Secondary Release of Exosomes From Astrocytes Contributes to the Increase in Neural Plasticity and Improvement of Functional Recovery After Stroke in Rats Treated With Exosomes Harvested From MicroRNA 133b-Overexpressing Multipotent Mesenchymal Stromal Cells

We previously demonstrated that multipotent mesenchymal stromal cells (MSCs) that overexpress microRNA 133b (miR-133b) significantly improve functional recovery in rats subjected to middle cerebral artery occlusion (MCAO) compared with naive MSCs and that exosomes generated from naive MSCs mediate the therapeu-tic benefits of MSC therapy for stroke. Here we investigated whether exosomes isolated from miR-133boverexpressing MSCs (Ex-miR-133b(+)) exert amplified therapeutic effects. Rats subjected to 2 h of MCAO were intra-arterially injected with Ex-miR-133b(+), exosomes from MSCs infected by blank vector (Ex-Con), or phosphate-buffered saline (PBS) and were sacrificed 28 days after MCAO. Compared with the PBS treatment, both exosome treatment groups exhibited significant improvement of functional recovery. Ex-miR-133b(+) treat-ment significantly increased functional improvement and neurite remodeling/brain plasticity in the ischemic boundary area compared with the Ex-Con treatment. Treatment with Ex-miR-13313(+) also significantly increased brain exosome content compared with Ex-Con treatment. To elucidate mechanisms underlying the enhanced therapeutic effects of Ex-miR-133b(+), astrocytes cultured under oxygen- and glucose-deprived (OGD) condi-tions were incubated with exosomes harvested from naive MSCs (Ex-Naive), miR-133b downregulated MSCs (Ex-miR-133b(-)), and Ex-miR-133b(+). Compared with the Ex-Naive treatment, Ex-miR-133b(+) significantly increased exosomes released by OGD astrocytes, whereas Ex-miR-133b(-) significantly decreased the release. Also, exosomes harvested from OGD astrocytes treated with Ex-miR-133b(+) significantly increased neurite branching and elongation of cultured cortical embryonic rat neurons compared with the exosomes from OGD astrocytes subjected to Ex-Con. Our data suggest that exosomes harvested from miR-133b-overexpressing MSCs improve neural plasticity and functional recovery after stroke with a contribution from a stimulated secondary release of neurite-promoting exosomes from astrocytes.