No abstract

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.

Abstract-We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1ϫ10 6 ) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (PϽ0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (nϭ12) treated with hMSCs compared with numbers in rats (nϭ12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (PϽ0.05) raised endogenous rat VEGF levels in the IBZ from 10.5Ϯ1.7 ng/mL in the control group to 17.5Ϯ1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (PϽ0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (PϽ0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.