Global signatures of the human mesenchymal stromal cell secretome: A comparative proteomic analysis of iPSC and tissue-derived MSC secretomes before and after inflammatory licensing

Abstract

Abstract Multipotent mesenchymal stromal cells (MSCs) are one of the most heavily studied cell therapies worldwide, with much of their therapeutic potential underpinned by their complex secretory profiles. Like MSCs themselves, their secretome varies significantly between donors, sources, and according to microenvironmental cues. As such, there remains a lack of consensus as to the global nature of the MSC secretome, its source-based heterogeneity, and the dynamic changes it undergoes in response to inflammatory licensing. A full understanding of these differences is essential in understanding the mechanisms that drive MSC-based tissue repair and in optimising the properties of MSCs for cell therapies. This study used liquid chromatography tandem mass spectrometry (LC/MS-MS) to characterise and compare the secretomes of 13 MSC lines, sourced from bone marrow (BM.MSCs), umbilical cord (UC.MSCs), and adipose tissue (AT.MSCs), alongside multiple batches of clinical and commercial grade induced pluripotent stem cell derived MSCs (iMSCs), all under both resting and inflammatory licensed conditions. We confirm, for the first time, that iMSCs successfully recapitulate the process of inflammatory licensing, validating their comparability to tissue-derived MSCs and providing important support for their application as an immunotherapy. We identify a global and dichotomous signature of the MSC secretome and inflammatory licensing, where resting secretomes are defined by prominent extracellular matrix (ECM) proteins and overrepresentation of pro-regenerative and wound healing processes, while licensed secretomes downregulate these factors in favour of chemotactic and immunosuppressive proteins and immunomodulatory processes. Furthermore, under both resting and licensed conditions, MSC secretomes separate based on source, with iMSC and UC.MSC secretomes more similar to each other, containing higher concentrations of proteins indicating proliferative potential and telomere maintenance, while adult tissue-derived, BM.MSC and AT.MSC secretomes contained more fibrotic and ECM proteins This dataset provides a detailed atlas of resting and licensed MSC secretomes and generates insights into the molecular mechanisms underlying the differences observed between MSC secretomes from different sources or functional states. Going forward, this will inform the design of more effective MSC-based therapies by identifying the most suitable MSC source for a particular application, and allow the development of tailored culture conditions and/or preconditioning methods to enhance the therapeutic potential of these cells, potentially identifying specific factors that can be adapted for pharmaceutical intervention.