Exosomes — beyond stem cells for restorative t.pdf

Stroke is the leading cause of adult-onset disabil-ity worldwide1.Most survivors of stroke experience some degree of spontaneous functional recovery,but the regain of function tends to be modest and is often insufficient for survivors to be independent in their daily lives.Consequently,disability after stroke imposes many social and economic burdens on soci-ety.Similarly,people with traumatic brain injury(TBI)can experience spontaneous recovery,but many are left permanently disabled,and no current medical interven-tion can aid or improve upon spontaneous restoration of neurological function.Patients who survive a stroke often improve rapidly in the following weeks(the acute phase),after which their recovery slows but can continue for months(the chronic phase)2.This spontaneous improvement of neurologi-cal function,which is also observed after TBI,suggests that limited remodelling can occur in the adult brain to compensate for injury and loss of tissue.Long-term neurological recovery is thought to depend on remodel-ling of neuronal circuitry in healthy tissue to compensate for dead and damaged tissue that is not recoverable.For example,in rodents,spontaneous remodelling of axons after experimental ischaemic stroke has been observed for at least 8 weeks without any intervention,and this remodelling was necessary for neurological recovery3.In this context,a possible therapeutic strategy to improve recovery from brain insults such as stroke and TBI is amplification of intrinsic restorative processes by targeting intact CNS tissue to promote neurite remod-elling in the brain and spinal cord,angiogenesis at the site of damage,and functional restorative changes in the glia(astrogliosis,oligodendrogenesis and induction of a reparative microglial phenotype).Initial attempts at such a strategy were cell-based therapies,particularly with mesenchymal stromal cells(MSCs)derived from bone marrow48.This approach has proved to be safe and has improved recovery of neuro-logical function in animal models;early-phase clini-cal evidence for MSC therapy is also promising7,9.The rationale for such cell-based therapies was the replace-ment of dead neurons by differentiation of grafted MSCs,but overwhelming evidence from preclinical studies demonstrates that MSC therapy and other cell-based therapies promote endogenous neuronal rewiring and increase angiogenesis and neurogenesis in the ischaemic brain by secreting factors that trigger signalling pathways that amplify brain remodelling4,10.Emerging data suggest that exosomes,a subtype of extracellular vesicles,that are released from the MSCs contribute substantially to the beneficial effects of cell therapies,including MSC therapy for stroke,TBI and other neurodegenerative diseases9,11,12.Exosomes beyond stem cells for restorative therapy in stroke and neurological injuryZhengGangZhang1*,BenjaminBuller1 and MichaelChopp1,2Abstract|Stroke is a leading cause of disability worldwide,and brain injuries devastate patients and their families,but currently no drugs on the market promote neurological recovery.Limited spontaneous recovery of function as a result of brain remodelling after stroke or injury does occur,and cell-based therapies have been used to promote these endogenous processes.Increasing evidence is demonstrating that the positive effects of such cell-based therapy are mediated by exosomes released from the administered cells and that the microRNA cargo in these exosomes is largely responsible for the therapeutic effects.This evidence raises the possibility that isolated exosomes could be used alone as a neurorestorative therapy and that these exosomes could be tailored to maximize clinical benefit.The potential of exosomes as a therapy for brain disorders is therefore being actively investigated.In this Review,we discuss the current knowledge of exosomes and advances in our knowledge of their effects on endogenous neurovascular remodelling events.We also consider the opportunities for exosome-based approaches to therapeutic amplification of brain repair and improvement of recovery after stroke,traumatic brain injury and other diseases in which neurorestoration could be a viable treatment strategy.1Department of Neurology,Henry Ford Hospital,Detroit,MI,USA.2Department of Physics,Oakland University,Rochester,MI,USA.*e-mail:zhazh neuro.hfh.eduhttps:/doi.org/10.1038/s41582-018-0126-4REVIEWSNature reviews|NeurologyIn this Review,we discuss recent advances that demonstrate the importance of exosomes in intercellular signalling in the brain and consider the use of exosomes for treatment of acute stroke and for amplification of brain remodelling to improve recovery of neurological function after stroke and TBI.We discuss evidence that shows how unmodified and modified exosomes can affect outcomes of stroke and TBI when used as therapy,and we examine cellular and molecular mechanisms that might underlie the benefits of exosome therapy.We also outline opportunities for and challenges in translation of exosome-based therapy to clinical applications.Properties and functions of exosomesExosomes are endosome-derived membrane-bound vesicles with diameters of 30150 nm,and they are released by cells in all living systems in physiological and pathophysiological conditions1315.The sizes of other subtypes of extracellular vesicles microve-sicles and apoptotic bodies overlap with those of exosomes,but each subtype has a distinct biogenesis pathway1618;the biogenesis of exosomes is reviewed in detail elsewhere1618.Exosomes are uniform lipid bilayer spheroids,and their membranes contain tetraspanin proteins,including CD63,CD81 and CD9,and the endosome membrane proteins flotillin and ALIX(also known as PDCD6-interacting protein).The endosome membrane proteins have been used as exosomal markers19,20,but distinguish-ing exosomes from other extracellular vesicles with overlapping size and density solely on the basis of these markers is difficult14.Exosome cargo.Exosomes carry proteins,lipids and nucleic acids,including mRNA,microRNA(miRNA)and long non-coding RNA.In physiological conditions,exosomes routinely transfer these functional biomole-cules from cell to cell to facilitate intercellular commu-nication14,17,18.Of the cargo that exosomes carry,miRNAs have been investigated most fully in terms of their func-tional importance,and studies have indicated that they are central to the therapeutic effects of exosomes.miRNAs derive from primary transcripts that are processed to form mature duplex miRNAs21,22.One or both of the mature RNA strands binds to argonaute 2 (Ago2)and is incorporated into the RNA-induced silencing complex(RISC),which targets mRNA for cleavage or translational repression21,23,24.Novel miRNAs arise frequently but are rarely lost25,and miRNA diver-sity within an organism correlates with morphological complexity even though genomic density does not26.This phenomenon might reflect the fact that miRNAs each have several or even hundreds of target genes,making them highly efficient regulators of gene net-works.miRNAs are therefore extremely powerful in the regulation of complex networks.Exosomes in the brain.Neurons contain large numbers of multivesicular bodies,where generation of exosomes initially occurs27,and brain cells secrete exosomes in physiological conditions28(Fig.1).These brain exosomes have an important role in the heterocellular communi-cation that regulates brain function27,29,30.Glutamatergic synaptic activity in cultured mature neurons increases exosomal release from these cells,and these neuron-derived exosomes carry the neuronal-specific protein L1 cell adhesion molecule(L1CAM)and the GluR2 and GluR3 subunits of glutamate receptors3133,suggesting that neuronal exosomes have a role in regulating neu-ronal function.Exosomes derived from neurons also communicate with cerebral endothelial cells to regulate the bloodbrain barrier(BBB)34.Oligodendrocytes and astrocytes also communicate with neurons and each other via exosome release27.Exosome release is regulated by potassium chloride con-centrations in astrocytes and by cytosolic calcium levels in oligodendrocytes.Glutamate from activated neurons also stimulates oligodendrocytes to release exosomes35,36.Multivesicular bodies from oligodendrocytes have been detected in the periaxonal space,and oligodendrocyte exosomes carry the myelin proteins proteolipid protein(PLP),23-cyclic-nucleotide-phosphodiesterase(CNP)and myelin basic protein(MBP)36,37;these observations suggest that oligodendrocyte exosomes coordinate the myelination of axons by oligodendrocytes38.In addition,oligodendrocyte exosomes improve neuronal viability in conditions of oxygenglucose deprivation(OGD)by transferring their cargo,which includes superoxide dis-mutase and catalase that provide resistance to oxidative stress,into neurons37.Astrocytes have diverse roles in brain physiology,from maintaining BBB integrity to synaptic regulation,and several lines of evidence show that many of their functions are mediated by exosomes.For example,some evidence suggests that synaptic plasticity depends in part on the release of miR-26-containing exosomes from astrocytes39,an effect that might result from miR-26-mediated inhibition of glycogen synthase kinase 340,a potent suppressor of axonal remodelling and synaptic plasticity.In addition,one study has shown that astro-cyte exosomes protect against OGD-induced neuronal death via transfer of exosomal cargo prion protein(PrP)into oxygenglucose-deprived neurons41.Furthermore,owing to the unique property of astrocytes that the end feet of a single cell often contact the BBB and the syn-aptic space,internalization and release of exosomes by astrocytes might bridge signals from neurons to the periphery and vice versa42.Much is left to discover about the functions of exosomes from astrocytes,given that Key pointsExosomesareinvolvedinmanyaspectsofnormalbrainphysiologyandfacilitatecommunicationbetweenbraincellsandbetweenthebrainandtheperiphery.Increasingevidencesuggeststhatexosomesfrommesenchymalstromalcells(MSCs)mediatethebeneficialeffectsofcelltherapyforstrokeandtraumaticbraininjury(TBI).TheeffectsofMSC-derivedexosomesalonehavethepotentialtoimproveneurologicaloutcomesinanimalmodelsofstroke,TBIandotherneurologicaldiseases.Ofthecargoinexosomes,microRNA(miRNA)isofprimeimportanceinmediatingthetherapeuticeffects.ComparedwithnaiveMSC-derivedexosomes,engineeredMSC-derivedexosomesthatcontainselectedmiRNAhavemorepotenttherapeuticeffectsinstrokeandTBI diversity is high in exosomes from astrocytes and distinct from that of the parent cells43.Given the numerous roles that astrocytes have in normal physi-ology and pathophysiology,the specificity of miRNA abundance in astrocyte exosomes could signify that the functions these exosomes have in the brain are as varied as those of the parent cell themselves.Exosomes as a potential therapySince the earliest investigations of MSC therapy for brain injuries,the cell therapy community has sought to isolate the specific paracrine factors that mediate recovery.The only constituents of the MSC secretome that have suc-cessfully been used to recapitulate the response to ther-apy with the parent cells are exosomes9,11,12,14,17,18,4446,and several studies have been conducted in which exosomes(or extracellular vesicles)were used to treat stroke,TBI or intracerebral haemorrhage(ICH)in animals(Table1).The therapeutic potential of MSC-derived exosomes in rodent models of stroke and TBI was documented for the first time in 2013 and 2015,respectively9,11,12.These studies showed that intravenous administration of MSC-derived exosomes to rats that had been sub-jected to focal cerebral ischaemia or TBI substantially increased neurovascular remodelling and improved the neurological,behavioural and cognitive outcomes dur-ing recovery9,11,12.These improvements were comparable to those observed with MSC therapy9,11,12.A subsequent head-to-head comparison of the two treatments con-ducted in an independent laboratory demonstrated that the treatment of stroke with MSCs or MSC-derived exosomes in mice produced equal improvements in motor function and coordination46.Improved recovery of sensorimotor function as a result of treatment with MSC-derived exosomes has also been demonstrated in a rat model of subcortical stroke47.In another study in a mouse model of TBI,intravenous administration of human MSC-derived exosomes substantially pre-served pattern separation and spatial learning ability48.AstrocyteEndothelial cellsabcLymphocyteNeuronMicrogliaOligodendrocyteCommunicationbetween CNSand peripheryInflammatoryextracellularvesicles releasedinto bloodExtracellularvesicles from peripheral immunecells aggravate CNS responseSuppression ofinflammation andpromotion ofaxon growthExosomesDendritic cellMacrophageMaintenance ofBBB integrityBBBbreakdownafter injuryTrophicsupportfor axonsFig.1|roles of exosomes in the brain in physiology,after an insult and in exosome therapy.a|In normal physiology,exosomes mediate communication between cells within the CNS and between the CNS and the periphery.Oligodendrocytes provide trophic support to axons,and neurons transfer miR-132 via exosomes to endothelial cells,where it helps to maintain bloodbrain barrier(BBB)integrity.Exosomes also mediate dynamic crosstalk between astrocytes and endothelial cells.b|After a neural insult,the BBB is disrupted,and exosomes and other extracellular vesicles from distressed or dying cells in the brain enter the bloodstream and the cerebrospinal fluid,where they stimulate inflammatory responses in peripheral immune cells in addition to microglia in the brain.Furthermore,exosomes from inflammatory peripheral immune cells can infiltrate the CNS,where they exacerbate the response to injury.c|When exosomes are introduced intravenously,they target several cell types,including peripheral and CNS immune cells,and help to induce an anti-inflammatory phenotype in these cells.Furthermore,they target neurons and glia,which in turn release their own exosomes that promote CNS repair,including axonal sprouting.Nature reviews|NeurologyReviewsImprovements in sensorimotor and cognitive function have also been reported as a result of treatment with MSC-derived exosomes in a rat model of ICH49,50.Following the initial studies in rodents,several studies of exosome therapy have involved large animal models of stroke and TBI.In a sheep model of hypoxicischaemic brain injury in ovine fetuses,inutero administration of MSC-derived extracellular vesicles reduced the fre-quency and duration of seizures and preserved the sen-sitivity of the baroreceptor reflex45.In a porcine model of polytrauma including TBI,administration of exo-somes derived from human MCSs 9 h after injury pro-moted neurological recovery51.Similarly,treatment with MSC-derived exosomes in adult rhesus monkeys with cortical lesions led to faster and fuller recovery than without treatment;the treated animals attained a grasping