The Human Mesenchymal Stem Cell Secretome Directs Macrophage Differentiation in an Oxygen-Dependent Manner

Abstract Background Chronic disease states can share inflammation as an underlying pathology. Macrophages are associated with chronic inflammation; in general, M1 phenotype macrophage activity inhibits cell proliferation and causes tissue damage whilst M2 macrophages promote proliferation and tissue repair. Human mesenchymal stem/stromal cells (hMSCs) are of particular interest for immunoregulatory diseases, through their ability to suppress T cell proliferation. Methods The application of a physiological oxygen environment to hMSC cultures modulates their gene expression and results in an altered secretome. Consequently, physiological, 2% O 2 hMSC SFCM was assessed in comparison to air oxygen (21% O 2 ) produced SFCM and applied to activated THP-1 cells in 10% and 21% O 2 to assess changes in macrophage differentiation. Results SFCM significantly suppressed proliferation and metabolic activity whilst promoting cell adherence compared to serum-free media (SFM) controls ( p < 0.001). Terminal differentiation phenotype varied in an oxygen-dependent manner, with 21% air oxygen SFCM leading to an increased M1:M2 macrophage differentiation ratio, with cells displaying distinct pancake-like morphology, increased TNFa secretion (21% O 2 = 922 ± 5 pg/mL, and 10% O 2 = 373 ± 2 pg/mL) and CD197 expression (21% = 69.1%±6.4, and 10% = 18.6 ± 0.4%). In contrast, physiological oxygen SFCM led to the production of a greater M2:M1 macrophage differentiation ratio; displaying an elongated spindle shape, increased IL-10 secretion (21% O 2 = 3780 ± 560 pg/mL, and 10% O 2 = 9115 ± 889), and CD3 and CD14 expression. SFCM collected under 2% O 2 suppressed pro-inflammatory transcriptional profiles through downregulation of TNFa, IL-1B, and IL-12B compared to 21% O 2 collected SFCM. Conclusion Collectively, SFCM induced terminal macrophage differentiation in an oxygen-dependent manner. This effect was not completely replicated by the application of isolated candidate anti-inflammatory cytokines (IL-4, IL-10, IL-13 or TGFb) or a combinational cytomix, highlighting the complex effects of conditioned media products and further work needed to determine mechanism of action.