off (Merck Millipore), reducing the initial volume of 43\ub14 folds for hASCs (n=3) and 46\ub18 folds for hDFs (n=3). CM protein concentration was assessed through Bradford Protein Assay (Bio Rad) and differences in the protein content between hASC and hDF CM were identified through nanoflow liquid chromatography\u2013tandem mass spectrometry (nLC-MS/MS), following standard procedures. The statistical analysis of the complete dataset of identified and quantified proteins was performed by Student\u2019s t-test, followed by hierarchical clustering analysis using MeV software. RESULTS: 1208 factors were identified in the CM of the two cell types, 976 of which were quantified. 36 secreted proteins resulted significantly different between hASC and hDF CM, with 15 uniquely or preponderantly present in hASC CM. Among these, several molecules with known immune functions, such as CCL2 and Metrnl, were recognized. Interestingly, between the proteins solely released by hDFs, the factor CB4 of the complement system is listed. A full description of the results will be presented in the poster. CONCLUSION: From a clinical perspective, the choice of CM over cell therapy presents substantial benefits, e.g. in terms of safety and handling. With this study, we lay the basis for the characterization of the factors -released as soluble components or vesicular cargos- involved in the immunoregulatory properties of hASC CM, in the light of giving a solid rationale to its use as novel pharmaceutical in the treatment of inflammatory diseases