Adipose derived stem cell extracellular vesicles modulate primary human macrophages to an anti-inflammatory phenotype in vitro

EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune-regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ), M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and ADSC-EVs were co-cultured with macrophages for 48 h. Flow cytometry and high-dimensional analysis clustered macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a ‘high inflammatory profile’ (CD36⁺⁺⁺CD206⁺⁺⁺CD86⁺⁺⁺; 16.5 ± 7.0%; p < 0.0001) to a cluster with a ‘lower inflammatory profile’ (CD36^+CD206+CD86+; 35  ± 21.5%; p < 0.05). M1-like macrophages shifted from a cluster with a ‘high inflammatory profile’ (CD206⁺⁺CD11b⁺⁺CD36⁺⁺CD163⁺⁺; 26.1 ± 9.4%; p = 0.0024) to a ‘lower inflammatory profile’ (CD206⁺CD11b⁺CD36^+CD163+; 72.8  ± 8.7%; p = 0.0007). There was no shift in M2-like clusters following ADSC-EV treatment. ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.