Abstract
Abstract
Objectives
Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs.
Methods
Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype.
Results
Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNAseq revealed that there were 2,085 highly significant upregulated genes in 3D matrices compared to TCP. Notably, >90% of highly expressed genes (including FOSB, FOS and TNFAIP6) were shared in all hydrogels. Gene ontology confirmed the TNF signalling pathway among the most significantly represented. Protein-protein interaction predictions identified TNF-alpha/NF-kappa B and AP1 pathways as differentially influenced by the hydrogel environment. Using inhibitors to these pathways, NFkB, but not AP1, impacted on the upregulation of TNFAIP6 and CXCL10 in 3D culture. Conditioned media from these studies was added to cultures of human monocytes with distinct changes in the resulting macrophage phenotype. MSCs in a 3D environment promoted a greater acquisition of the M2 repair macrophage phenotype and impacted on the numbers of pro-inflammatory M1 macrophages.
Conclusion
These data provide further evidence that the immunomodulatory action of human MSCs can be influenced by the surrounding structural environment. These observations have significance for understanding the events that following skeletal injury and the potential to be exploited in preconditioning MSCs for cell therapy.
