s41392-023-01704-0 by Biogenomics

Clinicalapplicationsofstemcell-derivedexosomes

FeiTan

1,2,3,4 ✉,XuranLi1,2,ZhaoWang1,JiaojiaoLi1,2,KhawarShahzad1,2 andJialinZheng 5,6

Althoughstemcell-basedtherapyhasdemonstratedconsiderablepotentialtomanagecertaindiseasesmoresuccessfullythan conventionalsurgery,itneverthelesscomeswithinescapabledrawbacksthatmightlimititsclinicaltranslation.Comparedtostem cells,stemcell-derivedexosomespossessnumerousadvantages,suchasnon-immunogenicity,non-infusiontoxicity,easyaccess, effortlesspreservation,andfreedomfromtumorigenicpotentialandethicalissues.Exosomescaninheritsimilartherapeuticeffects fromtheirparentalcellssuchasembryonicstemcellsandadultstemcellsthroughverticaldeliveryoftheirpluripotencyor multipotency.Afterathoroughsearchandmeticulousdissectionofrelevantliteraturefromthelast fiveyears,wepresentthis comprehensive,up-to-date,specialty-specificanddisease-orientedreviewtohighlightthesurgicalapplicationandpotentialof stemcell-derivedexosomes.Exosomesderivedfromstemcells(e.g.,embryonic,inducedpluripotent,hematopoietic,mesenchymal, neural,andendothelialstemcells)arecapableoftreatingnumerousdiseasesencounteredinorthopedicsurgery,neurosurgery, plasticsurgery,generalsurgery,cardiothoracicsurgery,urology,headandnecksurgery,ophthalmology,andobstetricsand gynecology.Thediversetherapeuticeffectsofstemcells-derivedexosomesareahierarchicaltranslationthroughtissue-specific responses,andcell-specificmolecularsignalingpathways.Inthisreview,wehighlightstemcell-derivedexosomesasaviableand potentalternativetostemcell-basedtherapyinmanagingvarioussurgicalconditions.Werecommendthatfutureresearch combineswisdomsfromsurgeons,nanomedicinepractitioners,andstemcellresearchersinthisrelevantandintriguingresearch area.

SignalTransductionandTargetedTherapy (2024)9:17 ;https://doi.org/10.1038/s41392-023-01704-0

INTRODUCTION

Stemcellsareapopulationofundifferentiatedcellswithunique abilitiestoself-renewandrecreatefunctionaltissues.Theyare primarilyclassiﬁedbytheirdifferentiationpotential,originand lineageprogression.Accordingtotheirpotency,stemcellscanbe totipotent,pluripotent,multipotent,oligopotentandunipotent.1 Stemcellsexistbothinembryosandadultcells.Embryonicstem cells(ESCs)andinducedpluripotentstemcells(iPSCs)arebest examplesofpluripotentstemcells,2 whereasadultmultipotent stemcellsareexempliﬁedbyhematopoieticstemcells(HSCs),3 mesenchymalstemcells(MSCs),4 neuralstemcells(NSCs),5 and endothelialstem/progenitorcells(EPCs)6 (Fig. 1a).Allthese subtypesofstemcellshavebeenextensivelytrialedforthe treatmentofhumandiseases.

Stemcell-basedtherapy,asamodalityofregenerative medicine,hasgeneratedtremendousattention,asitoffers newoptionsforpatientssufferingfrompreviouslyincurable diseases.Subsequently,thousa ndsofrelatedclinicaltrialshave beenregistered,coveringawidespectrumofmedical problems,suchasmusculoskeletalandneurologicaldisorders, immunediseases,hematologicaldysfunctions,anddegenerativeconditions. 7 However,sometrialshavefailedtoshowany bene ﬁ tintheclinic.Thisislikelyduetotheinevitable limitationsofstemcelltherap y,suchasinfusiontoxicity, immunogenicity,tumorigenicpo tentialsandethicalissues. 8 Exosome,secretedbyalmostallcelltypesincludingstemcells

(Fig. 1 a),hasbeenpositedasasaferandmoreversatile alternativetostemcelltherapy. 9

Exosomesarenanoscale,spherical,andlipidbi-layeredsingle membraneextracellularvesicles,whichactasintercellular messengers.10 Exosomeshavebeenregardedasminiature versionsoftheirparentalcells,partiallybecauseexosomesfrom acertaincelltypeprovidecell-speciﬁcoruniquesetsof biomolecules.Inaddition,thestemcellshavebeenfoundto functioninaparacrinefashionthroughtheirsolublesecretome includingexosomes.11 Inotherwords,stemcell-derivedexosomes (SC-Exo)inheritsimilartherapeuticeffectsfromtheirparentalcell oforigin,e.g.,anti-inﬂammation,immunomodulationandtissue regeneration.12 Collectively,stemcell-derivedexosomesarea potentsurrogateforstemcelltherapywithoutexhibitingthe disadvantagestheircellularcounterpartspresent13 (Table 1).

Priortoclinicalapplications,exosomesmustbepreparedand optimizedintermsofproduction,purification,andmodification (Sections2.3and2.4).Awiderangeofmedicalreviewsanalyzing theseupstreammeasuresofexosometherapyhavebeen publishedinrecentyears.Nevertheless,someresearchavenues remainunder-investigated:inparticular,systematicinvestigation dedicatedtodownstreamclinicalapplicationsislacking,especially fromasurgicalperspective.Tissuesthathavebeendamaged, whetherbydiseaseorasurgeon’sscalpel,respondbyinflammatoryandregenerativedynamics,14 makingsurgeryaperfectarena forstemcell-derivedexosometherapy.15 Stemcell-derived

1DepartmentofORL-HNS,ShanghaiFourthPeople’sHospital,andSchoolofMedicine,TongjiUniversity,Shanghai,China; 2PlasmaMedicineandSurgicalImplantsCenter,Tongji University,Shanghai,China; 3TheRoyalCollegeofSurgeonsinIreland,Dublin,Ireland; 4TheRoyalCollegeofSurgeonsofEngland,London,UK; 5CenterforTranslational NeurodegenerationandRegenerativeTherapy,TongjiHospitalafﬁliatedtoTongjiUniversitySchoolofMedicine,Shanghai,Chinaand 6ShanghaiFrontiersScienceCenterof NanocatalyticMedicine,TongjiUniversity,Shanghai,China Correspondence:FeiTan(iatrologist@163.com)

Received:23July2023Revised:15October2023Accepted:12November2023

Fig.1 Illustrationoftheupstreammeasuresofexosometherapy(figuregeneratedusingAutodesk3dsMax2023). a productionand purificationofexosomes(MSCsandNSCsareusedasexamplesformultipotentstemcells). b contentofnaturalexosomes. c modificationof exosomes.(BMbonemarrow,DCdendriticcell,IACimmunoaffinitychromatography,iPSCinducedpluripotentstemcell,MHCmajor histocompatibilitycomplex,miRNAmicroRNA,MSCmesenchymalstemcell,MVBmultivesicularbody,NSCneuralstemcell,SECsize-exclusion chromatography,UCumbilicalcord)

Table1. Thecomparisonbetweenstemcelltherapyandstemcell-derivedexosometherapy

TreatmentmodalityAdvantagesLimitations

Stemcelltherapymultilineagedifferentiationpotentialshort-livedviabilityandlowengraftmentafter injection applicabletothetreatmentforawiderangeofdiseasesstringentstorageandtransportrequirements extensiveaccumulationoflaboratoryandclinicaldatatumorigenicpotential easytoisolateandpossibleformass-productioninfusiontoxicity well-developedregulatoryguidelinesimmunogenicity ethicalissues

Stemcell-derivedexosome therapy comparabletherapeuticeffectstostemcellsbutmuch smaller batch-to-batchinconsistency moreconcentratedfunctionalcargos,e.g.,cytokinesnostandardizedprotocolforpurificationandstorage modifiableatitssurfaceandinitscargosrelativelylowyieldforlargescalemanufacturing versatiledeliverymodalitiesnoindustry-standardqualityspecifications stableforlong-termstorageandtransportinsufficientregulatorycontrol negligibleriskoftumorigenesisandimmuneresponse lackofethicalissues

exosomesinheritsimilartherapeuticeffectsfromtheirparental celloforigin,e.g.,tissueregeneration,anti-inﬂammationand immunomodulation.12,16–18

Inthiswork,wewilldissectrelevantpublicationsfromthelast ﬁveyearsinordertopresentacomprehensive,up-to-date, specialty-speciﬁcanddisease-orientedreview(Fig. 2).Ouraimis tobridgethegapthatcurrentlyexistsbetweensurgeons, nanomedicinepractitioners,andstemcellresearchers.

GENERALBACKGROUNDOFEXOSOMESANDEXOSOME THERAPY

Biogenesis,composition,anduptakeofexosomes

Exosomesdifferfromothertypesofprimaryextracellularvesicles (e.g.,apoptoticbodiesandmicrovesicles)intermsofsize,content, andproductionmechanism.19 Themostpopularlyaccepted mechanismofexosomeformation,i.e.,anendosomalroute,isas follows(Fig. 1a).Theinitialendosomesareproducedbycell membraneinvaginationduringwhichthebioactivesubstances begintoaccumulatewithintheearlysortingendosomes.Thelate sortingendosomesthenformmultivesicularbodies(MVBs)aftera secondindentation.Finally,theMVBsfusewiththecell membrane,releasingthecarriedexosomestotheoutside.Nonendosomalrouteofexosomebiogenesis,suchasplasma membranebudding,hasalsobeenreported.20

Asthethreemajorexosomedatabases(i.e.,ExoCarta,Vesiclepedia,andEVpedia)summarize,exosomescontainnumerous molecules,includingproteins,glycoconjugates,lipids,nucleic acids,metabolites,andotherbioactivesubstances(Fig. 1b).The examplesofeachcategoryandthecorrespondingfunctionshave beenthoroughlyreviewedelsewhere.21,22 Ontheonehand, exosomescompriseacomplexproteinnetworkincludingexternal proteins(e.g.,tetraspanins,antigen-presentingcomplexes,and adhesionmolecules)andinternalproteins(e.g.,heatshock proteins,ESCRTmachinery,cytokinesandchemokines,and membranetransporters).23 Ontheotherhand,asthemost abundantinhumanexosomalnucleicacids,microRNA(mRNA) couldparticipateinhematopoiesis,exocytosis,andnerveand vascularregenerationthroughexosome-mediatedcellular communication.24

Therearevariousuptakemechanismsonceexosomesreachthe recipientcell,allofwhichcanbecategorizedintomembrane fusion,receptorinteraction,andinternalization21 (Fig. 1b).Finally, theexosomalcargosarereleasedintothecytoplasm,theprocess ofwhichdependsonthesourceoftheexosome,natureofthe

cargo,andthemetabolicstateoftherecipientcell.25 Theentire lifecyclefromexosomebiogenesistouptakeandintracellular signalingcanbetrackedusing ﬂuorescent,luminescent,and radioactivetechniques.26 27

Sourceandclassiﬁcationofexosomes

Dependingonwhetherexosomeshavebeenartificiallymodified, theyarebroadlyclassifiedintonaturalexosomesandengineered exosomes(Section2.4).Dependingonthespeciesoforigin, exosomesaredividedintoanimal-derivedandplant-derived exosomes.Currently,exosomesaremainlyclassifiedaccordingto thetypeoftheirparentalcells.Almostalltypesofhumancellscan produceexosomes.Theseinclude,butarenotlimitedto, macrophages,dendriticcells(DCs),platelets,stemcells,andeven tumorcells28 (Fig. 1a).

Forexample,macrophage-derivedexosomescontributeto diseaseprogression(e.g.,diabetes,atherosclerosisandheart failure)29 anddiseasetreatment(e.g.,cutaneouswound,inﬂammatoryboweldisease,andfungalandviralinfection).30 However, theyseemtoplayparadoxicalrolesinsuppressingandpromoting tumors.31 LikeDCs,DC-derivedexosomes(Dex)couldalsointeract withimmunecells(e.g.,Tcells,Bcells,andNKcells)throughtheir surfaceproteinssuchasmajorhistocompatibilitycomplexes (MHCs).32 Somepreclinicalandclinicaltrialshavedemonstrated theeffectivenessandsafetyofDex-basedimmunotherapyfor cancers.33 Furthermore,tumor-derivedexosomes(Tex)notonly areinvolvedduringtumorproliferation,invasion,metastasis,and immunitybutalsocanbeusedasbiomarkersforcancerdiagnosis andtreatment.34 Lately,Texhasbeenusedasananti-tumordrug andanantigenpresenterforDCvaccination,servingasa promisingcell-freecancerimmunotherapy.35 Finally,theclinical applicationsofstemcell-derivedexosomeswillbediscussedin detailinthefollowingsections.

Exosomescanbefoundinallbody fluidssuchasblood,saliva, urine,plasma,tears,semen,amniotic fluid,andevenbreastmilk.36 Body fluid-derivedexosomesareahighlystablereservoirof diseasebiomarkers,assistingliquidbiopsyinvariousclinical settingssuchascancers,cardiovasculardiseases,andperinatal disorders.37 38 However,thecoexistingcontentsandavailabilityof eachtypeofbody fluidmightcreatechallengestoexosome isolation.

Production,isolationandpuriﬁcationofexosomes

Oneofthemajorobstaclespreventingexosome-basedtherapeuticsfromenteringclinicalpracticeisthelowyieldandefﬁciencyof

Fig.2 Illustrationofthedownstreamsurgicalapplicationsofexosometherapy(figuregeneratedusingAdobePhotoshop2023andAdobe Illustrator2023).Thetherapeuticeffectsofexosomesareahierarchicaltranslationthroughdisease-specifictissueresponses,tissue-specific cellularalterations,andcell-specificmolecularsignalingpathways

exosomes.Forexample,onlylessthan1 μgexosomalprotein couldbeharvestedfrom1mlculturemediuminalaboratory setting.39 Therearevariousmethodsofupscalingexosome production,whicharecategorizedintobiochemicalstrategies (e.g.,LPS,BMP-2,HIF-1α,andIFN-γ andTNF-α),physicalstrategies (hypoxia,thermalstress,andstarvation),mechanicalstrategies (shearstressand3Dculturing)andinstrumentalstrategies (hollow-ﬁberbioreactorsandstirredtankbioreactors).40

Exosomesareheterogeneousintermsofsize,content,surface markers,andsource,whichmakestheirisolationdifficult.The currentlyavailabletechniquesforexosomeisolationandpurificationarebasedontheirsize,surfacecharge,orimmunoaffinity26

(Fig. 1a).However,thereisno ‘one-ﬁts-all’ approachasthese techniquesallhaveadvantagesanddisadvantages.

Forexample,ultracentrifugationisdeemedthegoldstandard forexosomeextraction.Althoughitrequiresminimalreagentsand expertize,thetimeconsumption,highcost,lowefficiency,and lipoproteinco-separationhavelimiteditslarge-scaleuse.41 Immunoaffinitychromatographyisaseparationtechnologybased onthespecificbindingofantibodiesandligands.Itisrapidand provideshighpurity,specificity,andyield.However,theantigen/ proteincouplingusedneedstobeexpressedonthesurfaceof exosomes.19 Size-basedisolationtechniquesmainlyreferto ultrafiltrationandsize-exclusionchromatography,bothofwhich

arequickandsuitableforlarge-scaleapplications.Butpore clogging,exosomeloss,andlowpurityaremakingthismethod difficulttopopularize.42 Althoughnosingletechniqueisperfect, combiningtheabovetechniqueswithothers(e.g.,precipitationbasedandmicrofluidics-based)mightbeasolutiontosimultaneouslymeetmultiplerequirementsforexosomeisolationand purification.

Modiﬁcationofexosomes

Exosomescanbebiochemicallymodifiedtobroaden,change,or improvetheirtherapeuticeffects.Themodificationofexosomesis classifiedintointernalstrategies(e.g.,drugloading)andexternal strategies(e.g.,surfacemodification).Ontheonehand,exosomes maybeanidealtherapeuticcarriertodeliverdrugs,nucleicacids, andvaccinesduetotheiradvantagesinstability,non-immunogenicity,andtargetingrecipientcells.43 Therearevariouscargo loadingtechniquesincludingpre-productionloadingmethods (e.g.,transfection,co-incubation,andelectroporation)andpostproductionloadingmethods(e.g.,freeze-thawcycles,incubation, sonication,extrusion,andhypotonicdialysis)dependingon whethertheyareappliedbeforeorafterexosomebiogenesis10,26,44–46 (Fig. 1c).Forexample,Tianetal.loadeddoxorubicin inDexusingelectroporationforthetreatmentofbreastcancer.47 Kimetal.loadedpaclitaxelinRAW264.7-derivedexosomesusing incubationandsonicationtoovercomemultidrugresistancein cancercells.48 Ohnoetal.loadedantitumorlet-7amiRNAin HEK293-derivedexosomesusingtransfectiontomanagebreast cancer.49

Ontheotherhand,surfacemodificationofexosomesis exemplifiedbygeneticengineeringofexosomalmembraneor parentalcells,chemicalconnectionoftargetingligands,electrostaticinteraction,andmagneticnanoparticletechnology.10 The mainpurposeofsurfacemodificationistoselectivelydeliver exosomestotargetcellsforprecisetreatment.Forexample, Alvarez-Ervitietal.modifiedDCsusinggeneticengineeringto expressLamp2bandRVGpeptides,therebytargetingthecentral nervoussystem(CNS).50 Zhuetal.insertedtumor-targeting peptides,c(RGDyK),intotheexosomesurfaceusingachemical reactiontotargetglioblastoma.51 Nakaseetal.boundexosomes withacomplexformedbypH-sensitivefusionpeptideand cationiclipidusingelectrostaticinteraction,therebyachieving enhancedcytosolicdelivery.52

Characterizationandverificationofexosomes Exosomesneedtoundergocharacterizationandverification beforetherapeuticapplications.Currentmethodsusedfor exosomecharacterizationmainlyfocusonthesize,morphology, andcargoprofileofexosomes.43 Size-orientedverification includesnanoparticletrackinganalysis(NTA),dynamiclight scattering(DLS),andtunableresistivepulsesensing(TRPS), whereasmorphology-orientedanalysisincludesscanningelectron microscopy(SEM)andtransmissionelectronmicroscopy(TEM).19 Inaddition,cargoprofilingisfurthersubdividedintoproteomic, lipidomic,andgenomicanalysesincludingwesternblotting,ELISA, flowcytometry,massspectroscopy,andPCR.36 Sinceeachofthe abovecharacterizationmethodshasadvantagesanddisadvantages,itisauniversalpracticetocombineanalysesfromthree differentaspects,e.g.,apackageofTEM,NTA,andwestern blotting,toidentifyisolatedexosomes.

Storageofexosomes

Thecurrentlyusedpreservationmethodsforlong-termstorageof exosomesmainlyincludecryopreservation,lyophilization,and spray-drying.10 Temperatureandantifreezearethetwomost importantingredientsforcryopreservation.Storageat4°Cmight weakenthebiologicalactivityandreducetheproteincargoof exosomes,whereas 80°Cisconsideredtheoptimaltemperature causingtheleastimpactonexosomemorphologyandcontent.57,58 Non-permeabledisaccharideantifreeze,especiallytrehalose,representsthebestchoiceasitpreventsexosome aggregationandcryodamage.59 Heat-sensitivematerials,e.g., exosomesandvaccines,treatedbylyophilizationoffreezedryingcanbeeasilystoredandreconstitutedbysimplyadding water.ArecentstudyshowedthatlyophilizationwithcryoprotectantcouldretaintheactivityofexosomalproteinsandRNAfor approximately4weeksevenwhenstoredatroomtemperature.60 Finally,incontrasttofreeze-drying,spray-dryingisasingle-step process,therebyreducingtheneedforexpensiveequipmentand lengthymulti-stepmilling.However,coreparametersofspraydryingsuchasexosomefeedingrate,atomizationpressure,and outlettemperature,canallaffectexosomestabilityandcargo integrity.61

ORTHOPEDICANDTRAUMASURGERYANDSC-EXOTHERAPY Fracture

Fracturesarethemostcommontraumaticlarge-organinjuries, andapproximately10%healimproperly.62 Fracturehealing involvesananabolictissue-bulkingphaseandacatabolictissueremodelingphase,whicharecontrolledbyvariousfactorssuchas stemcells,innateandadaptiveimmunefunctions,andstability.63 Biopharmacologicaltreatmentforfracturescanbegivenlocally (e.g.,bonemorphogeneticprotein,BMP)orsystemically(e.g., parathyroidhormone,PTH).Asapromisingalternative,exosome therapyforfracturehealingmostlyutilizesbonemarrow-derived MSCsasacellularsupplier(Table 2).

ThepresumedmechanismofhowMSC-derivedexosomes promotefracturehealingisasfollows.Firstly,theprogressionof bonerepairneedsavarietyofcells,e.g.,inflammatorycellsinthe inflammationstage,endothelialandmesenchymalprogenitor cellsinthe fibrovascularstage,osteoblastsandchondrocytes duringboneformation,andosteoclastsduringcallusremodeling.62 Secondly,mostofthesecellscanuptakeexosomes, especiallyosteoblastsandvascularendothelialcells,64 whichare mostrelatedtofracturehealing.Lastly,uponexosomeabsorption, thegeneexpressionoftherecipientcellsismodified,thereby activatingvarioussignalingpathways(Fig. 3a),causingvarious cellularandtissueresponses(Fig. 3b)andultimatelyleadingto improvedfracturehealing.

Earlyresearchhasemployedvariousanimalmodelsoffracture healing.Inatransversefemoralshaftfracturemodel,exosomes werefoundtonotonlypromoteosteogenesisinwild-typemice, butalsorescueretardationoffracturehealinginCD9 / mice,a strainknowntohavealowerboneunionrate.65 Inafemoral nonunionmodel,exosomesenhancedfracturehealingby promotingosteogenesisandangiogenesispossiblyviatheBMP2/Smad1/RUNX2pathway.66 Inatibialdistractionosteogenesis model,exosomessecretedbyyoungMSCspromotedosteogenic capacityofolderMSCsandenhancednewboneformationin olderrats.67 Inaddition,EPC-derivedexosomesacceleratedbone regenerationduringdistractionosteogenesisbystimulating angiogenesis.68

Forexample,microscopy-basedmethods,suchasSEMand TEM,candirectlyvisualizethesurfacetopographyandinternal structure,respectively.However,TEMisnotsuitableforquick measurementofalargenumberofsamplesduetocomplicated operationandtedioussamplepreparation. 53 NTAfacilitatesfast detectionandreal-timeexosomeobservationwhilehavinga higherresolutionthan fl owcytometry.Themaindisadvantage ofNTAisitsdif fi cultyindistinguishingexosomesfrom contaminatedproteins. 54 Asamaturetechnique,western blottingcanqualitativelyandquantitativelydetecttheexpressionofexosomalproteinbiomark ers,especiallyexosomesfrom cellculturemedia.However,itistime-consumingandnot suitableforthedetectionofexosomesfrombiological fl uids. 55 , 56

Table2. Stemcell-derivedexosomesforthetreatmentofdiseasesinorthopedicsurgeryandrelatedspecialties

TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

micemodel;rescued retardationoffracturehealinginCD9/mice;promotedbonehealing inwild-typemice

FractureBM-MSC-exoN/Afemoralfracturemodel,wildtypeandCD9/ -

BM-MSC-exoHUVECs,MC3T3-E1cells;improvedproliferation&migrationratmodeloffemoralnonunion;enhancedosteogenesisand angiogenesisviaBMP-2/Smad1/RUNX2pathway

67 EPC-exoHUVECs;enhancedproliferation,migrationviamiR-126distractionosteogenesisratmodel;acceleratedboneregeneration withbettermechanicalpropertiesintibiaswithhighervascular density

youngBM-MSC-exoolderBM-MSCs;enhancedproliferation&osteogenic differentiation distractionosteogenesisratmodel;acceleratedboneregeneration withbettermechanicalpropertiesintibias

BM-MSC-exoMC3T3-E1cells;promotedproliferation&differentiationmice;exosomalmiR-136-5ppromotedfracturehealingbytargeting LRP4toactivateWnt/ β -cateninpathway

mice;exosomalmiR-25regulatedubiquitinationanddegradationof Runx2bySMURF1topromotefracturehealing

BM-MSC-exoMC3T3-E1cells;acceleratedosteogenicdifferentiation, proliferation,andmigration

obesity-inducedfracturemousemodel;exosomallncRNAH19 improvedfracturehealingviamiR-467/HoxA10axis

BM-MSC-exoBM-MSCs;high-fatdietinhibitedexosecretion&osteogenic markers

CBS-heterozygousmice;exosomallncRNA-H19absorbedmiR-106and restoredboneformationandmechanicalquality

BM-MSC-exoHUVECs,BM-MSCs;promotedangiogenesis&osteogenesis viaangiopoietin-1/Tie2-NOpathway

DMOG-stimulatedBM-MSC- exo HUVECs;promotedproliferationandtubeformationcalvarialdefectratmodel;improvedboneregenerationand neovascularizationbyactivatingAkt/mTORpathway

mice;miR-29a-loadedexopromotedangiogenesisandosteogenesis byincreasingtrabecularbonemass

BM-MSC-exoHUVECs;exosomalmiR-29apromotedproliferation, migration,andtubeformationbyvasohibin-1

118

UC-MSC-exoHUVECs;hypoxiaenhancedexoproductionviaHIF-1 α ; improvedproliferation&tubeformation femoralfracturemousemodel;hypoxicexopromotedfracturehealing bytransferringmiR-126toagreaterextentthannormoxicexo

N/A

Osteoporosisadipose-MSC-exoMLO-Y4cells;reducedhypoxia/serumdeprivation-induced osteocyteapoptosisandosteocyte-mediated osteoclastogenesis

119

UC-MSC-exoBM-MSCs;inhibitedapoptosisHLU-induceddisuseosteoporosisratmodel;actedviamiR-1263/ Mob1/Hipposignalingpathway

120

UC-MSC-exoosteoblasts;promotedcellproliferationandosteogenic differentiation estrogen-de ﬁ cientosteoporosismodelmice;improvedtibialdensity andreversedosteoporosis;miR-2110andmiR-328-3paremost importantosteogenesisregulatoryexosomalmRNAs

mice;exosomalmiR-100-5pamelioratedOAseveritybyprotecting articularcartilageandamelioratinggaitabnormalitiesviainhibitionof mTOR

79 MSC-exochondrocytes;promotedproliferationandinhibited apoptosis

OsteoarthritisIPFP-MSC-exochondrocytes;inhibitedapoptosis&autophagy,and enhancedmatrixsynthesis

mice;exosomallncRNA-KLF3-AS1protectedchondrocytesviamiR- 206/GIT1axis

N/A

80 synovial-MSC-exoprimarychondrocytes;miR-320c-enhancedchondrogenesis viaADAM19

81 chondrogenicMSC-exochondrocytes;increasedcellproliferationandmatrix synthesisviatargetingWnt5a mice;exosomalmiR-92a-3pinhibitedcartilagedegradationinOA animalmodel

82 synovial-MSC-exohumanprimarychondrocytes;enhancedproliferation& migrationviaWnt/YAPsignaling rats;miR-140-5p-oe-exopreventedOAbydecreasingjointwearand cartilagematrixloss

83 TGFβ -stimulatedMSC-exoC5.18cells;exosomalmiR-135bincreasedcellviabilityby regulatingspeci ﬁ cityprotein-1 rats;promotedcartilagerepairbydecreasingOARSIscoreand increasingnumberofchondrocytes

Table2. continued TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

iPSC-exo,MSC-exohumanchondrocytes;stimulatedproliferation&migrationcollagenase-inducedOAmice;iPSC-MSC-exoshowedastronger therapeuticeffectonOAthansynovialmembraneMSC-exo

BM-MSC-exochondrocytes;decreasedin ﬂ ammatoryfactors&glutamine metabolicproteins rats;increasedmice ’ sexercisecapacity,improvedchondrocyte functionandglutamatemetabolism,anddecreasedcartilagedamage andin ﬂ ammation,therebyalleviatingOAprogression

87 MSC-exochondrocytes;increasedproliferation,matrixsynthesisand regenerativeimmunephenotype ratosteochondraldefectmodel; ↑ CD163 + M2and ↓ CD86 + M1 macrophages,andreducedpro-in ﬂ ammatorycytokinesIL-1 β &TNFα

BM-MSC-exoosteoblasts;promotedcellproliferationandosteogenic differentiationbyreducingElf3 mice;exosomalmiR-206amelioratedin ﬂ ammationandincreased osteocalcinandBMP2infemoraltissue;

88 gingival-MSC-exoCD4 + T-cells;inhibitedIL-17AandpromotedIL-10collagen-inducedarthritismicemodel;reducedincidenceandbone erosionofarthritisviainhibitingIL-17RA-Act1-TRAF6-NFκ Bpathway

SpinalcordinjuryIGF-1stimulatedNSC-exoPC12cells;inhibitedapoptosisandpromotedneural proliferation&regeneration rats;reducedlesionsizeandpromotedfunctionalrecovery,causedby miR-219a-2-3p-dependentinhibitionofYY1

mice,subarachnoidinjection;enhancedfunctionalrecovery

miR-enclosedNSC-exoHT22cells;attenuatedneuronalapoptosisbyactivating autophagyviamiR-374-5p/STK-4axis

mice;enhancedrepairofneurologicalfunctionsvialncGm36569/miR- 5627-5p/FSP1axis

MSC-exoHT-22&HEK-293hypoxiccellmodel;suppressedneuronal ferroptosis

97 hypoxicpreconditionedBM- MSC-exo BV2microglia;hypoxiapromotedexoreleasefromMSC;exo uptakebyBV2dependedonoxygenstatus

BM-MSC-exomacrophages;takenupbyasubsetofM2macrophagesrats;bothMSCintravenousinfusionandfractionatedMSC-exo promotedM2macrophagepolarization,upregulatedTGFβ ,and reducedBSCBleakage

mice;promotedfunctionalbehavioralrecoverybyshiftingmicroglial M1/M2polarization;exosomalmiR-216a-5pregulatedviaTLR4/NFκ B/ PI3K/Aktpathway

EF-MSC-exoN/Arats;improvedneurologicalfunctionalrecoveryandreducedlesion volumebyinhibitingNLRP3in ﬂ ammasome

100

EPC-exomacrophages;promotedanti-in ﬂ ammatorymacrophagesmice;exosomalmiR-222-3ppromotedfunctionalrepairviaSOC3/ JAK2/STAT3pathway

101

mice;acceleratedmicrovascularregeneration,reducedspinalcord cavity,andimprovedfunctionalrecovery

NSC-exoSCMECs;enrichedinVEGF-Aandenhancedangiogenic activity

102

FTY720-loadedNSC-exoSCMECs;protectedbarrierfunctionofSCMECsunder hypoxicconditionsviaPTEN/Aktpathway rats;amelioratedhindlimbfunctionandreducedin ﬂ ammatory in ﬁ ltrationbydownregulatingBaxandaquaporin-4andupregulating claudin-5andBcl-2

104

BM-MSC-exopericyte;pre-Txwithexoreducedpericytepyroptosisand increasedpericytesurvivalrate ratmodelofT10SCI;improvedneuronsurvival,nerve ﬁ berextension, BSCBintegrity,reducedcaspase1&IL-1 β ,andacceleratedlocomotor functionalrecovery

105

rats;miR-146a-5p-modi ﬁ edexopromotedmorelocomotorfunctionof hindlimbsthanunmodi ﬁ edexobytargetingneurotoxicastrocytes

miR-modi ﬁ edUC-MSC-exoPC12cells;reducednegativeeffectsofneurotoxicastrocytes onPC12cellviabilityandneurites

107

106 placental-MSC-exoNSCs;promotedcellproliferation,andincreased phosphorylatedlevelsofMEK,ERKandCREB rats;promotedendogenousneuralstem/progenitorcellsproliferation, neurogenesis,andimprovedlocomotoractivityandbladder dysfunction

123

BM-MSC-exo-oe-NGFNSC;promoteddifferentiationofNSCsintoneurons&axonal regeneration mice;promotedrecoveryofspinalfunction&spinalcordregeneration

122 adipose-MSC-exoSchwanncells;promotedproliferation,migration, myelination,&neurotrophicfactors rats;improvedaxonregeneration&myelination,andrestored denervationmuscleatrophy

Sciaticnerveinjuryadipose-MSC-exoDRGneurons;increasedneuriteoutgrowthrats;enhancedaxonalregenerationandwalkingbehavior;discover ed neuralgrowthfactorstranscriptsinexo

Table2. continued

TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

124

rats;acceleratedfunctionalrecovery,axonregenerationand remyelination

LPS-treatedBM-MSC-exoRAW264.7cells;enhancedM2macrophagepolarizationvia TSG-6/NFκ B/NLRP3pathway

111

mousemodelofcardiotoxin-inducedmuscleinjury;promotedmuscle regeneration;exosomalmiR-494enhancedmyogenesisandmigration activity

Muscle&tendontearMSC-exoC2C12myoblasts,HUVECs;promotedmyogenesisand angiogenesis

112

Achillestendonrepairrabbitmodel;improvedmechanicalstrengthby upregulatingdecorinandbiglycan

adipose-MSC-exorabbitprimarytenocytes;enhancedproliferationand migration

114

adipose-MSC-exoN/Aratmodelofmassiverotatorcufftear;preventedatrophy,fatty in fi ltration,in fl ammation,andvascularizationofmuscles;elevated myo fi berregenerationandbiomechanicalproperties

115

adipose-MSC-exoN/Arabbitmodelofchronicrotatorcufftear;decreasedfattyin ﬁ ltration, promotedtendon-bonehealing,andimprovedbiomechanical properties

116

BM-MSC-exoHUVECs,U937cells;promotedproliferation&angiogenic tubeformation;reducedM1polarization rats;increasedbreakingloadandstiffnessofrotatorcuffafter reconstructioninrats,reducedangiogenesisaroundrotatorcuff endpoint,andpromotedtendon-bonehealing

rats;intradiscalinjectionofexoalleviatednucleuspulposusapoptosis andIVDdegenerationbasedonhistologyandMRI

127

126 ESC-exonucleuspulposuscells;exosomalmiR-302cinhibited pyroptosis rats;ameliorateddamageinIVDdegenerationviadownregulating NLRP3in ﬂ ammasome

128

BM-MSC-exonucleuspulposuscells;exosomalmiR-21alleviatedapoptosis viaPTEN/PI3K/Aktpathway

N/A

BM-MSC-exonucleuspulposuscells;alleviatedcompression-induced apoptosis&mitochondrialdamagebyinhibitingoxidative stress

Intervertebraldisc degeneration

Akt proteinkinaseB, BM bonemarrow, BMP bonemorphogeneticprotein, BSCB blood-spinalcordbarrier, CBS cystathionine β -synthase, CREB cAMPresponseelementbinding, DMOG dimethyloxaloylglycine, DRG dorsalrootganglion, EF epiduralfat, Elf E74-likefactor, EPC endothelialprogenitorcell, ERK extracellularsignal-regulatedkinase, ESC embryonicstemcell, exo exosome, FSP ﬁ broblast-speci ﬁ cprotein, GIT G-proteincoupledreceptorkinaseinteractingprotein, HIF hypoxia-induciblefactor, HLU hindlimbunloading, HUVEC humanumbilicalveinendothelialcell, IGF insulingrowthfactor, IL interleukin, IPFP infrapatellarfatpad, IVD intervertebraldisc, KLF Krüppel-likefactor, LPS lipopolysaccharide, LRP lipoproteinreceptorrelatedprotein, MEK mitogen-activatedproteinkinase, miR microRNA, mTOR mechanistic targetofrapamycin, NFκ B nuclearfactor-kappaB, NGF nervegrowthfactor, NLRP nucleotide-bindingdomain-likereceptorprotein, NP nucleuspulposus, NSC neuralstemcell, oe overexpressing, PI3K phosphoinositide3-kinase, PTEN phosphatase&tensinhomolog, Runx runt-relatedtranscriptionfactor, SCMEC spinalcordmicrovascularendothelialcell, SMURF smadubiquitinationregulatoryfactor, TGF transforminggrowthfactor, Tie tyrosinekinasereceptor, TLR Toll-likereceptor, TNF tumornecrosisfactor, TSG TNFstimulatedgene, Tx treatment, UC umbilicalcord, VEGF vascularendothelialgrowthfactor, YAP yes-associatedprotein, YY yinandyang

Fig.3 Mechanismsofstemcell-derivedexosometherapy(ﬁguregeneratedusingAdobePhotoshop2023andAdobeIllustrator2023). a activationandregulationofvarioussignalingpathways. b disease-speciﬁccellularandtissueresponses

SignalTransductionandTargetedTherapy(2024)9:17

Asamajorcargoofexosomes(Section2.1),RNAcanalter recipientcellgeneexpressionandphenotypicfunction,with microRNA(miRNA)andlongnon-codingRNA(lncRNA)beingthe mostwidelystudied.69 Fromtheperspectiveofanexosomal miRNA,onegroupdiscoveredthatmiR-136-5pfrombonemarrow MSC-derivedexosomespromotedosteoblastproliferationand differentiationinvitro,therebypromotingfracturehealing invivo.70 Thiswasachievedbyinhibitingthedownstreamtarget geneofmiR-163-5p,low-densitylipoproteinreceptor-related protein4(LRP4),throughtheWnt/β-cateninpathway.Theother groupfoundthatMSC-derivedexosomalmiR-25couldregulate theubiquitinationanddegradationofRunt-relatedtranscription factor2(Runx2)bySmadubiquitinationregulatoryfactor1 (SMURF1)topromotefracturehealinginmice.71 FromalncRNA perspective,especiallythebone-speciﬁclncRNAH19,aChinese grouprevealedthatalthoughahigh-fatdietreducedosteogenic differentiationandweakenedfracturehealing,thiscouldbe reversedbyMSC-derivedexosomallncRNAH19viamiR-467/ HoxA10axisinanobesity-inducedfracturemodel.72 Inaddition, anAmericangroupdemonstratedthatexosomallncRNAH19not onlyimprovedosteogenesisbutalsoangiogenesisthroughthe angiopoietin1/Tie2-NOsignalingpathwayinanimmunocompromisednudemousemodel.73

InsteadofusingnaturallyderivedexosomesfromMSCs,some researchershaveconductedpre-isolationmodiﬁcationofexosomestoachievebetterresults.Liangetal.preconditionedMSCs withlowdosesofdimethyloxaloylglycine(DMOG),asmall angiogenicmolecule,topreparetheexosomesforanenhanced angiogenesisandboneregenerationinacritical-sizedcalvarial defectmodelbytargetingtheproteinkinaseB/mechanistictarget ofrapamycin(Akt/mTOR)pathway.74 Alternatively,Luetal.loaded MSC-derivedexosomeswithmiR-29a,whichshowedarobust abilityinpromotingangiogenesisandosteogenesisbytargeting vasohibin1.75 Furthermore,umbilicalcordMSC-derivedexosomes demonstratedcomparableresultstotheirbonemarrowcounterpartsduringfracturehealing.76 Inaddition,exosomesderived fromMSCsunderhypoxiaexhibitedbettereffectsonbone fracturehealingthanthoseundernormoxia.Mechanistically, hypoxiapreconditioningenhancedtheproductionofexosomal miR-126throughtheactivationofhypoxia-induciblefactor1(HIF1α).Variousstudieshaveshownthathypoxiapreconditioning representsaneffectiveandpromisingoptimizationofthe therapeuticeffectsofMSC-derivedexosomesforbonefracture healing.

Osteoarthritis

Osteoarthritis(OA)isthemostcommonjointdiseaseandmost frequentreasonforactivitylimitationinadults,affecting approximately240millionpatientsglobally.77 Thepathologyof OAhasevolvedfrombeingviewedascartilage-onlytoamultitissuediseasethataffectsallcomponentsofthewholejoint, includingbone,synovium,muscle,ligament,andperiarticular fat.78 Clinicaltrialshavesuccessfullyrevealedsystemiccompoundsthatarreststructuralprogression(e.g.,cathepsinKand Wntinhibitors)orreduceOApain(e.g.,nervegrowthfactor inhibitors).AsapotentialtreatmentoptionforOA,mostMSCderivedexosometherapyusedchondrocytesasatargetininvitro models.TheseMSCscouldoriginatefromvarioustissues,suchas bonemarrow,synovium,gingiva,andinfrapatellarfatpads(IPFPs). Somestudiesfocusingonchondrogenesisdemonstrateda particularinterestintheroleofmiRNA.Wuetal.foundthatIPFP MSC-derivedexosomesprotectarticularcartilagefromdamage andameliorategaitabnormalityinOAmicebymiR100-5pregulatedinhibitionofmTOR-autophagypathway.79 Sinceitis easytoretrievehumanIPFPfromOApatientsbyarthroscopic operationwithinaclinic,thistypeofexosometherapymight simplifyandacceleratetheprocessfrombenchtobedside.Liu etal.discoveredthatMSC-derivedexosomescouldpromote

proliferationandinhibitapoptosisofchondrocytesvialncRNAKLF3-AS1/miR-206/GIT1axisinOA.80 Thecellularworkconducted byKongetal.showedthatsynovialMSC-derivedexosomalmiR320ccouldenhancechondrogenesisbytargetingADAM19.81 In addition,Maoetal.suggestedthatexosomalmiR-92a-3pfrom chondrogenicMSCscouldenhancechondrogenesisandsuppress cartilagedegradationviatargetingWnt5a.82 Incontrasttothese studiesusingoriginalexosomes,fewgroupsmodifiedexosomes priortotheirsystemicadministration.Taoetal.modified exosomesbytransfectingsynovialMSCswithmiR-140-5pand foundthatexosomalmiR-140-5p-overexpressioncouldenhance cartilagetissueregenerationandpreventOAofthekneeinarat model.83 Meanwhile,Wangetal.usedTGF-β1tostimulateMSCs, andtheresultantexosomalmiR-135bincreasedchondrocyte proliferationbyregulatingspecificityprotein-1.84 Inacomparative study,Zhuetal.demonstratedthatexosomesfromiPSC-derived MSCscouldprovideastrongertherapeuticeffectonOAthan synovialmembraneMSC-derivedexosomes.85

Otherstudieshavefocusedonnotonlychondrogenesisbut alsoanti-inflammationandimmunemodulationduringOA treatment.Forexample,MSC-derivedexosomesinhibitedinflammatoryfactors,glutaminemetabolicactivity-relatedproteins, glutamine,andGSH/GSSGratioinvitro,whileimprovingmice’s chondrocytefunction,tissueinflammation,andexercisecapacity invivo,therebyalleviatingOAprogression.86 Usingaholistic approach,recentstudieshaveshiftedtheattentionawayfrom cartilagetowardsothertissues(e.g.,bone)inadiarthrodialjoint. Firstly,bonemarrowMSC-derivedexosomalmiR-206promoted proliferationandosteogenicdifferentiationofosteoblastsinOA byreducingE74-likefactor3(Elf3),andamelioratedinflammation andincreasedexpressionofosteocalcinandBMP2inmouse femoraltissues.87 Secondly,MSCexosome-treatedosteochondral defectsdemonstratedaregenerativeimmunephenotype,characterizedbyahigherinfiltrationofCD163+ M2macrophagesover CD86+ M1macrophages,withaconcomitantreductioninproinflammatorysynovialcytokinesIL-1β andTNF-α 88 Lastly,gingival MSC-derivedexosomesprovedtobeimmunosuppressivein preventingcollagen-inducedarthritis.89 Comparedwithparental cells,theseexosomeshadthesameorstrongereffectsin inhibitingIL-17AandpromotingIL-10,reducingincidencesand boneerosionbyarthritis,viainhibitingtheIL-17RA-Act1-TRAF6NF-κBsignalingpathway.

Currently,thereisnosingle ‘onesize fitsall’ drugthatmaybe suitableforallOApatients.Disease-modifyingOAdrugs (DMOADs)mightbecomethenext-generationOAtreatment.90 It isveryvaluableandrelevantthatMSC-derivedexosometherapy forOAcoincideswithDMOADs:botharecapableoftargeting inflammatorycytokines,matrix-degradingenzymes,andtheWnt pathway.Thus,emergingapproachesforDMOADdevelopment, suchasmiRNA-basedmodalityandtargetingcellularsenescence, mightalsobeusedtorefineMSC-basedexosometherapyforOA.

Spinalcordinjury

Traumaticspinalcordinjury(SCI)isadevastatingglobalhealth issuethatposesasignificantfunctionalandeconomicburden bothonthepatientandsociety.91 ThepathophysiologyofSCI includesprimaryinjuriescausedbymechanicaltraumaand secondaryinjurycascadecharacterizedbyapoptosis,edema, ischemia,inflammatorycellinfiltration,andexcitotoxicity.92 Despitesurgicalintervention,clinicalstudiesinvolvingpharmacotherapycanbebroadlyclassifiedaseitherneuroprotectiveor neuroregenerative.93 Targetingeacheventoftheabovemechanisticchain,bothMSC-andNSC-derivedexosometherapycould exertabeneficialinfluenceonspinalcordprotectionand regeneration.

Somegroupshavetargetedneuronalcelldeath.Maetal. revealedthatinsulin-likegrowthfactor1(IGF-1)-stimulatedNSCderivedexosomescouldinhibitneuronalapoptosiswhile

promotingfunctionalrecoveryafterSCIthroughamiR-219a-2-3p/ YY1pathway.94 Alternatively,Zhangetal.discoveredthat subarachnoidinjectionofNSC-derivedexosomescouldsuppress neuronalcellapoptosisbyactivatingautophagyviamiR-374-5p/ STK-4axisforenhancedfunctionalrecoveryinSCI.95 Shaoetal. exploredotherformsofcelldeath(e.g.,ferroptosis)usingMSCderivedexosomes,andfoundthatexosomallncGm36569could inhibitneuronalcellferroptosisviamiR-5627-5p/FSP1axis, therebydecreasingneuronaldysfunction.96

Somegroupshavetargetedanti-inflammationandimmunomodulation.Nakazakietal.discoveredthatfractionatedintravenousinfusionofMSC-derivedexosomescouldtargetM2 macrophagesandupregulateTGF-β,therebystabilizingmicrovesselsandimprovingfunctionalrecovery.97 Similarly,Liuetal. demonstratedthatinadditiontohypoxiaincreasingexosome productionfrombonemarrowMSCs,preconditionedexosomal miR-216a-5pcouldalsorepairtraumaticSCIbyshiftingmicroglial M1/M2polarizationviatheTLR4/NF-κB/PI3K/Aktpathway.98 Huangetal.valuablyprovedthatepiduralfatMSC-derived exosomescouldattenuateNLRP3inflammasomeandimprove functionalrecoveryinSCI.99 ComparedtoMSC-derivedexosomes, exosomesderivedfromEPCscouldprovidecomparableantiinflammatoryeffect.Yuanetal.showedthattheexosomalmiR222-3pfromEPCscouldpromoteanti-inflammatorymacrophages viatheSOC3/JAK2/STAT3pathwayandimprovemousefunctional repairafterSCI.100

Somegroupshavetargetedangiogenesisandblood-spinalcord barrier(BSCB)integrity.Forexample,Zhongetal.usedunmodified NSC-derivedexosomes,foundthattheywerehighlyenrichedin VEGF-A,andcouldthereforeenhancetheangiogenicactivityof spinalcordmicrovascularendothelialcells(SCMECs).101 Incomparison,Chenandco-workersmodifiedNSC-derivedexosomeswith FTY720,animmunemodulatorandmicrovascularregulator,to protectthebarrierfunctionofSCMECsviathePTEN/Aktpathway, therebyamelioratinghindlimbfunction.102 Itiswell-knownthat theconnectionbetweenthemicrovascularendotheliumofthe spinalcordandthepericyteiscrucialinmaintainingthestructural integrityofBSCB.103 Thus,Zhou’steamattemptedtoverifytherole ofexosometherapyinpericytehomeostasis.104 Theyprovedthat bonemarrowMSC-derivedexosomescouldreducepericyte pyroptosisandincreasepericytesurvivalrateinvitro,while improvingBSCBintegrityandlocomotorrecoveryinvivo. Finally,somegroupshavetargetedotheraspectsduring neuroprotectionandneuroregeneration,suchasneurotoxic astrocytesandendogenousNSCsustainability.Laietal.proved thathumanumbilicalcordMSC-derivedexosomescouldfacilitate recoveryofspinalcordfunctionbytargetingneurotoxic astrocytes.105 Inaddition,miR-146a-5p-modifiedexosomes exertedamorepowerfuleffectthanunmodifiedexosomes.Li etal.discoveredthatexosomesderivedfromnervegrowthfactor (NGF)-overexpressingbonemarrowMSCscouldenhanceneuronal differentiationofNSCsandaxonalregeneration.106 Zhouetal. demonstratedthatplacentalMSC-derivedexosomescould promotetheactivationofproliferatingendogenousNSCs,thereby improvingbothlocomotoractivityandbladderdysfunction,107 whichisafrequentsequelaethatcouldfurtherworsenthequality oflifeofSCIpatients.108

Muscleandtendontear

Muscleandtendontearscanresultfromeitheracutetrauma(e.g., fractures,Section3.1)orchronicoveruse(e.g.,sportsinjury).109 Healingofmusclestrainandtendontearfollowsthetypical woundhealingcourse,involvingtheinﬂammatory,proliferative, andremodelingphases.Multiplenon-surgicalstrategieshave beentrialedtoimprovehealing,includingcell-basedandgrowth factor-basedtherapies.110 Thefollowingproof-of-conceptstudies indicatethatMSC-derivedexosomescouldbecomethenextgenerationmusculoskeletaltreatment.

Ontheonehand,somegroupshavefocusedonindividual componentsofthemuscle-tendon-boneunit.Nakamuraetal. claimedthatMSC-derivedexosomescouldimproveinvitro myogenesisinC2C12myoblastsandangiogenesisinHUVECs, whileacceleratinginvivoskeletalmuscleregenerationina cardiotoxin-inducedmuscleinjurymodel.111 Thesebeneﬁtswere atleastinpartmediatedbymiRNAssuchasmiR-494.Chenetal. discoveredthatexosomesfromadiposeMSCscouldenhancethe proliferationandthemigrationofprimarytenocytes,whilealso improvingmechanicalstrengthofrepairedtendonsbyupregulatingdecorinandbiglycaninarabbitAchillestendonrupture model.112

Ontheotherhand,somegroupshaveregardedthemuscletendon-boneunitasasinglefunctionalsystemandusedrotator cufftearasthediseasemodel,whichisthemostcommon shoulderconditionforwhichpatientsseektreatment.113 One groupofresearcherspublishedtwoconsecutivestudiesusing adiposeMSC-derivedexosomes.Inaratmodelofmassiverotator cufftear,exosometherapycouldpreventtheatrophy,inﬂammation,andvascularizationofmuscles.114 Inarabbitmodelof chronicrotatorcufftear,exosometherapycouldpreventfatty inﬁltrationandimprovebiomechanicalproperties.115 Another groupreportedthatbonemarrowMSC-derivedexosomescould increasethebreakingloadandstiffnessoftherotatorcuffafter reconstruction,induceangiogenesisaroundtherotatorcuff endpoint,andpromotegrowthofthetendon-boneinterface.116

Otherorthopedicdiseases

Osteoporosisisametabolicbonediseasecharacterizedbylow bonedensityandweakeningofbonearchitecture,whichincrease theriskoffractures.Itresultsfromosteoclasticboneresorption undercompensatedbyosteoblasticboneformation.117 Inacellular study,adiposeMSC-derivedexosomescouldantagonizehypoxia/ serumdeprivation-inducedosteocyteapoptosisandosteocytemediatedosteoclastogenesis.118 Furtheranimalstudiesrevealed thatumbilicalcordMSC-derivedexosomescouldinhibitbone marrowMSCapoptosisandpreventdisuseosteoporosisviamiR1263/Mob1/Hippopathway,119 andimprovetibialdensityand reverseestrogen-deﬁcientosteoporosisviamiR-2110andmiR328-3p.120

ComparedtoSCI,damagetoperipheralnerve(e.g.,sciaticnerve injury)isconsiderablymorecommon.Thesubsequentnerve regenerationiscontrolledbytheinterplaybetweenneuronsand Schwanncells,andfurthercomplicatedbyinflammatorycell infiltration.121 ItwasshownthatadiposeMSC-derivedexosomes couldtargetneuronsbyincreasingneuriteoutgrowthinvitroand axonalregenerationandwalkingbehaviorinvivo.122 Adipose MSC-derivedexosomescouldtargetSchwanncellsbypromoting theproliferation,migrationandsecretionofneurotrophicfactors invitroandrestoredenervationmuscleatrophyinvivo.123 LPSpreconditionedMSC-derivedexosomescouldtargetinflammatory cellsbyenhancingM2macrophagepolarizationinvitroand accelerateperipheralnerveregenerationinvivo.124

Intervertebraldisc(IVD)degenerationisamajorcauseoflower backpainwhichistheleadinginjuryintotalglobalyearslived withdisability.Itsmolecularprocessesincludeextracellularmatrix (ECM)degeneration,inﬂammation,oxidativestress,apoptosis, senescenceandreducedautophagy.125 Theemergingavenuesof exosometherapyattempttosolvesomeoftheseissues.Cheng etal.demonstratedthatintradiscalinjectionofbonemarrowMSCderivedexosomescouldinhibitnucleuspulposuscell(NPC) apoptosisandalleviateIVDdegenerationviaexosomalmiR-21.126 Ontheotherhand,Chenetal.discoveredthathumanESC-derived exosomescouldinhibitNLRP3inﬂammasometoalleviate pyroptosisinnucleuspulposuscellsbydeliveringmiR-302c.127 Inadditiontocelldeathandmitochondrialdamage,oxidative stressinNPCswasalsofoundtobeinhibitedbyMSC-derived exosomes.128 SinceIVDdegenerationandOAshareacommon

moleculardiseasespectrum125 thepositiveresultsofOAtreatmentusingMSC-derivedexosomes(Section3.2)couldbeusedas areferenceforIVDdegenerationresearch.

Osteonecrosis,aka.,avascularnecrosis,ofthefemoralhead (ONFH)isadisablingconditionaffectingayoungerpopulation, whichoftenresultsintotalhiparthroplasty.129 Glucocorticoid (GC)-inducedosteonecrosisisoneofthemostcommoncausesof ONFH,whosepathogenesisismanifestedintwoaspects: compromisedbloodsupplytothefemoralheadanddampened osteogenicactivity.Liuetal.showedthatexosomesfromiPSCderivedMSCscouldpreventGC-inducedONFHbypromoting angiogenesisandosteogenesisviathePI3K/Aktpathway.130 Zuo etal.demonstratedthatmiR-26a-overexpressingexosomes derivedfromHSCscouldprovidesimilartherapeuticeffects.131

NEUROSURGERYANDSC-EXOTHERAPY

Ischemicstroke

Strokesarethesecondhighestcauseofdeathandthethird leadingcauseofdisabilityglobally,withischemicstrokebeingthe mostcommonsubtype.132 Thekeyeventsduringtheischemic cascadeincludeneuronaldysfunction,excitotoxicity,neurochemicalinjury,andneuroinﬂammation.133 Intermsoftreatment,anew generationofclinicaltrialsisnowunderway,whichuses cytoprotectivedrugs,suchasimmunomodulators,IL-6receptor antagonists,Rhokinaseinhibitors,andfreeradicalscavengers.134 Targetingeacheventoftheabovepathophysiology,nearlyall subtypesofSC-exodemonstratedpotenttherapeuticeffectson strokerecovery(Table 3).

Somegroupshavetargetedneuroprotectionandneurogenesis. Firstly,SC-exotherapycouldinhibitneuronalcelldeath.Luoetal. foundthatNSC-derivedexosomescouldinhibitapoptosiswhile promotingtheproliferationofSH-SY5Ycellsbothundernormal andoxygen-glucosedeprivation(OGD)conditions.135 Thiswas alsotestedinamiddlecerebralarteryocclusion(MCAO)modelas areducedinfarctionareaandneuronalapoptosisviaexosomal miR-150-3p.OtherinvitroandinvivostudiesshowedsimilarantiapoptoticeffectsusingEPC-derivedexosomes.136,137 Zhangetal. discoveredthattheexosomalanti-apoptoticeffectcouldbe improvedbypreconditioningtheparentalNSCswithinterferon gamma(IFN-γ).138 Secondly,SC-exotherapycouldprotectcellsof theCNS.Kangetal.revealedthatexosomesderivedfrombone marrowMSCscouldrescueOGD-inducedinjuryinneuralcellsby suppressingNLRP3inﬂammasome-mediatedpyroptosis.139 Exosomessourcedfromhypoxiccultureshadamorepronounced neuroprotectiveeffectthantheircounterpartsfromnormal cultures.Similarly,Lietal.discoveredthatexosomesderived fromhumaniPSC-derivedneuralprogenitorcellsexhibiteda neuroprotectiveeffectonOGDneuronsandneuriteoutgrowth.140 Thisprotectionofneuronalfunctionunderischemicconditions wasregulatedthroughthePTEN/Aktpathway.Inaddition,Sun etal.provedthatNSC-derivedexosomescouldalsoprotect astrocytes,whichbecomesupportingreactiveastrocytes(RAs) afterstrokes.141 Thirdly,SC-exotherapycouldimprovepost-stroke neurogenesis.Weietal.suggestedthatZeb2/Axin2frombone marrowMSC-derivedexosomescouldimprovepost-stroke neurogenesis,neuralplasticity,andspatialmemoryandnerve function,likelyviatheSOX10,Wnt/β-catenin,andendothelin-3/ EDNRBpathways.142 Wangetal.illustratedthatmiR-126-modiﬁed EPC-derivedexosomescouldalleviateacutebraininjuryand promotefunctionalrecoveryafterstrokebyenhancing neurogenesis.143

Somegroupshavetargetedtheinhibitionoftheneuroinflammation.Firstly,unmodifiedSC-exotherapyexhibitedanantiinflammatoryeffectthroughexosomalmiRNAs.Dongetal. showedthatbonemarrowMSC-derivedexosomescouldinduce BV2microgliadeactivationandM2polarizationinvitro,while reducinginfarctsizeandimprovingneuronalfunctioninvivovia

transferringmiR-23a-3p.144 Similarly,Zhangetal.unveiledthat umbilicalcordMSC-derivedexosomalmiR-146a-5pcouldattenuatemicroglia-mediatedneuroinflammationafterOGDinvitro, whileimprovingbehavioraldeficitsandmicrogliaactivation invivoviatheIRAK1/TRAF6signalingpathway.145 Secondly,the anti-inflammatoryeffectofSC-exotherapycouldbeenhancedby modifyingtheexosomes.Yoonandco-workersestablishedtumor susceptibilitygene(TSG)101-overexpressinghumanNSCs,thereby increasingexosomesecretion.146 Theengineeredexosomesnot onlyattenuatedLDHreleaseandproinflammatoryfactorsinvitro, butalsoreducedinfarctionvolume,inhibitedDNA-damage pathway,andupregulatedneurotrophicfactorsinvivo.Furthermore,Tian’steambrokenewgroundbyingeniouslyattaching RGDpeptideontoanNSC-derivedexosomemembrane,which targetedthelesionregionoftheischemicbrainafterintravenous administration,therebysuppressingtheinflammatoryresponse aftercerebralischemiabyinhibitingtheMAPKpathway.147 Interestingly,Gaoetal.usedinducedNSCs(iNSCs)reprogrammed frommouse fibroblastsforstroketreatment.Theyshowedthat iNSC-derivedexosomes,bearingsimilartherapeuticeffectswith NSC-derivedones,couldnotonlypromoteneurogenesisbutalso inhibitneuroinflammation.148

Finally,somegroupshavetargetedotheraspectsduringstroke recovery,suchasneurochemicalinjuryandoxidativestress.Zhu etal.loadedbrain-derivedneurotrophicfactor(BDNF)into exosomesderivedfromNSCstoconstructengineeredexosomes.149 InamodelofH2O2-inducedoxidativestress,exosome therapysignificantlyenhancedNSCsurvival.InaratMCAOmodel, exosometherapynotonlyinhibitedmicroglialactivation,butalso boostedthedifferentiationofendogenousNSCsintoneurons. Collectively,BDNF-basedmodificationofNSC-derivedexosomes hasimprovedeffectsinthetreatmentofischemicstroke.Onthe otherhand,miR-210-modifiedEPC-derivedexosomescould protectneuronsfromhypoxiaandreoxygenation(H/R)-induced apoptosis,oxidativestress,anddecreasedviability,thereby supportingthetreatmentofischemicstroke.150,151 Theexosomal miR-17-5pfromACE2-enrichedEPC-derivedexosomescould amelioratecerebralischemicinjuryinagedmice.152 Inan intriguingstudyconductedbyXuandco-workers,combination ofNSC-exoandEPC-exowithmiR-210andmiR-123overexpressionexertedbettertherapeuticeffectsonischemicstrokeby protectingH/RinjuredneuronsthroughtheBDNF-TrkBandNox2/ ROSpathways.153

Incontrasttoischemicstroke,hemorrhagicstrokeposesa deadlierthreatandworsedisabilityinmostsurvivors.154 miR-137 overexpressionwasfoundtoboosttheneuroprotectiveeffectsof EPC-derivedexosomesagainstapoptosis,ferroptosis,andmitochondrialdysfunctioninoxyhemoglobin-treatedSH-SY5Ycells,an invitrohemorrhagicstrokemodel,partiallythroughtheCOX2/ PGE2pathway.155

Traumaticbraininjury

Approximately70millionpatientssufferfromtraumaticbrain injury(TBI)globallyeachyear,whichposesseriousphysical, psychosocialandeconomicthreats.156 TBIcanbecategorizedas primaryinjuries(e.g.,axonaldeath,neuroinﬂammation,neurochemicalchange,andmetabolicdysfunction)andsecondary injuries(e.g.,ischemicandhypoxicdamage,cerebraledema, raisedintracranialpressure,hydrocephalus,andinfection).157 Each patientwithaTBIhasauniquesetofcircumstancesdependingon variablessuchasthelocationandseverityoftheinjury,making medicalandsurgicaltreatmentquitechallenging.158 Therefore, systemictherapyusingSC-exomaybecomea ‘one-size-ﬁts-all’ optionformanagingTBI.

Aseriesofanimalstudiespublishedinitiallyfocusedonthe functionalrecoveryandmacroscopicaspectsofMSC-derived exosometherapy.InaratTBImodel,exosome-treatedanimals showedsigniﬁcantimprovementinspatiallearningand

Table3. Stemcell-derivedexosomesforthetreatmentofdiseasesinneurosurgeryandrelatedspecialties

TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

ratMCAOmodel;reducedinfarctionareaandneuronapoptosis, exosomalmiR-150-3penhancedneuroprotectiveeffectsbytargeting CASP2

IschemicstrokeNSC-exoSH-SY5Ycells;inhibitedapoptosisandpromoted proliferationbothinnormalandOGDconditions

EPC-exoN/AratMCAOmodel;reducedinfarctsize,neurologicaldefectscore,and percentageofapoptoticcells,butincreasedCD31andVEGF

ratMCAOmodel;promotedbehavioralandstructuraloutcomes; in ﬂ ammatoryfactorIFNγ preconditionedexoweremorepotent

IFNγ inducedNSC-exoNSCs;increasedcellproliferation&survival,andreduced cellapoptosis

BM-MSC-exoOGDN2acells,ratprimarycorticalneurons; neuroprotectiveagainstNLRP3in ﬂ ammasome-mediated pyroptosis

iPSC-exoratprimarycorticalneurons;improvedneuronalsurvival andneuriteoutgrowthviaPTEN/Aktpathway

mice;reducedinfarctvolume

NSC-exo,iCM-exoprimarymousecorticalastrocytes,neuronalcells; protectedafterOGDischemia;NSC-exo>iCM-exo

142

OGDratneuron;increasedneuritebranching&elongationratMCAOmodel;improvedpost-strokeneurogenesis,neuralplasticity, andspatialmemoryandnervefunction,likelyviaSOX10,Wnt/ β -catenin,andendothelin-3/EDNRBpathways

Zeb2/Axin2enrichedBM- MSC-exo

143

miR-126-EPC-exoN/AdiabeticmouseMCAOmodel;improvedacutebraininjuryand functionalrecoveryafterstrokebypromotingneurogenesis

144

BM-MSC-exoBV2microglia;inducedmicrogliadeactivationandM2 polarization ratMCAOmodel;reducedinfarctsizeandimprovedneuronalfunction viatransferringmiR-23a-3p

145

UC-MSC-exoBV2microglia;attenuatedmicroglia-mediated in fl ammationafterOGD mice;reducedinfarctvolume,behavioralde fi cits,andameliorated microgliaactivation;exosomalmiR-146a-5preduced neuroin fl ammationviaIRAK1/TRAF6pathway

TSG101-oe-NSC-exoN2Acells;attenuatedLDHreleaseandproin ﬂ ammatory factors, ratMCAOmodel;reducedinfarctionvolume&in ﬂ ammatorycytokines, inhibitedDNA-damagepathway,andupregulatedneurotrophicfactors 146

RGDNSC-exoReN&BV2cells;showedintrinsicanti-in ﬂ ammatoryactivitymice;targetedischemicbrainregionsandsuppressedpostischemia in ﬂ ammatoryresponse;exosomalmiRsinhibitedMAPKpathway 147

149

ratMCAOmodel;inhibitedtheactivationofmicroglia,promotedthe differentiationofendogenousNSCsintoneurons,andimproved behavioralfunction

BDNF-NSC-exoH 2 O 2 -inducedoxidativestressinNSCs;reducedapoptosis andincreasedneurogenicdifferentiation

150

153

N/A

miR-210-EPC-exoH/RinjuredSH-SY5Ycells;protectedfromapoptosis& oxidativestress

mouseMCAOmodel;exosomalmiR-17-5pinhibitedapoptosis, oxidativestress&braindysfunctionviaPTEN/PI3K/Aktpathway

ACE2-enrichedEPC-exoH/Rinjuredmousebrainmicrovascularendothelialcells; inhibitedsenescence

159

ratMCAOmodel;reducedinfarctvolume&neurologicalde ﬁ citsscore viaNox2/ROS&BDNF/TrkBpathways

152 NSC-exo + EPC-exoH/RinjuredSH-SY5Ycells;protectedfromapoptosis& oxidativestress

TraumaticbraininjuryMSC-exoN/Arats;improvedspatiallearning&sensorimotorfunctionand neurovascularplasticity

160

BM-MSC-exoN/Arats;improvedspatiallearning,and3D>2Dcultureconditions; enhancedsensorimotorrecovery;increasedendothelialcells& neurons,andreducedneuroin ﬂ ammation

161

MSC-exoN/Aprimarymotorcortexmonkeymodel;animalsreturnedtopre- operativegrasppatterns&latencytoretrievefoodrewardinthe ﬁ rst 3 –5weeksofrecovery

Table3. continued

TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

162

MSC-exoN/AcombinedTBI&HSswinemodel;attenuatedseverityofneurologic injuryandallowedforfasterneurologicrecovery

163

adiposeMSC-exoprimaryratmicroglia&neuron;suppressedmicroglia activationbyinhibitingNFκ B&MAPK rats;promotedfunctionalrecovery,suppressedneuroin ﬂ ammation, reducedneuronalapoptosis,andincreasedneurogenesis;exomainly takenupbymicroglia/macrophages

164

BM-MSC-exoBV2microglia;promotedM1toM2phenotypeand upregulatedanti-in ﬂ ammatorycytokines mice;reducedcorticaltissueapoptosisandinhibited neuroin ﬂ ammation,possiblybyexosomalmiR-181bviaIL-10/STAT3 pathway

166

165 MSC-exoN/Arats;improvedangiogenesis&neurogenesis,andsensorimotor& cognitivefunction,reducedneuroin ﬂ ammation&hippocampal neuronalcellloss;100µg&1daywereoptimal

179

NSC-exoNSCs;exosuperiortoparentalcellsrats;improvedneurobehavioralperformance,inhibitedastrocyte neuroin ﬂ ammation,enhanceddoublecortinneurogenesis,while maintainingSOX2&Nestinstemness

Alzheimer ’ sdiseaseBM-MSC-exoN/Aearly-stageADmice;reducedA β plaqueburden&dystrophicneurites; carriedneprilysin

180

BM-MSC-exoprimaryneuron;reducedA β -inducediNOSexpressionmice;rescuedsynapticimpairmentandimprovedcognitivebehavior

182

181 heatshock-inducedNSC- exo HC2S2cells;exhibitedgreaterneuroprotectionagainst oxidativestressandA β -inducedneurotoxicity N/A

183

184

185

186

NSC-exoN/AADtransgenicmice;enhancedmitochondrialfunction,sirtuin1 activation,synapticactivity,decreasedin ﬂ ammatoryresponse,and rescuedcognitivede ﬁ cits

ADtransgenicmice;improvedbrainglucosemetabolismandcognitive function;upregulatedsynapse-relatedgenes&downregulatedHDAC4 expression

MSC-exoSH-SY5YwithFADmutations;reducedA β expressionand restoredneuronalmemory

N/A

AF-MSC-exoBV2microglia,SH-SY5Ycells;mitigatedneuroin ﬂ ammatory microglialinjuryandrecoveredneurotoxicityfromA β

UC-MSC-exoBV2microglia;reducedin ﬂ ammatoryreaction&induced alternativemicroglialactivation mice;alleviatedneuroin ﬂ ammationandreducedA β depositionby modulatingmicroglialactivation;increasedspatiallearning&memory function

188

MSC-exoN/Amice;stimulatedneurogenesisinsubventricularzoneandalleviated A β -inducedcognitiveimpairment

189

193

5xFADmousemodel;BBBbreakdownoccurredat4monthsofage, whichcouldbemimickedwithaninvitroBBBmodel

NSC-exo5xFADprimarycerebralendothelialcells;reversedAD- causedBBBde ﬁ ciency

RVG-taggedMSC-exoN/AtransgenicAPP/PS1mice;improvedCNS-targeteddelivery;reducedA β deposition&astrocytes,andimprovedcognitivefunction;RVG-exo werebetter

Parkinson ’ sdiseaseBM-MSC-exoSH-SY5Y&SK-N-SHcells;exosomalTSG-6attenuated MPP + -inducedneurotoxicityviaSTAT3/miR-7/NEDD4axis N/A

194

6-hydroxydopamine-inducedPDmice;protecteddopaminergiccell viabilityviaexosomalmiR-182-5p,miR-183-5p,&miR-9

197

EAEratmodel;reducedin ﬂ ammationanddemyelinationofCNSby regulatingpolarizationofmicrogliafromM1toM2;decreased neurobehavioralscoresandpreventedweightloss

198

NSC-exoSH-SY5Y&BV2cells;anti-oxidativestress,anti- in ﬂ ammatory&anti-apoptoticeffects

MultiplesclerosisBM-MSC-exoHAPImicroglia;downregulatedTNFα &iNOSand upregulatedIL-10,TGFβ andarginase-1

BM-MSC-exoN/A2micemodels:EAE&CPZ;improvedneurologicaloutcome,increased OPCdifferentiation&remyelination,decreasedneuroin ﬂ ammationvia TLR2pathway

Table3. continued TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

199

VasculardementiaNSC-exoN/Arats;exo-derivedMIATimprovedlearningability&memoryviamiR- 34b-5p/calbindin-1axis

200

201

202

203

204

205

N/A

NSC-exorescuedcellularviabilityinHIV-damagedneurons,and inhibitedapoptosisandin ﬂ ammatoryfactorsecretion

HIV-associated neurocognitivedisorders

NSC-exoN/Amice;exosomalmiR-124improvedexercise&fearbehavior,reversed cognitiveimpairment,andreducedneuroin ﬂ ammation

Radiation-inducedcognitive dysfunction

NSC-exoN/Amice;protectedhostneurons,enhancedneurotrophicfactors& synapticsignaling,andreducedneuroin ﬂ ammation

EpilepsyBM-MSC-exoN/Amice;reducedhippocampalin ﬂ ammation,andpreventedabnormal neurogenesis&memorydysfunction

L5spinalnerveligationratmodel;increasedpawwithdrawalthreshold andlatency,reducedapoptosisandin ﬂ ammationinspinaldorsalhorn

MechanicalallodyniaBM-MSC-exomicroglia;downregulatedNOTCH2whichistargetedby exosomalmiR-150-5p

Spinabi ﬁ daapertaNSC-exoBM-MSCs;promotedneuronaldifferentiationofMSCsratembryomodel;exosomalNetrin1promotedneuronal differentiationofMSCs&NSCsbyupregulatingHand2/Phox2b

DepressionBM-MSc-exoN/Arats;suppressedapoptosis&boostedproliferationinhippocampal tissuesbyupregulatingexosomalmiR-26a 206

207

StressUC-MSc-exoN/Amiceacutebraindisordermodel;increasedadiponectin,improved cognitivefunctionandhippocampalneurogenesisthatwas suppressedbystreptozotocininjection

208 NSC-exoNSCs;HFDdownregulatedCREB/BDNF/TrkBsignalingmice;intranasaladministrationrestoredCREBtranscriptionalactivity, rescuedbothBDNF&HFD-dependentmemoryde ﬁ cits

BrainageingNSC-exoNSCs;rescuedIRS-1/FoxOactivationandcounteracted reducedproliferationandsenescence mice;intranasaladministrationcounteractedHFD-dependent impairmentofadulthippocampalneurogenesisbyrestoringbalance betweenproliferatingandsenescentNSCs

210

209 hypothalamicNSC-exoN/ANSC-alation-inducedmousemodel;exosomalmiRNAsreduced hypothalamicin ﬂ ammation,andsloweddownageing,independentof foodintake

211

MSC-exoprimaryratbrainendothelialcells;rescuedOGD-induced injury&inhibitedTLR4/NLRP3/caspase-1/NFκ Bpathway N/A

Deephypothermic circulatoryarrest

A β amyloidbeta, AD Alzheimer ’ sdisease, AF amniotic ﬂ uid, BBB bloodbrainbarrier, BDNF brain-derivedneurotrophicfactor, BM bonemarrow, CREB cAMPresponseelementbinding, CNS centralnervoussystem, EAE experimentalautoimmuneencephalomyelitis, EDNRB endothelinreceptortypeB, exo exosome, FAD familialAlzheimer ’ sdisease, FoxO ForkheadboxO, H/R hypoxiaandreoxygenation, HAPI highlyaggressive proliferatingimmortalized, HDAC histonedeacetylase, HFD highfatdiet, HS hemorrhagicshock, iCM inducedpluripotentstemcell-derivedcardiomyocyte, IFN interferon, IL interleukin, iNOS induciblenitricoxide synthase, iPSC inducedpluripotentstemcell, IRAK interleukin1receptorassociatedkinase, IRS insulinreceptorsubstrate, LDH lactatedehydrogenase, MAPK mitogen-activatedproteinkinase, MCAO middle cerebralarteryocclusion, MIAT myocardialinfarctionassociatedtranscript, miR microRNA, MPP + 1-methyl-4-phenylpyridinium, MSC mesenchymalstemcell, NEDD4 neuronallyexpresseddevelopmentallydownregulated4, NFκ B nuclearfactor-kappaB, NLRP NOD-,LRR-andpyrindomain-containingprotein, NSC neuralstemcell, oe overexpressing, OGD oxygen-&glucose-deprived, SOX Sry-Boxtranscriptionfactor, STAT signaltransducerandactivatoroftranscription, TGF transforminggrowthfactor, TLR Toll-likereceptor, TNF tumornecrosisfactor, TRAF TNFreceptorassociatedfactor, TrkB tropomyosinreceptorkinaseB, TSG TNF stimulatedgene, UC umbilicalcord, Zeb zinc ﬁ ngerE-boxbindinghomeobox

sensorimotorfunction.159 Inaddition,exosometreatmentsignificantlyincreasedthenumberofnewbornendothelialcellsin thelesionboundaryzone,andnewbornimmatureandmature neuronsinthedentategyrus.InanotherratTBImodelwithsimilar findings,exosomesderivedfromMSCsculturedina3Dsystem providedbetteroutcomesthanthoseinaconventional2D condition.160 InamonkeymodelofTBItotheprimarymotor cortex,exosome-treatedanimalsreturnedtopre-operativegrasp patternsandlatencytoretrieveafoodrewardinthe first 3–5weeksofrecovery.161 Inanevenmorecomplicatedand clinicallyrealisticlargeanimalmodel,inwhichbothTBIand hemorrhagicshockwereinvestigated,exosometherapyattenuatedtheseverityofneurologicinjuryandenabledfaster neurologicrecovery.162

Incomparison,studiescompletedinrecentyearsshednew lightonthemolecularmechanismsunderlyingSC-exotherapyfor TBI.Chenetal.reportedthatadiposeMSC-derivedexosomes couldpromotefunctionalrecovery,suppressneuroinflammation, reduceneuronalapoptosis,andincreaseneurogenesis.Thiswas achievedthroughtheuptakeofexosomesspecificallybymicroglia andsuppressionoftheiractivationbyinhibitingtheNF-κB& MAPKpathways.163 Wenetal.showedthatbonemarrowMSCderivedexosomescouldreducecellapoptosisincorticaltissueof mousemodelsofTBI,inhibitneuroinflammation,andpromotethe transformationofmicrogliatotheanti-inflammatoryphenotype. ThiswasrealizedbytheactionofmiR-181bontheIL-10/STAT3 pathway.164 Abedietal.provedthatNSC-derivedexosomescould improveneurobehavioralperformance,inhibitastrocyteneuroinflammation,enhanceneurogenesis,whilemaintainingNSCstemness.165 Avaluableadditional findingwasthatexosomesseemed tobesuperiortotheparentNSCsintermsofsensorimotor functionalrecovery.Finally,adose-responseandtherapeutic windowdemonstratedthatMSC-derivedexosomescouldimprove angiogenesisandneurogenesis,andsensorimotorandcognitive function,whilereducingneuroinflammationandhippocampal neuronalcellloss.166 Although100µgand1daymightbethe optimaldoseandtherapeuticwindowrespectively,exosomes exhibitedawiderangeofeffectivedosesfortreatmentofTBI withinatherapeuticwindowofatleast7dayspost-injury.

TBIandSCIaretwoofthemostsevereCNStraumas,whichare increasinglyrecognizedasglobalhealthpriorities.Theemerging evidencepresentedinSections3.3and4.2aremutuallybeneficial forthesetwocloselyrelatedresearchsubspecialties.Henceforth, futureresearchonSC-exotherapyforTBIandSCIcouldbeeither mechanism-based(e.g.,theroleofbrain-gutaxis167 transcriptional factors168 inflammasome169 andthecomplementsystem170)or modification-based(loadingexosomeswithdrugs,e.g.,immunomodulators171 antioxidants172 circularRNAs173 andmicroRNAs174).

Alzheimer’sdisease

UnlikeTBIandSCI,whicharetraumaticinnature,Alzheimer ’s disease(AD)andParkinson’sdisease(PD)arethemostcommon neurodegenerativediseases(NDD).ThehallmarksofNDDinclude, butarenotlimitedto,pathologicalproteinaggregation,synaptic andneuronalnetworkdysfunction,aberrantproteostasis,cytoskeletalabnormalities,alteredenergyhomeostasis,DNAandRNA defects,in ﬂammation,andneuronalcelldeath.175 ADisthemost commonformofdementiagloballyandaccountsfor25million cases.176 Currently,onlytwoclassesofdrugsareapprovedfor symptomaticADtreatment,includingcholinesteraseinhibitors andNMDAantagonists.Althoughseveraltherapeuticsare activelyundergoingclinicaltrials,noneofthemarenearcurative forAD.177 Thechallengesofbrain-drugdelivery,e.g.,thebloodbrainbarrier(BBB)andpharmacokineticdrawbacks,areverylikely tobesolvedbynanosizedexosomes,whichareadditionally packagedwithpotentbiomolecules.MostSC-exotherapy involvesamyloid- β (Aβ ),whichispositionedatthecenterofAD pathophysiology.178

Theinitialworkfocusedontheclearanceofaggregationofthe pathologicalprotein,Aβ peptide.Theintracerebralinjectionof MSC-derivedexosomesbyEliaandco-workersreducedAβ plaque burdenanddystrophicneuritesinboththecortexand hippocampusintheearlystagesofapreclinicalmodelofAD.179 Inaddition,usingimmunoblotting,theauthorsconﬁrmedthe presenceofNeprilysin,aneutralendopeptidasecapableofAβ degradation,intheexosome’slysatesanditsmRNA.

Someteamshavefocusedonrelievingsynapticdysfunction andoxidativestress.Wangetal.foundthatMSC-derived exosomescouldrescuesynapticimpairmentandimprove cognitivebehaviorinAPP/PS1mice,whilealleviatingexogenous Aβ-inducedinduciblenitricoxidesynthase(iNOS)expression.180 InsteadofusingMSC-derivedexosomes,Lietal.administered NSC-derivedexosomesandenhancedmitochondrialfunction, sirtuin1activation,synapticactivity,andrescuedcognitive deﬁcits.181 Usingalternativemethods,Huberetal.noticedthat heatshock-inducedexosomesderivedfromNSCsexhibited greaterneuroprotectionagainstoxidativestressaswellas Aβ-inducedneurotoxicity.182

Someteamshavecenteredtheirresearcharoundenergy homeostasis.Chenetal.foundthatMSC-derivedexosomescould improvebrainglucosemetabolismandcognitivefunctioninAD transgenicmiceusing 18F-FDGPET/CTimagingandNORtesting, respectively.183

Someteamshavefocusedonmicroglialneuroinflammation.In Zavatti’scellularstudy,itwasfoundthatamniotic fluidMSCderivedexosomescouldmitigateneuroinflammatorymicroglial phenotypeandrecoverneurotoxicityfromAβ usingLPSstimulatedBV2microgliaandSH-SY5Yneuroblastomacellsas models,respectively.184 Dingetal.showedthatumbilicalcord MSC-derivedexosomescouldalleviateneuroinflammationand reduceAβ depositionbymodulatingmicroglialactivation,thereby increasingspatiallearningandmemoryfunctioninADmice.185

Someteamshavefocusedonneuronalcelldeathand neurogenesishopingtocounteractADprogression.RezaZaldivarandco-workersgaveMSC-derivedexosomestoADmice andtheSC-exotherapystimulatedneurogenesisinthesubventricularzoneandalleviatedAβ-inducedcognitiveimpairment.186 TheseeffectsarecomparabletothoseshownintheMSCs.

SometeamshavefocusedontheBBB,thedysfunctionofwhich leadstoincreasedpermeability,microbleeds,impairedglucose transport,anddegenerationofpericytesandendothelialcells.187 Liuetal.indicatedthatBBBbreakdownin5xFAD(familial Alzheimer’sdisease)miceoccurredat4monthsofage,andmore importantly,treatmentwithNSCs-derivedexosomesreversedADcausedBBBdeﬁciency.188

Finally,somegroupshavefocusedonimprovingthetechnicality ofSC-exotherapyforAD.Whenexosomesareinjectedintravenously,theycouldbetrackedinotherorgansinsteadofthetargeted regionsinthebrain.Cuietal.conjugatedMSC-derivedexosomes withCNS-specificrabiesviralglycoprotein(RVG)totargetthemto thebrainoftransgenicADmice.189 Themodifiedexosomesnotonly exhibitedincreaseddeliverytothecortexandhippocampus,but alsosignificantlyimprovedlearningandmemorycapabilitieswith reducedAβ deposition.Ontheotherhand,Gaoetal.obtainediNSCs throughsomaticcellreprogramming,whichopenedanewwindow forsourcingtherapeuticexosomes.TheydemonstratedthatiNSCderivedexosomes,bearingcomparabletherapeuticeffectswith NSC-derivedones,couldmitigatevariousADphenotypes,e.g., cognitivefunction,Aβ deposition,neuroinflammation,andneuroregeneration,inapreclinicalmousemodel.190

Parkinson’sdisease

Parkinson’sdiseaseisthesecondmostcommonneurodegenerativediseaseamongtheelderly,affectingmorethan6million patientsworldwide.191 PDiscausedbythenecrosisofdopaminergicneuronsinthesubstantianigraandthepresenceofprotein

inclusionsnamedLewybodies.Themolecularpathophysiology includes α-synucleinproteostasis,mitochondrialdysfunction, oxidativestress,calciumimbalance,andneuroinﬂammation.192

InastudyusingbonemarrowMSC-derivedexosomes,Huang etal.discoveredthatexosome-derivedTNF-stimulatedgene-6 (TSG-6)couldattenuate1-methyl-4-phenylpyridiniumion(MPP + , metaboliteofaneurotoxinMPTP)-inducedneurotoxicity.Inthis invitroPDmodelusingSH-SY5YandSK-N-SHcells,theexosomal anti-PDprogressioneffectwasfoundtobemediatedthroughthe STAT3/miR-7/NEDD4axis.193

InastudyusingNSC-derivedexosomes,Leeetal.revealedthat SC-exotherapycouldhelptopreventtheneuropathologyand progressionofPD.194 WorkinginvitroonSH-SY5YandBV2cells, NSC-derivedexosomescouldreducetheintracellularreactive oxygenspecies(ROS)andassociatedapoptoticpathways.Working invivoon6-hydroxydopamine-inducedPDmice,NSC-derived exosomescoulddownregulatepro-inflammatoryfactorsand significantlyreducedopaminergicneuronalloss.Thepresenceof NSC-specificmicroRNAs,suchasmiR-182-5p,miR-183-5p,miR-9 andlet-7,wasconfirmedandfoundtobeinvolvedincell differentiation,neurotrophicfunction,andimmunemodulation.

Multiplesclerosis

Multiplesclerosis(MS)isthemostcommonnon-traumatic, neurodegenerative,anddisablingCNSdiseaseaffectingyoung adults.ThepathologicalhallmarkofMSistheformationof demyelinatinglesionsinthebrainandspinalcord,withan inﬂammatoryandautoimmuneinvolvement.195 Currentlylicensed disease-modifyingtherapiesincludeinterferon-based,immunomodulatory,immunosuppressive,andimmunereconstitution drugs.196 Afewpreliminarystudieshavehighlightedthepotential ofMSC-derivedexosomesforMStreatment.

Inananimalexperimentusingexperimentalautoimmune encephalomyelitis(EAE)rats,Lietal.showedthatSC-exotherapy significantlydecreasedneuralbehavioralscores,reducedthe infiltrationofinflammatorycellsintotheCNS,anddecreased demyelination.197 Inaddition,exosometreatmentupregulated M2-relatedcytokineswhiledownregulatingM1-relatedonesby regulatingthepolarizationofmicroglia.

Inananimalstudyusingtwomousemodelsofdemyelination (theEAEmodelandthecuprizonedietmodel),Zhangetal.found thatSC-exotherapycouldpromoteremyelinationbyactingboth directlyonoligodendrocyte(OL)progenitorsandindirectlyon microglia.198 MSC-derivedexosomescouldimproveneurological outcomes,increasethenumbersofnewlygeneratedandmature OLs,decreaseAβ precursorproteindensity,decreaseneuroinﬂammationbyshiftingfromM1toM2phenotype,andinhibitthe TLR2/IRAK1/NF-κBpathway.

Otherneurosurgicalandrelateddiseases

ThesurgicalpotentialofMSC-andNSC-derivedexosometherapyin fourmajortypesofneurosurgicalorneurologicaldiseaseshasbeen thoroughlydiscussedabove.Inadditiontovasculardisruption-, trauma-,neurodegeneration-,and autoimmune-relateddisorders, otherdiseaseshavebeenprovensuitabletargetsforSC-exotherapy recently(Table 3).Theseinclude,butarenotlimitedto:1,dementia, suchasvasculardementia.199 HIV-associatedneurocognitivedisorders200 andradiation-inducedcognitivedysfunction201,202;2, functionaldisorders,suchasepilepsy203 andmechanicalallodynia204;3,congenitalabnormalities,suchasspinabiﬁdaaperta205;4, neuropsychologicalconditions,suchasdepression206 andstress207; 5,brainaging208–210;6,iatrogenicbrainproblems,suchasdeep hypothermiccirculatoryarrest.211

PLASTICSURGERYANDSC-EXOTHERAPY

Intheinflammatorystage,exosomescouldinhibitthe proliferationofperipheralbloodmononuclearcellsandpromote thetransformationofregulatoryTcellsinvitro,andreducethe numberoflymphocyticinfiltrationsintheskin.214 Inaddition, exosomescouldreduceIgE,eosinophilandmastcellcount,and downregulateinflammatorycytokines.215 Intheangiogenicstage, educatedexosomes(e.g.,atorvastatinanddeferoxamine)could promoteangiogenesisindiabeticwoundsviatheAkt/eNOSand PTEN/PI3K/Aktpathways.216–218 EPC-derivedexosomescould acceleratecutaneouswoundhealingbypromotingangiogenesis219 throughtheErk1/2pathway220 andp53pathway.221 Inthe proliferativestage,stemcell-derivedexosomescouldpromotethe proliferationandmigrationof fibroblastsandkeratinocytes.Some wereachievedthroughthePI3K/Akt222 Akt/HIF-1α223 ERK1/2224 andWnt/β-catenin225 pathways,whileothersthroughinhibitionof LATS2226 PPARγ 227 andAIFnucleustranslocation228 Inthe final remodelingstageofwoundhealing,granulationtissueisreplaced bypermanentscar,duringwhichabnormalwoundhealingmight occur(e.g.,keloidsandhypertrophicscars).MSC-derivedexosomescouldsuppress fibroblast-myofibroblasttransitionviathe TGF-β/Smad2pathway229,230 andincreasecollagensynthesisin earlystageandreduceinlatestage231 therebyreducingscar formation.

Furthermore,ESC-derivedexosomeswerefoundtoexertsimilar therapeuticeffectforwoundhealingtoMSC-derivedones.Chen etal.usedhumanESC-derivedexosomestohelphealingof pressureulcer.232 Theynoticedthatexosomescouldameliorate endothelialsenescencebyactivatingNrf2andrecoveragingrelatedangiogenicdysfunction,therebyacceleratingwound healing.232 Inaddition,Baeetal.revealedthattheexosomal mmu-miR-291a-3pfromESCscouldinhibitcellularsenescencein humandermal ﬁbroblaststhroughtheTGF-β receptor2pathway, therebyacceleratingtheexcisionalskinwoundhealingprocess.233

Inadditiontowoundhealing,otherplasticsurgery-related diseaseshavebeenproventobesuitabletargetsforSC-exo therapy(Table 4).Theseinclude,butarenotlimitedto:1,skin grafting,suchasskin ﬂaps234;2,tissueloss,suchascraniofacial defect235;3,autoimmuneskindiseases,suchasscleroderma236;4, skininfections,suchasleishmaniasis237;5,hairtransplantation, suchasforalopecia238;6,skinaging239

GENERALSURGERYANDSC-EXOTHERAPY

Asamajorsubspecialtyofgeneralsurgery,hepatobiliarysurgery hasattractedtremendousattentiontoSC-exotherapy.Firstly, acuteliverinjury(ALI)/acuteliverfailure(ALF)isararebut challengingsyndromemanifestedbyhepaticdysfunction,coagulopathy,encephalopathy,andmultiorganfailure.About60%of caseswithALFrequireandundergoorthotopiclivertransplantationorresultindeath.240 Inonestudy,Lin’steamfocusedonthe celldeathaspectofALI,andfoundthatMSC-derivedexosomes couldprotectagainstferroptosisviastabilizationofSLC7A11in carbontetrachloride-inducedALI.241 Alternatively,Shao’steam focusedonthepre-isolationmodiﬁcationoftheexosomes,and revealedthatexosomesderivedfromumbilicalcordMSCscould ameliorateIL-6-inducedALIthroughexosomalmiR-455-3p.242 Secondly,incontrasttoALI,liver ﬁbrosisoccurswhentheliver sustainsachronicinjury,whichmayprogressintocirrhosis,liver failure,hepatocellularcarcinoma,andevendeath.243 Maetal. discoveredthatMSC-originatedexosomalcircDIDO1couldsuppresshepaticstellatecellactivationbymiR-141-3p/PTEN/Akt

Woundhealingoccursinallpartsofthehumanbody,with cutaneouswoundsbeingthemostcommon.Thehighestwoundrelatedexpenseswereattributedtosurgicalwoundsfollowedby diabeticulcers.212 Theoverallbutoverlappingphasesofwound healingincludehemostasis,inﬂammation,angiogenesis,proliferationandremodeling,eachofwhichisgovernedbydistinctcell typesandmodulatedbyvarioussignalingpathways.213 Morethan halfofrelevantworkusingSC-exotherapytoboostcutaneous woundhealingisMSC-based(Table 4).

Table4. Stemcell-derivedexosomesforthetreatmentofdiseasesinplasticsurgeryandrelatedspecialties

TargetdiseaseExosomeInvitromodel& ﬁ ndingsInvivomodel& ﬁ ndingsRefs.

214

eczemamousemodel;acceleratedwoundclosurewithmorenewepidermis& dermisandlessscar;reducedintegralscoreofskininjuryandno.of lymphocytein ﬁ ltrationinskin

WoundhealingUC-MSC-exoPBMCs;inhibitedcellproliferation,promotedTreg transformation&formationofendothelialtube

215

adipose-MSC-exoN/AHDM-inducedmousemodel;reducedIgE,eosinophil&mastcellcount,and downregulatedin ﬂ ammatorycytokines

218

221

educatedBM-MSC-exoHUVECs;promotedangiogenesisviaAkt/eNOSpathwaymice;promotedcutaneouswoundhealing

streptozotocin-induceddiabeticwoundratmodel;exosomalmiR-221-3p facilitatedwoundrepairbyenhancingangiogenesisviaAkt/eNOSpathway

216 atorvastatin-treatedBM-MSC- exo HUVECs;promotedproliferation,migration,tube formation,andVEGFlevel

streptozotocin-induceddiabeticwoundratmodel;acceleratedcutaneous woundhealingbypromotingangiogenesis

deferoxamine-stimulatedBM- MSC-exo HUVECs;activatedthePI3K/AktpathwayviamiR-126- mediatedPTENdownregulation

streptozotocin-induceddiabeticwoundratmodel;acceleratedcutaneous woundhealingbypromotingangiogenesisviaErk1/2pathway

EPC-exoHMECs;enhancedproliferation,migration&tubule formation

EPC-exoN/Astreptozotocin-induceddiabeticwoundmousemodel;exosomalmiR-221-3p acceleratedcutaneouswoundhealingviap53pathway

adipose-MSC-exoHDFs;improvedproliferation&migrationmice;promotedwoundhealingviaPI3K/Aktsignalingpathway

225

mice;promotedwoundhealing,whichwaseliminatedbyinhibitionofp Akt andHIF

N/A

adipose-MSC-exoHaCaTcells;promotedproliferation&migrationby activatingAkt/HIF-1 α pathway

iPSC-exo,MSC-exoHDFs,HaCaTcells;acceleratedproliferationviaERK1/2 pathway

N/A

MALAT1-adipose-MSC-exoHaCaTcells&HDFs;promotedwoundhealingbymiR- 124viaWnt/ β -cateninpathway

MSC-exoBJcells;promoted ﬁ broblastsmigrationrats;exosomalmiR-135apromotedcutaneouswoundhealingbyinhibiting LATS2expression

228

diabeticmice;acceleratedwoundhealingviadownregulatingPPAR γ

EPC-exoHaCaTcells;promotedproliferation&migration,and inhibitedapoptosis

mice;attenuatedfull-thicknessskinwoundingbyenhancingepidermalre- epithelializationanddermalangiogenesisviasuppressingAIFnucleus translocation

UC-MSC-exoH 2 O 2 -treatedHaCaTcells;increasedproliferation& migration,andsuppressedapoptosis

UC-MSC-exoN/Amice;suppressedmyo ﬁ broblastdifferentiationbyinhibitingTGFβ /Smad2 pathwayduringwoundhealing;miR-21,-23a,-125b,-145responsiblefor preventingscarformation

N/A

UC-MSC-exoHDFs;suppresseddermal ﬁ broblasts-myo ﬁ broblasts transitionviaTGFβ /Smad2/3pathway

mice;exorecruitedtowoundarea,andacceleratedcutaneouswoundhealing; increasedcollagenI&IIIinearlystageandinhibitedcollageninlatestageto reducescarformation 231

adipose-MSC-exoprimaryHDFs;stimulatedproliferation,migration,and collagensynthesis

232

D-galactose-inducedagingmice;exosomalmiR-200aacceleratedwound closureandenhancedangiogenesisviaNrf2activation

ESC-exoHUVECs;amelioratedsenescence,proliferation,and migration

233

ESC-exoHDFs;inhibitedcellularsenescenceviaTGFβ receptor2 pathway mice;exosomalmmu-miR-291a-3pacceleratedexcisionalskinwoundhealing process

234

I/Rinjuryratmodel;increased ﬂ apsurvival&capillarydensity,anddecreased in ﬂ ammatoryreaction&apoptosis;H 2 O 2 -conditionedexowerebetter

ﬂ apadipose-MSC-exoHUVECs;increasedcellproliferation,migrationwithmore cord-likestructures

Skin

235

CraniofacialdefectSCAP-exoHUVECs;improvedangiogeniccapacityandcell migration mice;promotedcraniofacialsofttissueregenerationbyenhancingCdc42- mediatedvascularization

SclerodermaUC-MSC-exoN/A

broblastactivationandcollagendepositionindermal

mice;attenuatedmyo

β /Smadsignalingpathway

ﬁ brosisbydownregulatingtheTGF-

237

L.majorpromastigotes&amastigotes;inhibitedfor4 –10folds;combinations superiortoexoalone

ﬁ cialwoundmodel;healed72% woundin24h

LeishmaniasisUC-MSC-exo + Aloe-EmodinL929&J744cells,arti

depilation-inducedmicehairregenerationmodel;promotedhairfollicle growthbyactivatingWnt/ β -cateninpathway

AlopeciaNSC-exodermalpapillacells;exosomalmiR-100promotedcell proliferation

238 AIF apoptosis-inducingfactor, Akt proteinkinaseB, BM bonemarrow, eNOS endothelialnitricoxidesynthase, EPC endothelialprogenitorcell, ESC embryonicstemcell, exo exosome, HDF humandermal ﬁ broblasts, HDM housedustmite, HIF hypoxia-induciblefactor, HMEC humanmicrovascularendothelialcell, HUVEC humanumbilicalveinendothelialcell, I/R ischemia-reperfusion, LATS largetumorsuppressor, MALAT metastasisassociatedlungadenocarcinomatranscript, miR microRNA, MSC mesenchymalstemcell, NSC neuralstemcell, PBMC peripheralbloodmononuclearcell, PI3K phosphoinositide3-kinase, PTEN phosphatase&tensinhomolog, SCAP stemcellsfromapicalpapilla, TGF transforminggrowthfactor, Treg regulatoryTcell, UC umbilicalcord, VEGF vascularendothelialgrowthfactor

pathwayinhumanliver fibrosis.244 Inaddition,Wangetal.found thatexosomesderivedfrom3DhumanESCspheroidscould attenuatehepaticstellatecellactivationandinhibitliver fibrosis throughinactivationoftheSmadpathwaybyexosomalmiR-67663p.245 Forthosewithend-stageliver fibrosisneedingaliver transplant,liverischemiareperfusioninjury(IRI)isaserious complicationforgraftdysfunctionandorganrejection.246 Yang etal.demonstratedthatbonemarrowMSC-derivedexosomes couldrelievehepaticIRI,reducehepatocyteapoptosis,and decreaseliverenzymelevelsbyenhancingautophagy.247 Du etal.showedthatexosomesfromiPSC-derivedMSCscould protectliveragainsthepaticIRIviaactivatingsphingosinekinase andthesphingosine-1-phosphatepathway.248 Thirdly,nonalcoholicfattyliverdisease(NAFLD)isknowntoadverselyaffect strokerecovery.Usingatype2diabetesmellitusmousemodel, Venkatetal.demonstratedthatHSC-derivedexosomescould simultaneouslyreduceliverdysfunctionandimproveneurological andcognitivefunction.249 Lastly,acutepancreatitisisan unpredictableandpotentiallylethaldisease,theprognosisof whichmainlydependsonwhetheritdevelopsintomultipleorgan dysfunctionsyndrome.250 Chenetal.revealedthatexosomesfrom iPSC-derivedMSCscouldimprovemyocardialinjurycausedby severeacutepancreatitisthroughtheAkt/Nrf2/HO-1pathway.251 Peripheralarterydiseaseaffects200millionpatientsworldwide and,initsmostseverestage,cancausecriticallimbischemia, subjectingpatientstoincreasedriskofcardiovascularevents, amputationanddeath.252 Asacell-freetherapy,placentaMSCderivedexosomeinfusioncouldenhanceangiogenesisina murineauricleischemicinjurymodelusinglaserDopplerblood flowanalysis.253 Mechanistically,MSC-derivedexosomesnotonly promotetube-likestructureformationinvitro,butalsomobilize endothelialcellsintosubcutaneousMatrigelpluginvivo,mainly throughexosomalpro-angiogenicmicroRNAs,suchasmiR-30b.254 Inaddition,HSC-derivedexosomescouldrepairischemichindlimb inmicebyimprovinglimbperfusion,capillarydensity,motor functionandtheiramputation.255 Thiswasmostlikelycausedby internalizationofexosomalmiR-126-3pbyendothelialcells relativetosmoothmusclecellsand fibroblasts.HumaniPSCderivedexosomesdemonstratedsimilarneoangiogeniceffect throughtheexosomalmiR-199b-5p.256 Ontheotherhand, endovascularre-canalizationisincreasinglybeingusedto reestablishblood flowtoischemicareasandrestoretissueloss organgreneforpatientswithperipheralarterydisease.257 Three independentteamsallprovedthatEPC-derivedexosomescould promotevascularrepairandacceleratereendothelializationinrat modelsofballoon-inducedvascularinjurybyenhancingendothelialcellfunction.258–260 Inaddition,Kongetal.demonstrated similarprotectiveeffectofEPC-derivedexosomesagainstballoon injurybyinhibitingneo-intimalhyperplasia.261 Thiswasachieved throughpromotionofreendothelializationandsuppressionof restenosisratherthanthroughthedirectinhibitionofproliferation andmigrationofsmoothmusclecells.

McCullohandhisco-workerspublishedanintriguingstudyon theSC-exotherapyfornecrotizingenterocolitis(NEC)whichhas anoverallmortalityofover30%forprematureinfantsrequiring surgery.262 Theauthorscomparedthetherapeuticeffectof exosomesderivedfromfourdifferenttypesofstemcells,i.e., amniotic ﬂuidMSCs,bonemarrowMSCs,amniotic ﬂuidNSCsand neonatalentericNSCs.263 Wheninjectedataconcentrationofat least4×108,alltypesofSC-exowereshowntoreducethe incidenceandseverityofexperimentalNECaseffectivelyastheir parentalstemcells.

Sepsisisadeadlyandpotentiallypreventablecomplicationin generalsurgery,inwhichmicrovasculardysfunctionleadsto multi-organfailureandmortality.264 Usingamurinesepsismodel bycecalligationandpuncture(CLP),Zhouandco-workers demonstratedthatEPC-derivedexosomescouldimprovesepsis outcome.265 Thiswasmanifestedbyreducedlungandrenal Table4.

vascularleakage,improvedorganfunction,andincreasedsurvival throughtheexosomalmiR-126-5pandmiR-126-3p.Similarly,Liu etal.exhibitedprotectiveeffectofEPC-derivedexosomeson sepsis-inducedorgandamageandimmunesuppressionbythe exosomalmiR-382-3pthroughtheIκBα/NF-κBpathway.266

CARDIOTHORACICSURGERYANDSC-EXOTHERAPY

Theworld’sleadingmortalityisischemicheartdisease(IHD)which isprimarilycausedbyobstructivecoronaryatherosclerosis.The ruptureofanatheroscleroticplaqueisthemostcommontrigger ofacutearterialthrombosiscausingmyocardialinfarction(MI). Prolongedoxygendeprivationtothemyocardiumcanleadto cardiomyocytedeath.Althoughtimelyreperfusionisessential, myocardialIRImightoccur,thusmitigatingthebeneﬁcialeffects ofreperfusion.Despitemoderncoronaryreperfusion,themortality andmorbidityassociatedwiththedevelopmentofheartfailureas aconsequenceofacuteMIremainsubstantial,highlightingthe importanceofnext-generationcardioprotectivetherapies,suchas SC-exo.267

TheworkconductedbyXingetal.focusedonatherosclerosis. TheadiposeMSC-derivedexosomalmiR-342-5pwasshownto protectendothelialcellsagainstatherosclerosisbytargeting PPP1R12BinaH2O2-challengedHUVECmodel.268 Thework conductedbyPengetal.andGaoetal.focusedonMI.The exosomalmiR-25-3pfromMSCscouldalleviateMIbytargeting pro-apoptoticproteinsandEZH2inanOGDcardiomyocytes modelandleftanteriordescendingarteryligationanimal model.269 Similarly,humaniPSC-derivedexosomescouldimprove recoveryfromMIwithoutincreasingthefrequencyofarrhythmogeniccomplicationsinaswinemodel.270 Theworkconductedby Wenetal.andSantosoetal.focusedonthedeathof cardiomyocytes.MSC-derivedexosomescouldamelioratecardiomyocyteapoptosisinhypoxicconditionsthroughmiR-144by targetingthePTEN/Aktpathway271 whereasiPSC-derivedexosomescouldregulateautophagyinhypoxiccardiomyocytes.272 BothKatsuretal.andChenetal.focusedonmyocardialIRI.The formerteamfoundthatexosomesderivedfromnoncardiomyocyte-relatedcells,i.e.,CTX0E03NSCs,couldreduce infarctsizewhiledelayingcardiomyocytemitochondrialpermeabilitytransitionporeopeningthroughtheJAK/STATpathway.273 ThelatterteamdiscoveredthatMSC-derivedexosomalmiR-1433pcouldsuppressmyocardialIRIbyregulatingautophagyviathe CHK2-Beclin2pathway.274 Finally,Chenandco-workersproved thatbonemarrowMSC-derivedexosomescouldattenuatecardiac hypertrophyand ﬁbrosisinpressureoverload-inducedremodeling,therebyprovidingapromisingpotentialtreatmentforheart failure.275

IncomparisontoMSC-derivedexosomes,exosomesderived fromESCsexhibitedcomparabletherapeuticeffectforcardiac conditions.Intermsofprotectionofcardiomyocytes,Khanetal. discoveredthattheESC-derivedexosomalmiR-294couldimprove cardiomyocytesurvival,promoteneovascularizationandinhibit fibrosisafterMI,therebyaugmentingpost-MIcardiacfunction.276 Inaddition,TavakoliDargagnietal.demonstratedthatESCderivedexosomescouldalleviatedoxorubicin-inducedcardiotoxicitybyinhibitingTLR4-NLRP3-mediatedpyroptoticcelldeathin cardiomyocytes.277 Similarly,Singla’steamshowedthatESCderivedexosomescouldimprovecardiacremodelingbyenhancinganti-inflammatoryM2macrophagesandreducing inflammation-inducedpyroptosis.278 Intermsofmanagementof heartfailure,Pangetal.exhibitedthatESC-derivedexosomes couldattenuateheartfailure,improvecardiacfunctionand promotemyocardialangiogenesisthroughtheFGF2signalingin atransverseaorticconstriction-inducedheartfailuremodel.279 Usingacoronaryarteryocclusion-inducedheartfailuremodel, Kervadecetal.showedthatexosomessecretedbyESC-derived cardiovascularprogenitorscouldrecovercardiacfunctionssuchas

reducedleftventricularend-systolicandend-diastolicvolumes.280 Finally,thesameresearchgrouplaterdemonstratedthat exosomesderivedfrommorereadilyavailablecellsources,e.g., iPSCs,werecapableofcardioprotectiveeffectssimilartothose offeredbyESC-derivedones.281

OtherimportantsubtypesofSC-Exo,suchasiPSC-exo,HSC-exo andEPC-exo,havealsoexhibitedcardio-protectiveeffects.For example,exosomessecretedbyiPSCscouldexertcytoprotective effectsonmaintainingintracellularCa2+ homeostasisand promotingcardiomyocytesurvival,therebyimprovingrecovery fromMI.282 HSC-exocouldreducethecardiacinjury-related indicesandthedegreeofcardiac fibrosiswhileelevatingthe ejectionfractioninananimalmodelofheartfailure.283 Inaddition, systemicinfusionofHSC-derivedexosomescouldimprove ischemiccardiomyopathyinaratmodelofacuteMI,with additionalbenefitsintreatingthesideeffectssuchaskidney damage.284 ModificationofHSCsusingsonichedgehog(Shh),an angiogenicfactor,couldpreservecardiacfunctionafteracuteMI bydeliveryofexosomalShhtoischemicmyocardium.285 Keetal. provedthatEPC-derivedexosomescouldenhancetheproliferationandangiogenesisofcardiac fibroblastsbyactivating mesenchymal-endothelialtransitionanddecreasingtheexpressionofHMGB1286 andlaterrevealedthattheexosomalmiR-2185pandmiR-363-3pfromEPC-derivedexosomescouldameliorate MIbytargetingthep53/JMYpathway.287 Inaninterestingstudy byYueetal.,IL-10deficiency-inducedsystemicinflammationwas foundtocompromisethereparativepropertiesofEPC-derived exosomesonmyocardialrepairbyupregulatingintegrin-linked kinase(ILK)enrichmentinexosomes,andILK-mediatedactivation ofNF-κBpathwayinrecipientcells.288

Intermsoftreatmentofthoracicdisorders,Liuetal.provedthat humanESCs-derivedexosomescouldalleviateinflammation, preventexcessivecollagendepositionandpreservealveolar architectureinthelungsofmicewithbleomycin-induced pulmonary fibrosis.289 Thiswasachievedbytheexosomalmi-175ptargetingthrombospondin-2.Similarly,Zhouetal.demonstratedthattheexosomalmiR-302a-3pfromiPSC-derived exosomescouldsuppressM2macrophagesviaTET1,thereby mitigatingpulmonary fibrosis.290 Liuetal.showedthatEPCderivedexosomescouldinhibitpulmonaryarterysmoothmuscle cellsproliferationandtheirresistancetoapoptosisbyregulating theMitofusin-2andRas-Raf-ERK1/2pathways,therebyactingasa potentialtherapeuticcandidateforthetreatmentofpulmonary arterialhypertension.291 Twoindependentteamsbothrevealed thathumanEPC-derivedexosomescouldimproveoutcomesof theLPS-inducedacutelunginjurypartiallythroughthedeliveryof miR-126intotheinjuredalveolus.292,293 Zhangetal.foundthat EPC-derivedexosomescouldimprovethebioactivityofpulmonarymicrovascularendothelialcellsandprotectthemfrom hyperoxicinjuryinthedevelopinglungvasculature,thereby contributingtothetreatmentofbronchopulmonarydysplasia.294 Moreover,Montay-Grueletal.demonstratedthathumanESCderivedexosomescouldimprovetheadverselatenormaltissue complicationsassociatedwithexposureofthelungstoionizing radiation,suchasthoseencounteredduringpostoperative treatmentoflungcancer.295

UROLOGYANDSC-EXOTHERAPY

Chronickidneydisease(CKD)isasyndromecharacterizedby persistentchangesinkidneystructure,function,orboth,affecting 10 14%oftheglobalpopulation.296 Themostcommon pathologicalfeatureand finalmanifestationofCKDissomeform ofrenal fibrosis.Kidney fibrosisoccurswhenwoundhealingis deregulated,whichleadstoexcessiveaccumulationofECM proteins,suchascollagenand fibronectin.Intheirstudy,Liuetal. discoveredthatbonemarrowMSC-derivedexosomescould alleviatevascularcalcification,adetrimentalindicatorofmorbidity

andmortalityinCKD.297 ThiswasachievedthroughexosomalmiR381-3pbytargetingNFAT5,whichwasfurtherverifiedinsevere arterialcalcificationindialysispatients.Inalternativestudies, severalgroupsdemonstratedthecapacityofbonemarrowMSCderivedexosomesintreatingrenal fibrosis,eachwithadistinct mechanisticinterpretation.Inacellularstudy,Yinetal.foundthat exosomescouldpreventTGF-β1-inducedepithelial-mesenchymal transitionofrenaltubularepithelialcellsbytransportingNedd4L, whichactivatesautophagy.Ina5/6subtotalnephrotomyrat model,Liuetal.revealedthatexosomescouldimproverenal functionandreduce fibroticsizebyregulatingtheSmurf2/Smad7 axis.298 Inaunilateralureteralocclusion-inducedinterstitial fibrosis mousemodel,Luetal.demonstratedthatexosomescould improverenal fibrosisbyreducingthepolarizationofM1andM2 macrophagesbyactivatingEP2receptors.299

Acutekidneyinjury(AKI)andCKDarecloselyconnected,with eachariskfactorfordevelopingtheother.RenalIRIisaleading causeofAKIandacutekidneyfailure.300 Limetal.provedthat exosomesfromiPSC-derivedMSCscouldcorrectserumcreatinine level,tubularnecrosis,apoptosis,inﬂammatorycytokineproduction,andoxidativestressinAKImicebyactivatingtheERK1/ 2signalingpathway.301 InasimilarworkbyZhangetal.,the exosomalmiR-21-5pfromEPCswerefoundtoalleviatesepsisinducedAKIbyinhibitingRUNX1expressioninCLPrats.302

OTORHINOLARYNGOLOGYANDHEAD&NECKSURGERYAND SC-EXOTHERAPY

Hearinglossisthemostcommonsensoryde ﬁ citworldwide, affectingnearly20%oftheglobalpopulation. 303 Thecausesof sensorineuralhearingloss(SNHL)canbeverydiverse,suchas presbycusis,ototoxicmedicati on-induced,noise-induced,and idiopathicsuddenSNHL.Tsai ’ steamdemonstratedthatumbilicalcordMSC-derivedexosomescouldrescuethelossofouter haircellsandrepaircochleardamageincisplatin-induced hearingloss. 304 TheunderlyingmechanismforthecochleaprotectiveeffectsismediatedbythemiRNAs(e.g.,miR-125a-5p andmiR-125b-5p)andremodelingfactors(e.g., ﬁ bronectinand galectin-3).

CochlearIRIisoneofthemainreasonsforidiopathicsudden andnoise-inducedSNHL,whichcanleadtoirreversibledamageof sensoryhaircellsandbipolarcochlearspiralganglion.305 Its pathophysiologyincludesoxidativestress,excesscelldeathand dysregulatedinﬂammation.Haoetal.discoveredthatexosomes derivedfrommiR-21-overexpressingNSCscouldpreventhearing lossfromIRIbyinhibitingtheinﬂammatoryprocessinthemouse cochlea.306 Thiswasevidencedbyareducedauditorybrainstem responsethreshold,upregulatedIL-10anddownregulatedTNF-α andIL-1β.

Hypothyroidismisaverycommondiseasewhichcouldresult fromthyroidectomyorradioactiveablationtotreathyperthyroidismorthyroidcancer.307 Stemcelltherapyandstemcell-derived exosometherapyhaveemergedasapromisingmanagementfor hypothyroidismthroughthyroidregeneration.Usinganinvitro culturesystemofthyroidlobes,Degosserieetal.suggestedthat EPC-derivedexosomescouldfacilitatethyrocyteorganizationinto thyroidfolliclesandlumenexpansion(i.e.,folliculogenesis),which waspromotedbylaminin-α1.308

Temporomandibularjoint(TMJ)disordersarethesecondmost commonmusculoskeletalconditionaffecting31%ofadultsand 11%ofchildren.309 Likeothersynovialjointsinthebody,TMJis alsopronetoOA,sharingcommonpathophysiologicalprocesses (Section3.2).Zhangandco-workersprovedthatMSC-derived exosomescouldalleviateTMJOAinanimmunocompetentrabbit modelbyattenuatinginﬂammationandrestoringmatrixhomeostasis.310 Theexosome-mediatedjointrepairwasattributedto adenosineactivationofAkt,ERKandAMPKsignaling,aswellas enhanceds-GAGsynthesis.

OPHTHALMOLOGYANDSC-EXOTHERAPY

Acquiredopticneuropathyisamajorcauseofblindnessinadults, andhasvariousetiologies,suchasvascular,inﬂammatory, traumatic,toxic,compressive,andnutritionaletiologies.Retinal ganglioncell(RGC)lossisthehallmarkofopticneuropathies,via multiplecelldeathpathways.311 MeadandTomarevfoundthat bonemarrowMSC-derivedexosomescouldpromotesurvivalof RGCs,andregenerationoftheiraxons,inaratopticnervecrush model.312 Theexosomalneuroprotectiveandneuritogeniceffects wereaccomplishedthroughexosomalmiRNAs,demonstratinga cell-freepotentialfortraumaticanddegenerativeoculardiseases. Retinaldegenerativediseases(e.g.,age-relatedmaculardegenerationandretinitispigmentosa)aretheleadingcauseofbilateral irreversiblevisionlossworldwide.313 Itischaracterizedby progressivedegenerationofphotoreceptors,RGCsorretinal pigmentepithelium(RPE)cells.Bian’steamdiscoveredthat exosomesderivedfromNSCscouldpreservephotoreceptors, visualfunctionandpreventthinningoftheouternuclearlayerin anRCSretinaldegenerationratmodel.314 Thiswasachievedby markedinactivationofmicroglialinﬂammationviaexosomal targetingofTNF-α,IL-1β andCOX-2.Intwoconsecutivestudies, Gao’steamshowedthatESC-derivedexosomescouldalleviate retinaldegenerationbyenhancingtheproliferationandretrodifferentiationofretinalMüllercellsasreplacementretinal neuronalprecursors.Ononehand,thiswasaccomplishedby regulatingtheexpressionofOct4inMüllercellsthroughexosomal HSP90.315 Ontheotherhand,activationoftheWntsignaling pathwaybydeliveringBDNFproteintoMüllercellsalsoplayedan importantrole.316 Furthermore,usingaratmodelofinherited retinaldegeneration,Parketal.demonstratedthatbothsubretinal andintravitrealinjectionofhumanHSC-derivedexosomescould providefunctionalrescueofadegeneratingretina.317

Failedhealingofcornealdefectoftenleadstocornealblindness whichhasbeenreportedassecondonlytocataractintheleading causesofblindness.318 Themostsevereandrecalcitrantcases wouldneedcornealtransplantation.Wangetal.compared exosomesderivedfromiPSCsandMSCsastherapeuticproviders forthetreatmentofcornealepithelialdefects.319 Itwasfoundthat bothtypesofexosomescouldpromoteproliferation,cellcycle progressionandmigrationwhileinhibitingapoptosisinvitro,and acceleratecornealepitheliumdefecthealinginvivo.More importantly,theiPSC-derivedexosomeshadastrongertherapeuticeffectthantheMSC-derivedexosomes.

Cornealtransplantationisoneofthemostsuccessfulformsof solidorgantransplantation.However,graftrejectioncanoccurin upto90%ofhigh-riskrecipients.320 Bothinnateandadaptive immunityarethepredominantreasonforgraftfailure.Immunosuppressivedrugshaveshownonlypartialeffectiveness.Jiaetal. showedthatMSC-derivedexosomescouldcrossbiological barriersandprolonggraftsurvivaltimeinaratmodelofcorneal allograftrejection.321 Thiswaslikelycausedbyinhibitionofthe inﬁltrationofCD4+ andCD25+ TcellsandthereductionofIFN-γ andCXCL11viatheTh1signalingpathway.

OBSTETRICSANDGYNECOLOGYANDSC-EXOTHERAPY

Primaryovarianinsufﬁciency(POI),orprematureovarianfailure (POF),isdeﬁnedaslossofovarianfunctionbeforetheageof40. Non-geneticcausesofPOIincludeautoimmunedisorders, metabolicconditions,infections,andiatrogenicprocedures(e.g., chemotherapy,radiotherapy,andsurgery).WomenwithPOIsuffer fromvariouscomplications,suchasosteoporosis,infertility, cardiovasculardisordersanddepression.322 Althoughpromptly initiatinghormonereplacementtherapyiscriticaltocontrolthese symptomsandcomplications,itfailstorestoreovarianfunction. Currently-testedexperimentaltherapiesincludemitochondrial activation,invitroactivation,stemcelltherapy,andexosome therapy.323

TheworkconductedbyLietal.showedthatumbilicalcord MSC-derivedexosomescouldimproveovarianfunctionina cyclophosphamide(CTX)-inducedPOImousemodel.324 TheSCexotherapynotonlyrestoredovarianfunction-relatedhormone levelsandthenumberofovarianfollicles,butalsoimprovedthe reproductiveabilityofPOImice.Inaddition,theexosomes promotedtheproliferationofovariangranulosacells(GCs)by regulatingtheHippopathway,andtheeffectwasneutralizedbya YAPinhibitor.Similarresultswereobtainedusingexosomesfrom iPSC-derivedMSCs.325 TheworkperformedbyDingetal. illustratedthatumbilicalcordMSC-derivedexosomescould restoreovarianphenotypeandfunctioninaPOImousemodel, promoteproliferationofCTX-damagedhumanGCsandoocytes, andalleviateROSaccumulationbydeliveringexosomalmiR-17-5p andtargetingitsdownstreammRNASIRT7.326 Itwasfurther elucidatedthatmiR-17-5pdown-regulatedPARP1, γH2AX,and XRCC6expressionbyinhibitingSIRT7.Lastly,theworkcompleted byYangetal.revealedthatbonemarrowMSC-derivedexosomes couldrecovertheestruscycle,increasethenumberofbasaland sinusfollicles,increaseestradiolE2andanti-Mullerianhormone levels,andreducefolliclestimulatinghormoneandluteinizing hormonelevelsinachemotherapy-inducedPOFratmodel.327 Mechanistically,thiswasachievedbyexosomalmiR-114-5pthat targetsPTEN.

FROMPRECLINICALSTUDIESTOCLINICALTRIALSOF EXOSOMETHERAPY

Manypreclinicalstudies,asdiscussedinSections3to11,have conﬁrmedtheadvantagesofMSC-derivedandNSC-derived exosomestotreatmanydiseasesspanningthesubspecialtiesof surgicalpractice.Withoutrestrictingthescopetostemcellderivedexosomesonly,manyclinicaltrialshavedemonstratedthe roleofexosomestobetwofold:biologicalmarkersand therapeuticagents.AsearchonClinicalTrials.govusing ‘exosome therapy’ , ‘exosometreatment’,and ‘exosome’ askeywords generated188records.However,only60(32%)ofthesedirectly relatetointerventionalstudiesusingexosomesastherapeutic agents(Table 5).Therest,especiallyoncology-relatedtrials,mostly usedexosomesasbiomarkers,suchaskeyplayersduringdisease pathogenesis(e.g.,NCT04288141,NCT04154332),diagnostic markersandguidancebeforetreatment(e.g.,NCT04629079, NCT03791073,NCT05451342,NCT03432806),monitoringindices andpredictivetoolsfortreatmentefﬁciency(e.g.,NCT05427227, NCT04499794,NCT04852653,NCT05370105,NCT03800121, NCT05370105,NCT05328089),andprognosticindicatorsafter treatment(e.g.,NCT06026735,NCT05705583,NCT05411445, NCT04167722,NCT05575622).Theclinicalapplicationsofexosomesasbiomarkershavebeenextensivelyexploredinother reviews328–332 andarethereforebeyonddiscussioninthisreview. Intermsoftheclinicalcharacteristicsofthe60clinicaltrialson exosometherapy(Table 5),thereareseveralhighlightsworth mentioning.

Firstly,thespectrumofdiseasescoveredisverybroad,asboth surgicalandmedicalconditionsareincluded.Theseincludemany surgicaldisordersdiscussedinSections3to11,suchasorthopedic diseases(osteoarthritisofthekneeinNCT05060107,boneloss inNCT04998058,andintervertebraldiscdegenerationin NCT04849429),neurosurgicaldiseases(ischemicstrokein NCT03384433andAlzheimer’sdiseaseinNCT04388982),plastic surgicaldiseases(cutaneouswoundhealinginNCT02565264and NCT05475418),generalsurgicaldiseases(livercirrhosisin NCT05871463),cardiothoracicdiseases(myocardialinfarctionin NCT05669144),andophthalmologydiseases(retinitispigmentosa inNCT05413148).Inotherwords,somepreclinicalstudieshave notyetdevelopedintoclinicaltrials.Theseinclude,butarenot limitedto,exosometherapyforfracture333 spinalcordinjury334 traumaticbraininjury335 acuteliverinjury336 andhearing

loss304,306 whichmightserveasfuturedirectionsforexosome therapy-relatedclinicaltrials.

Secondly,anincreasingnumberofclinicaltrialsfocusedontwo medicalconditions,i.e.,COVID-19(16trials,27%)andcancer(5 trials,8%).MSC-derivedexosomescanmanageviralinfectionand lungdamageinCOVID-19throughbothreparativeactionsand regenerativeeffects.337 Theformermanifestsasblockageofviral entryandreplication,andsuppressionofthecytokinestorm, whereasthelatteraspreventionofinflammation,and fluidclearanceandrestorationoflungpermeability.Inaddition, exosomescanbeengineeredintoadrug(e.g.,CD24,apotent immuneregulator)deliveryplatform338 andevenavaccine339 to combatCOVID-19.IncontrasttotheMSC-derivedexosomesfor COVID-19management,exosomesusedforcancertreatment mainlyrelyonnon-stemcellsandcargoengineering.Thisis partiallybecauseMSC-derivedexosomesdemonstratecontroversialeffectsontumorigenesisandmetastasis.340,341 Althoughthe exactinteractionbetweenMSC-derivedexosomesandtumorcells remainsopentodebate,scientistshaveovercomeseveral obstaclesbymodifyingexosomalcargostodeliverantioncogenic nucleicacidsandanticancermedications,andexosomalmembranesforspecifictumortargeting.342–346

Finally,amongthe40clinicaltrialsusingstemcell-derived exosomesfordiseasetreatment,38(95%)usedMSCand2(5%) usediPSCasthecellularsourceforexosomes.However,thisdiffers significantlyfromthepreclinicalstudiesdiscussedinSections3to 11.ThelackofuseofNSC-derivedexosomesinclinicaltrialsmight bepartiallybecauseofthesupplyconstraintsoftheirparental cells.347 Currently,NSCscanbeobtainedfromthreesources348 (Fig. 1a):1,isolationfromprimaryCNStissues(e.g.,adultandfetal brain);2,differentiationfrompluripotentstemcells(e.g.,iPSCsand ESCs);3,reprogrammingofsomaticcells(e.g., fibroblastsand bloodcells)toiNSCs.349 Recentstudieshavedeveloped fibroblastderivediNSCs,openinganewwindowforobtainingexosomes fromNSC-likecells.TheseiNSC-exocouldnotonlypromotecell survivalandproliferationnolessthanNSC-exoinvitro350,351 but alsoenhancerecoveryafterischemicstroke148 andmitigateADlikephenotypesinpreclinicalmodels.190 Therefore,iNSCsmight beanexcellentcellularsourcetoproduceclinical-gradeexosomes inclinicaltrials.

Insummary,theprogressionfrompreclinicalstudiestoclinical trialsofexosometherapyhasbeenexpeditious.However,issues likeinsufﬁcientclinicalindicationsforexosometreatmentand limitedsourcesforparentalstemcellsremaintobeaddressed.In addition,oncethelimitations(Table 1)inupscalingofmanufacturing,compliancewithgoodmanufacturingpractice,and regulatoryframeworkareovercome329 stemcell-derivedexosome therapywillsoonbeincorporatedintoclinicalpracticeandserve atthepatient’sbedside.

CONCLUSIONANDFUTUREPERSPECTIVES

Exosomeshavebeenpursuedrecentlyasacell-freealternativeto stemcell-basedtherapy.ESC-,iPSC-,HSC-,MSC-,NSC-andEPCderivedexosomesareofparticularinterest,partiallyduetothe pluripotencyormultipotencyoftheirparentalcells.Aftergoing throughproductionandpurificationwithorwithoutmodification, stemcell-derivedexosomeshavedemonstratedtremendous potentialintreatingnumerousdiseasesencounteredduring surgicalpractice.Theseareexemplifiedbydisordersinorthopedic surgery(e.g.,fracture,osteoarthritis,andspinalcordinjury); neurosurgery(e.g.,ischemicstroke,traumaticbraininjury,and Alzheimer’sdisease);plasticsurgery(e.g.,woundhealing);general surgery(e.g.,acuteliverinjury);cardiothoracicsurgery(e.g., myocardialinfarction);urology(e.g.,chronickidneydisease);head andnecksurgery(e.g.,sensorineuralhearingloss);ophthalmology (e.g.,acquiredopticneuropathies),andgynecology(e.g.,primary ovarianinsufficiency).Mechanistically,thediversetherapeutic

Table5. Clinicaltrialsofexosometherapy

CategoryofconditionsSpeciﬁcdiseaseNCTnumberSourceofexosomeGenderAgePhasesEnrollmentCountry

Behavior&mental disorders depression&anxietyNCT04202770MSCall>18N/A300USA

Blood&lymphconditionscoagulopathyNCT02594345redbloodcellall18–80N/A18Germany

Digestivesystemdiseasesperianal ﬁstulaNCT05499156MSCall18

701/280Iran

NCT05402748MSCall18–701/280Iran livercirrhosisNCT05871463MSCall18

75215Iran

IBSNCT04879810MSCall>18N/A4USA

DiseasesatorbeforebirthELBWbirthNCT05490173MSCall1

EyediseasesdryeyediseaseNCT05738629MSCall18

NCT04213248MSCall18

3daysN/A10Russia

701/212China

701/227China macularholesNCT03437759MSCall<80early144China retinitispigmentosaNCT05413148MSCall18

Gland&hormone-related diseases T1DMNCT02138331MSCall18

Heart&blooddiseasesMINCT05669144MSC,mitochondriaall35

702/3135Turkey

602/320Egypt

801/220Iran aorticdissectionNCT04356300MSCall20

Mouth&toothdiseasesperiodontitisNCT04270006MSCall18

80N/A60China

50early110Egypt oralmucositisNCT01668849plant,grapeall20

MusculoskeletaldiseasesmeniscalinjuryNCT05261360MSCall30

OA,kneeNCT05060107MSCall30

85160USA

50230Turkey

70110Chile degenerativediscdiseaseNCT04849429PRPall18

60130India bonelossNCT04998058MSCall>351/220Brazil

NeoplasmsmetastaticpancreascancerNCT03608631MSC + KRASG12D siRNA all>18128USA

coloncancerNCT01294072plant,curcuminall>20135USA

NSCLCNCT01159288tumorantigenloadedDC all18–70241France

bladdercancerNCT05559177chimericexosomal tumorvaccines all18–85early19China

HCCNCT05375604CDK-004all>1819USA

NervoussystemdiseasesfocalepilepsyNCT05886205iPSCall18–70early134China craniofacialneuralgiaNCT04202783N/Aall>18N/A100USA ischemicstrokeNCT03384433MSCall40–801/25Iran

ADNCT04388982MSCall>501/29China

Nutritional&metabolic diseases familial hypercholesterolemia

NCT05043181LDLRmRNAdeliveryall18–45130China

RespiratorytractdiseasesCOVID-19NCT04276987MSCall18–75124China

NCT05787288MSCall18–75early1240China

NCT05808400MSCall18–80early180China

NCT05216562MSCall18–752/360Indonesia

NCT04602442MSCall18

NCT04491240MSCall18

NCT04493242MSCall18

65290Russia

651/230Russia

852102USA

NCT04798716MSCall>181/255USA

NCT05387278MSCall18–75120USA

NCT04389385COVID-19speciﬁcT cell all18–75160Turkey

NCT04747574EXO-CD24all18–85135Israel

NCT04969172EXO-CD24all18

NCT04902183CovenD24all18

802155Israel

80290Greece

NCT04384445Zoﬁnall>181/220USA

NCT04657406Zoﬁnall>18N/AN/AUSA

NCT05228899Zoﬁnall>181/230USA

Table5. continued

CategoryofconditionsSpeciﬁcdiseaseNCTnumberSourceofexosomeGenderAgePhasesEnrollmentCountry

ARDSNCT04602104MSCall18–701/2169China NCT05354141MSCall18–653970USA NCT05947747EXO-CD24all>18290Israel COPDNCT05643729Zoﬁnall40–801/220USA pulmonaryinfection,drugresistant NCT04544215MPCall18–751/260China

Skin&connectivetissue diseases

atopicdermatitisNCT05969717iPSCall18–70early120China psoriasisNCT05523011MSCall>21110Singapore dystrophicepidermolysis bullosa NCT04173650MSCall>61/210USA

skinagingNCT05813379MSCfemale35–651/220Iran chroniculcerNCT04134676MSC-CMall18–80138Indonesia cutaneouswoundNCT02565264plasmaallallearly15Japan

NCT05475418MSCall18–60N/A5China alopeciaNCT05658094MSCall25–65N/A20Iran

Urinarytract&sexual organsconditions PCOSNCT03493984plant,ginger&aloefemale18–40N/AN/AUSA

DataobtainedfromClinicalTrials.govusing ‘exosometherapy ’ , ‘exosometreatment ’,and ‘exosome’ askeywords,asof2023-09-08.Thecategorizationof diseasesfollowsthesystembyClinicalTrials.gov AD Alzheimer ’sdisease, ARDS acuterespiratorydistresssyndrome, CM conditionedmedium, COPD chronicobstructivepulmonarydisease, DC dendriticcell, DM diabetesmellitus, ELBW extremelylowbirthweight, HCC hepatocellularcarcinoma, IBS irritablebowelsyndrome, iPSC inducedpluripotentstemcell, LDLR low-densitylipoproteinreceptor, MI myocardialinfarction, MPC mesenchymalprogenitorcell, MSC mesenchymalstemcell, NSCLC non-smallcelllungcancer, OA osteoarthritis, PCOS polycysticovarysyndrome, PRP platelet-richplasma, siRNA smallinterferingRNA

effectsofstemcell-derivedexosomesareachievedthrough disease-specificcellularandtissueresponses(e.g.,tissueregeneration,anti-inflammation,anti-celldeath,immunomodulation, andanti-oxidativestress)andtissue-specificmolecularsignaling pathways(e.g.,Wnt/β-catenin,PTEN/PI3K/Akt/HIF-1α,MAPK,and JAK/STATpathways).Collectively,stemcell-derivedexosome therapyhasbeenproventobeapotentandversatilesurrogate tostemcelltherapyinthesurgicalarena.

Futureemphasisofclinicalapplicationsofstemcell-derived exosomesshouldbeplacedonvariousnodesofthistherapeutic pipeline.Firstly,targetingthepretherapeuticlarge-scaleproduction ofexosomes,ahigh-throughputcellularsourceaswellasa reproducibleandscalableproductionandisolationprotocolare required.Comparedtothestaticsystemgrowingmonolayercells, dynamicsystemintheformofbioreactor,e.g.,hollow-fiber bioreactorandstirredtankbioreactor,canincreasetheefficiency byproducingcopiouscellsandexosomesinashortperiodoftime. However,thephenotypeofparentalcellsandderivedexosomes mightchangeduetophysicalandshearstressencounteredina reactor.Thus,theworkingparametersofthebioreactormustbe optimizedtofacilitatelarge-scaleproductionofstemcell-derived exosomes.Secondly,targetingthetherapeuticmodalityofexosomes,deliverymethodsotherthansystemicadministrationneedto beexplored.Whendeliveredthroughthevenoussystem,exosomes arerapidlyclearedfrombloodcirculationandaccumulateinthe liver,spleenandlungs,whichcanbeovercomebylocaldelivery. Variousbiomaterialshavebeenrecentlyusedtoprotect,assistand augmentlocallydeliveredexosomestomaximizetheirtherapeutic effects.Thesebiomaterialscouldbedesignedaccordingtotheir sources(e.g.,natural,synthetic,andhybridpolymers),format(e.g., scaffold,patch,spray,andmicroneedle),andresponsiveness(e.g., temperature,pH,andprotein),therebyallowingdisease-specific customization.Lastly,targetingthetherapeuticindicationsof exosometherapy,morediseasesthantheonesdiscussedinthis reviewshouldbeincludedintofuturepreclinicalstudiesandclinical trials.Forexample,airwayinflammatoryconditions(e.g.,allergic rhinitisandasthma)couldbesuitablecandidatesforexosome

therapy,consideringtheimmunomodulatoryeffectofMSC-derived exosomes.Disordersthatarebesttreatedusingsurgicalimplants (e.g.,cochlearimplant,intraocularlenses,andcontraceptive intrauterinedevices)couldbemanagedintheformofimplantbasedlocalreleaseofexosomes.Inadditiontoprimarydiseases, secondaryconditionsincludingsurgicaloperation-andgeneral anesthesia-relatedcomplications(e.g.,cognitiveimpairment,wound paresthesia,andmalignanthyperthermia)mightbecometherapeutictargetsofexosometherapy.Collectively,effortstoupscale exosomeproductioninconjunctionwithmultimodalexosome deliverywillacceleratetheclinicalapplicationsofstemcell-derived exosomesinarapidlyexpandingdiseasespectrum.

ACKNOWLEDGEMENTS

ThisworkissponsoredbytheFundamentalResearchFundsfortheCentral Universities.ItwasalsosupportedbytheNationalNaturalScienceFoundationof China(No.82271192).

AUTHORCONTRIBUTIONS

F.T.:conceptualization,formalanalysis,investigation,resources,writing,visualization, fundingacquisition;X.L.:software,methodology;Z.W.:datacuration;J.L.:validation; K.S.:resources;J.Z.:projectadministration,supervision.Allauthorshaveread, discussedandapprovedthe ﬁnalversionofthisarticle.

ADDITIONALINFORMATION

Competinginterests: Theauthorsdeclarenocompetinginterests.

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s41392-023-01704-0

Signal Transduction and Targeted Therapy

Clinicalapplicationsofstemcell-derivedexosomes

INTRODUCTION

GENERALBACKGROUNDOFEXOSOMESANDEXOSOME THERAPY

Storageofexosomes

ORTHOPEDICANDTRAUMASURGERYANDSC-EXOTHERAPY Fracture

Osteoarthritis

Spinalcordinjury

Otherorthopedicdiseases

NEUROSURGERYANDSC-EXOTHERAPY

Traumaticbraininjury

Multiplesclerosis

GENERALSURGERYANDSC-EXOTHERAPY

CARDIOTHORACICSURGERYANDSC-EXOTHERAPY

UROLOGYANDSC-EXOTHERAPY

OTORHINOLARYNGOLOGYANDHEAD&NECKSURGERYAND SC-EXOTHERAPY

OPHTHALMOLOGYANDSC-EXOTHERAPY

OBSTETRICSANDGYNECOLOGYANDSC-EXOTHERAPY

FROMPRECLINICALSTUDIESTOCLINICALTRIALSOF EXOSOMETHERAPY

CONCLUSIONANDFUTUREPERSPECTIVES

ACKNOWLEDGEMENTS

AUTHORCONTRIBUTIONS

ADDITIONALINFORMATION

REFERENCES