Extracellular vesicles released from human amniotic fluid stem cells modulate macrophages.

BACKGROUND

Human amniotic fluid stem cells (hAFSCs) secrete extracellular vesicles (EVs) that modulate fetal immunity. Because fetal and neonatal macrophages largely rely on innate responses, development of a targeted method to switch their inflammatory phenotype would fill a critical therapeutic gap in perinatal medicine. We therefore elucidated whether the hAFSC-EV surface marker CD44 functions as a "delivery code" for preferential uptake by inflammatory macrophages.

METHODS AND RESULTS

hAFSCs and hAFSC-EVs were isolated and characterized by performing flow cytometry, transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The hAFSC-EVs were co-cultured with macrophages derived from the human monocytic leukemia cell line THP-1. The uptake of hAFSC-EVs was evaluated using fluorescence microscopy and flow cytometry. The effects of hAFSC-EVs on macrophages were analyzed by western blotting and real-time quantitative polymerase chain reaction. Our analyses revealed that inhibiting CD44 expression with a functional antibody blocked hAFSC-EV uptake by macrophages and led to changes in the cellular phenotype. Furthermore, inhibiting the uptake of CD44-positive hAFSC-EVs suppressed the expression of some pro-inflammatory cytokines. These findings suggest that CD44-positive hAFSC-EVs play a crucial role in the anti-inflammatory effects of hAFSCs.

CONCLUSIONS

Our study provides insights into the specific delivery of hAFSC-EVs into inflammatory macrophages and sheds light on the potential therapeutic applications of these EVs for regulating inflammatory macrophage phenotypes.