Macrophage-Based Transcriptional Assays for the Comparative Assessment of the Anti-Inflammatory Paracrine Activity of Canine Adipose-Derived Mesenchymal Stromal Cells

Therapies based on mesenchymal stromal cells (MSCs) have high potential in the field of regenerative medicine due mainly to their immunomodulatory properties. However, their clinical translation is hampered by a lack of sufficiently standardised potency tests. Since macrophages constitute key mediators of the effects of MSCs, macrophage-based assays potentially provide a relevant in vitro tool for the evaluation of the activity of MSC products. This study involved the coculturing of canine adipose-derived mesenchymal stromal cells (ASCs) with macrophages derived from human THP-1 and U937 monocyte cell lines, murine RAW264.7 macrophages and primary human macrophages. The M2 polarisation was assessed following stimulation with IL-4/IL-13 in THP-1 and U937 macrophages. The mRNA expression of the pro- and anti-inflammatory markers was analysed using qPCR. The ASC transwell coculture altered the LPS-induced inflammatory mRNA expression in a strongly model- and marker-dependent manner. The U937-derived macrophages exhibited the most consistent suppression of the tested inflammatory transcripts and the RAW264.7 cells provided a practical readout for selected inflammatory markers, whereas the THP-1 macrophages evinced the suppression of TNFA but not IL1B or PTGS2 under the selected stimulation conditions. IL-4/IL-13 induced moderate but statistically non-significant changes in IL10 and TGFB1 in the U937-derived macrophages but no reproducible response in the THP-1-derived macrophages. In a subsequent U937 coculture experiment, ASC-derived paracrine factors altered selected M2-associated transcripts at specific time points. The results thus provided support for macrophage-based transcriptional readouts as an early-stage tool for comparing responder macrophage models and detecting the selected anti-inflammatory paracrine effects of canine ASCs; the U937 cells were found to be particularly suitable for the study of inflammatory polarisation and the RAW264.7 cells for the purpose of standardised screening.