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.

Multipotent human bone marrow derived mesenchymal stem cells (hMSCs) improve functional outcome after experimental traumatic brain injury (TBI). The present study was designed to investigate whether systemic administration of cell-free exosomes generated from hMSCs cultured in 2-dimensional (2D) conventional conditions or in 3-dimensional (3D) collagen scaffolds promote functional recovery and neurovascular remodeling in rats after TBI. Wistar rats were subjected to TBI induced by controlled cortical impact; 24 hours later tail vein injection of exosomes derived from hMSCs cultured under 2D or 3D conditions or an equal number of liposomes as a treatment control were performed. The modified Morris water maze, neurological severity score and footfault tests were employed to evaluate cognitive and sensorimotor functional recovery. Animals were sacrificed at 35 days after TBI. Histological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation. Compared with liposome-treated control, exosome-treatments did not reduce lesion size but significantly improved spatial learning at 33-35 days measured by the Morris water maze test, and sensorimotor functional recovery, i.e., reduced neurological deficits and footfault frequency, observed at 14-35 days post injury (p < 0.05). Exosome treatments significantly increased the number of newborn endothelial cells in the lesion boundary zone and dentate gyrus, and significantly increased the number of newborn mature neurons in the dentate gyrus as well as reduced neuroinflammation. Exosomes derived from hMSCs cultured in 3D scaffolds provided better outcome in spatial learning than exosomes from hMSCs cultured in the 2D condition. In conclusion, hMSC-generated exosomes significantly improve functional recovery in rats after TBI, at least in part, by promoting endogenous angiogenesis and neurogenesis and reducing neuroinflammation. Thus, exosomes derived from hMSCs may be a novel cell-free therapy for TBI, and hMSC-scaffold generated exosomes may selectively enhance spatial learning.

Cell-based therapy, e.g., multipotent mesenchymal stromal cell (MSC) treatment, shows promise for the treatment of various diseases. The strong paracrine capacity of these cells and not their differentiation capacity, is the principal mechanism of therapeutic action. MSCs robustly release exosomes, membrane vesicles (~30–100 nm) originally derived in endosomes as intraluminal vesicles, which contain various molecular constituents including proteins and RNAs from maternal cells. Contained among these constituents, are small non-coding RNA molecules, microRNAs (miRNAs), which play a key role in mediating biological function due to their prominent role in gene regulation. The release as well as the content of the MSC generated exosomes are modified by environmental conditions. Via exosomes, MSCs transfer their therapeutic factors, especially miRNAs, to recipient cells, and therein alter gene expression and thereby promote therapeutic response. The present review focuses on the paracrine mechanism of MSC exosomes, and the regulation and transfer of exosome content, especially the packaging and transfer of miRNAs which enhance tissue repair and functional recovery. Perspectives on the developing role of MSC mediated transfer of exosomes as a therapeutic approach will also be discussed.

We previously demonstrated that multipotent mesenchymal stromal cells (MSCs) with overexpressed 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 therapeutic benefits of MSC therapy for stroke. Here, we investigated whether exosomes isolated from miR-133b-overexpressed MSCs (Ex-miR-133b+) exert amplified therapeutic effects. Rats subjected to 2 hours (h) of MCAO were intra-arterially injected with Ex-miR-133b+, exosomes from MSCs infected by blank vector (Ex-Con), or phosphate-buffered solution (PBS), and were sacrificed 28 days post MCAO. Compared with the PBS treatment, both exosome treatment groups exhibited significant improvement of functional recovery. Ex-miR-133b+ treatment significantly increased functional improvement, and neurite remodeling/brain plasticity in the ischemic boundary area compared with the Ex-Con treatment. Treatment with Ex-miR-133b+ 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) conditions were incubated with exosomes harvested from naïve MSCs (Ex-Naive), miR-133b down-regulated 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-overexpressed MSCs improve neural plasticity and functional recovery after stroke with a contribution from a stimulated secondary release of neurite promoting exosomes from astrocytes.