Extracellular Vesicle Transcriptome in Ischemic Stroke Rat with Intra-arterial Transplantation of Human Neural Stem Cells (microRNA)

Extracellular vesicles (EVs) can cross the blood–brain barrier and enter the systemic circulation, potentially acting as peripheral biomarkers of stroke neuropathology. Here, we investigated alterations in EV RNA cargoes extracted from rat brain and plasma before and after stroke induction via middle cerebral artery occlusion and subsequent human neural stem cells (hNSCs) transplantation. EV RNA coexpression profiles were assessed, and digital source tracking was used to determine EV origin. The therapeutic effects of intra-arterial delivery of hNSCs on ischemic rat brains were quantified, focusing on functional recovery, resolution of ischemic lesions, and the microenvironment. Stroke induced distinct EV secretion patterns, with a notable increase in EV secretion from non-neuronal cells. hNSCs transplantation caused minimal immune rejection and transplanted cells survived in the brain for over a week. Stem cell-derived EVs were detected in peripheral blood, indicating prolonged systemic distribution after transplantation. Gene regulatory network analyses identified specific EV miRNAs that play crucial roles in neurogenesis, wound healing, angiogenesis, and blood–brain barrier integrity. An integrated analysis of EV RNAs in brain and plasma samples revealed that stroke increased correlations in RNA expression between brain and plasma and that hNSCs transplantation reversed the effect. Brain- and plasma-derived EVs carry similar molecular information after stroke, suggesting that plasma-derived EV RNAs reflect stroke pathophysiology. Intra-arterial transplantation of hNSCs improved outcomes after stroke in rats, by promoting endogenous neurogenesis and maintaining blood–brain barrier integrity. The identified EV miRNAs provided a new mechanism by which hNSCs transplantation regulates neural regeneration through the miR-204-5p/EFNB3 axis. https://onlinelibrary.wiley.com/doi/10.1002/mco2.70400 Overall design: Eighteen adult male Sprague-Dawley rats, aged 8-12 weeks, were subjected to transient middle cerebral artery occlusion (tMCAO, 90 minutes occlusion ). Three days after tMCAO, rats were injected with either a suspension of 2×10^5 hNSCs or saline randomly (9 rats in each group) using a 31-gauge needle. Three animals per group were sacrificed for sample collection (brain and plasma) at 4, 10, and 31 days post-tMCAO. Brain and serum EVs were extracted for RNA isolation and sequencing. Three healthy adult male Sprague-Dawley rats were sacrificed for brain and plasma sample collection, serving as healthy controls. Temporal expression profiles of brain- and plasma-derived EV-RNA before and after stroke were analyzed. Comparative analysis between treatment and control group at different time points post-stroke were also conducted.