Introduction: Mesenchymal stromal cells (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate different cell populations involved in the advance of the disease, such as macrophages. These immune cells possess a broad spectrum of inflammatory responses. In addition, previous data have shown that the MSC secretome influences macrophage phenotype and functional capacities. Furthermore, culturing MSC with physiomimetic cues from the extracellular matrix (ECM) have shown to improve their repairing actions upon transplantation. Physiomimetic culture of cells relies mainly on ECM-derived biomaterials, such as, decellularised scaffolds and lung ECM hydrogels which provide a similar biomechanical milleau to the organ. Despite the recent advances in MSC-based therapies, there is scarce information regarding the changes on the secretome content attributed to these culture platforms, and especially, how the secretome profile could influence macrophage activity in favour of therapy. In this setting, the aim of this study was to assess the macrophage activity exerted by the secretome isolated from physiomimetically cultured lung-resident mesenchymal stromal cells (LMSC).
Methodology: LMSC from human donors were cultured on in-house developed devices that enable lung-mimetic strain. Medium from LMSC cultured in either lung ECM scaffolds and in lung ECM hydrogels whilst subjected to cyclic stretch, and on tissue culture plates (TCP) was analysed for typical cytokines, chemokines and growth factors. RNA was analysed for the gene expression of relevant mechano regulators CTGF and CYR61. Human monocytes were differentiated to macrophages by adding PMA and assessed their phagocytic capacity of bioparticles in the presence of LMSC secretome. Macrophages were also polarized to M1 and M2 phenotypes by adding LPS or IL-4 plus IL-10, respectively. M0 (quiescent), M1 and M2 macrophages were exposed to the medium of LMSC from the different culture conditions and analysed for surface markers by flow cytometry.
Results: CYR61 gene expression showed decreases when cultured on the aforementioned lung-mimetic environments compared to TCP. Furthermore, CTGF and CYR61 displayed a marked reduction when cultured in lung ECM hydrogels. The secretome content was plotted in UMAPs where the scaffold clusters mostly to itself while there is a large overlap between the hydrogel and the TCP samples. Additionally, stretch elicited different changes on HGF, MCP-1, IL-6 and TNF-α according to the environment where LMSC had been cultured. Similarly, phagocytosis showed a differential increase on TCP due to the stretch which was not observed in the physiomimetic culture.
Conclusion: Mechanical features of the lung ECM orchestrate key outcomes on LMSC, hence providing new insights into preconditioning of MSC for therapy.