Analysis of cellular responses following interaction with extracellular vesicles derived from HEK293T and human adipose derived stem cells

Extracellular vesicles (EVs), particularly exosomes, are crucial mediators of intercellular communication that influence immune responses, cell proliferation, and angiogenesis. Their therapeutic potential has been explored for the treatment of chronic, non-healing wounds, especially in diabetic patients with chronic inflammation, impaired cellular proliferation, and poor angiogenesis. We investigated the role of exosomes derived from human embryonic kidney 293T (HEK293T) cells and human adipose-derived stem cells (hADSCs) on NIH3T3 fibroblast migration. Exosomes were characterized for size, concentration, and surface markers. Western blotting and scratch assays were used to assess ERK1/2 activation and cell migration. Characterization revealed that HEK293T exosomes were more abundant (3.1 × 10¹¹ ± 4.9 × 10⁹ particles/mL), larger (178 ± 4 nm), and exhibited stronger surface marker protein expression than exosomes from hADSCs (2.4 × 10⁹ ± 4.1 × 10⁸ particles/mL, 153 ± 5 nm). Both types of exosomes significantly enhanced NIH3T3 fibroblast migration with migration indices of 63.2 ± 4.3% and 48.9 ± 2.7% via ERK1/2 signaling, with HEK293T exosomes showing stronger effects because of their higher protein content. Western blot analysis revealed that robust ERK1/2 activation by both exosome types was crucial for fibroblast migration, with scratch assays highlighting their pro-migratory effects. Inhibition of the ERK1/2 pathway significantly reduced migration, with HEK293T exosomes showing a reduction to 9.6 ± 3.2% and hADSC exosomes to 2.6 ± 0.2% when treated with the MEK1/2 inhibitor PD98059. HEK293T exosomes induced more pronounced cell migration, and hADSC exosomes showed improved efficacy under stress, suggesting that both exosome types are promising candidates for targeted regenerative therapies in chronic wound healing.