Here, for the first time, we test a novel hypothesis that systemic treatment of stroke with exosomes derived from multipotent mesenchymal stromal cells (MSCs) promote neurovascular remodeling and functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo) followed by tail vein injection of 100 μg protein from MSC exosome precipitates or an equal volume of vehicle phosphate-buffered saline (PBS) (n=6/group) 24 hours later. Animals were killed at 28 days after stroke and histopathology and immunohistochemistry were employed to identify neurite remodeling, neurogenesis, and angiogenesis. Systemic administration of MSC-generated exosomes significantly improved functional recovery in stroke rats compared with PBS-treated controls. Axonal density and synaptophysin-positive areas were significantly increased along the ischemic boundary zone of the cortex and striatum in MCAo rats treated with exosomes compared with PBS control. Exosome treatment significantly increased the number of newly formed doublecortin (a marker of neuroblasts) and von Willebrand factor (a marker of endothelial cells) cells. Our results suggest that intravenous administration of cell-free MSC-generated exosomes post stroke improves functional recovery and enhances neurite remodeling, neurogenesis, and angiogenesis and represents a novel treatment for stroke.

Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes.

To test, in vivo, the hypothesis that exosomes from multipotent mesenchymal stromal cells (MSCs) mediate microRNA 133b (miR-133b) transfer which promotes neurological recovery from stroke, we employed knock-in and knock-down technologies to up-regulate or down-regulate the miR-133b level in MSCs (miR-133b+MSCs or miR-133b−MSCs) and their corresponding exosomes, respectively. Rats were subjected to middle cerebral artery occlusion (MCAo) and were treated with naïve MSCs, miR-133b+MSCs, or miR-133b−MSC at one day after MCAo. Compared with controls, rats receiving naïve MSC treatment significantly improved functional recovery, and exhibited increased axonal plasticity and neurite remodeling in the ischemic boundary zone (IBZ) at day 14 after MCAo. The outcomes were significantly enhanced with miR-133b+MSC treatment, and were significantly decreased with miR-133b−MSC treatment, compared to naïve MSC treatment. The miR-133b level in exosomes collected from the cerebral spinal fluid was significantly increased after miR-133b+MSC treatment, and was significantly decreased after miR-133b−MSC treatment at day 14 after MCAo, compared to naïve MSC treatment. Tagging exosomes with green fluorescent protein demonstrated that exosomes-enriched extracellular particles were released from MSCs and transferred to adjacent astrocytes and neurons. The expression of selective targets for miR-133b, connective tissue growth factor and ras homolog gene family member A, were significantly decreased in the IBZ after miR-133b+MSC treatment, while their expression remained at similar elevated levels after miR-133b−MSC treatment, compared to naïve MSC treatment. Collectively, our data suggest that exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons, which regulate gene expression, subsequently benefit neurite remodeling and functional recovery after stroke.

Object Transplanted multipotent mesenchymal stromal cells (MSCs) improve functional recovery in rats after traumatic brain injury (TBI). Here, we test a novel hypothesis that systemic administration of cell-free exosomes generated from MSCs promotes functional recovery and neurovascular remodeling in rats after TBI. Methods Wistar rats were subjected to TBI followed by tail vein injection of 100 μg protein of exosomes derived from MSCs or an equal volume of vehicle phosphate-buffered saline (n = 8/group) 24 hours later. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, neurological severity score and footfault tests were performed. Animals were sacrificed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Results Compared with saline-treated controls, exosome-treated TBI rats showed significant improvement in spatial learning at 34-35 days measured by the Morris water maze test (p < 0.05), and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14-35 days post injury (p < 0.05). Exosome treatment significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn immature and mature neurons in the dentate gyrus as well as reduced neuroinflammation. Conclusions We, for the first time, demonstrate that MSC-generated exosomes effectively improve functional recovery, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing inflammation in rats after TBI. Thus, MSC-generated exosomes may provide a novel cell-free therapy for TBI and possibly other neurological diseases.

Background and Purpose Multipotent mesenchymal stromal cell (MSC) harvested exosomes are hypothesized as the major paracrine effectors of MSCs. In vitro, the miR-17-92 cluster promotes oligodendrogenesis, neurogenesis and axonal outgrowth. We therefore investigated whether the miR-17-92 cluster enriched exosomes (Exo-miR-17-92+) harvested from MSCs transfected with a miR-17-92 cluster plasmid enhance neurological recovery compared to control MSC derived exosomes (Exo-Con). Methods Rats subjected to 2 hours of transient middle cerebral artery occlusion (MCAO) were intravenously administered Exo-miR-17-92+, Exo-Con, or liposomes, and were sacrificed 28 days post MCAO. Histochemistry, immunohistochemistry and Golgi-Cox staining were used to assess dendritic, axonal, synaptic and myelin remodeling. Expression of phosphatase and tensin homolog (PTEN) and activation of its downstream proteins, protein kinase B (PKB or Akt), mechanistic target of rapamycin (mTOR), and glycogen synthase kinase 3 beta (GSK-3β) in the peri-infarct region were measured by means of Western blots. Results Compared with the liposome treatment, both exosome treatment groups exhibited significant improvement of functional recovery, but Ex-miR-17-92+ treatment had significantly more robust effects on improvement of neurological function, and enhancements of oligodendrogenesis, neurogenesis and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone (IBZ) than the Ex-Con treatment. Moreover, Ex-miR-17-92+ treatment substantially inhibited PTEN, a validated miR-17-92 cluster target gene, and subsequently increased the phosphorylation of PTEN downstream proteins, Akt, mTOR and GSK-3β compared to Ex-Con treatment. Conclusions Our data suggest that treatment of stroke with tailored exosomes enriched with the miR-17-92 cluster increases neural plasticity and functional recovery after stroke, possibly via targeting PTEN to activate the PI3K/Akt/mTOR/GSK-3β signaling pathway.