Fig. 1

Experimental protocol schematic. a EVs characterization using various techniques: EVs imaged by electronic microscope (left); histogram representing a distribution graph of size and concentration of the particles of the EVs using NanoSight (middle); and CD63, CD81, and ALIX as positive markers and albumin as negative marker expression using western blot (right). Supernatant has been used as control. b In vitro study. Human neural progenitor cells were plated and cultured, using a seeding density of 1 × 10⁴ cells/cm² and grown to 70% confluence. At 7 days, the cells were differentiated to oligodendrocytes and neurons, with medium changes every 2–3 days for 14 days. At 21 days, cells were subjected to normoxia or OGD and the following day the cells received various doses of EVs (10 μg, 20 μg, 30 μg, 50 μg, 100 μg, or 200 μg) for 72 h. After fixing, proliferation and marker expression were analyzed. c Biodistribution study of EVs. Rats were subjected to subcortical infarct by endothelin-1 injection. Twenty-four hours later, EVs were labeled with DiI prior to administration and the rats received treatment (50 μg, 100 μg, or 200 μg of EVs). At 48 h, histological studies for biodistribution of EVs were performed. d In vivo study. Subcortical stroke was induced using endothelin-1. Each group received one of various doses of MSC-derived EVs or a saline solution as treatment 24 h after surgery. Functional deficit and MRI scans were evaluated at 48 h and at 28 days after surgery. The blood levels of EVs were analyzed 24 h prior to surgery, and at 48 h and 28 days after surgery. The histological and molecular analyses were performed at 28 days. Abbreviations: EVs, extracellular vesicles; MRI, magnetic resonance image; OGD, oxygen and glucose deprivation