s41419-022-05034-x by Biogenomics

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Mesenchymalstemcell-derivedextracellularvesiclesfor immunomodulationandregeneration:anextgeneration therapeutictool?

MengKou1,10,LiHuang1,10,JinjuanYang1,ZhixinChiang2,ShaoxiangChen1,JieLiu3,4,LiyanGuo1,XiaoxianZhang1,XiaoyaZhou1, XiangXu 5,XiaomeiYan6,YanWang7,JinqiuZhang1,AiminXu 3,4,Hung-fatTse4,8 andQizhouLian 1,3,4,8,9 ✉

©TheAuthor(s)2022

Mesenchymalstemcells(MSCs)canbewidelyisolatedfromvarioustissuesincludingbonemarrow,umbilicalcord,andadipose tissue,withthepotentialforself-renewalandmultipotentdifferentiation.Thereiscompellingevidencethatthetherapeuticeffect ofMSCsmainlydependsontheirparacrineaction.Extracellularvesicles(EVs)arefundamentalparacrineeffectorsofMSCsandplay acrucialroleinintercellularcommunication,existinginvariousbody ﬂuidsandcellsupernatants.SinceMSC-derivedEVsretainthe functionofprotocellsandhavelowerimmunogenicity,theyhaveawiderangeofprospectivetherapeuticapplicationswith advantagesovercelltherapy.WedescribesomecharacteristicsofMSC-EVs,anddiscusstheirroleinimmuneregulationand regeneration,withemphasisonthemolecularmechanismandapplicationofMSC-EVsinthetreatmentof ﬁbrosisandsupport tissuerepair.WealsohighlightcurrentchallengesintheclinicalapplicationofMSC-EVsandpotentialwaystoovercomethe problemofqualityheterogeneity.

CellDeathandDisease (2022)13:580;https://doi.org/10.1038/s41419-022-05034-x

FACTS

● MSC-derivedEVshavelow-immunogenicityandstrong potentialfortherapeuticapplications.

● MSC-derivedEVswereusedtotreattissue ﬁbrosisand promotetissueregeneration.

● MSC-derivedEVsareproposedasanoveltherapeuticagentto mediateimmunomodulationandpromoteregeneration.

OPENQUESTIONS

● HowcanMSC-derivedEVsmediateimmunomodulationand regeneration?

● HowcanMSC-derivedEVsbeusedtoaidregenerationof ﬁbrotictissue?

● HowcanmassmanufacturingofMSC-derivedEVsbeachieved andtheproblemofqualityheterogeneityovercome?

● WhatarethechallengesofMSC-derivedEV-basedimmunomodulationandregenerationinclinicalpractice?

INTRODUCTION

Mesenchymalstemcells(MSCs)existinvarioustissuessuchas bonemarrow(BMSCs),umbilicalcordblood(UC-MSCs)and umbilicalcordtissue,placentaltissue(hPMSCs),adiposetissue (ADSCs),andmenstrualblood(MenSCs).Thesecellshavemultidirectionaldifferentiationpotential[1]tobecomeosteoblasts, chondrocytesoradipocytesinvitro[2],andhaveauniquefunction ofcytokinesecretion[3].Cellmodelshavebeenappliedin proliferation,transplantation,anddifferentiationstudies,andin identiﬁcationofimmuneresponsesinvitro[4].Numerousstudies haveshownthatMSCshavegreatpotentialinimmuneregulation andregeneration[5].TheU.S.FDAhasapprovednearly60clinical trials[6],mainlyfocusedonHematopoieticStemCellTransplantation(HSCT)[7],tissuehealing,AutoimmuneDisease(AID),and genetictherapyvectors[8].Recently,MSCshavebeenwidelyused inclinicalstudiesasaregenerativeagentandtotreatavarietyof conditionsincludingosteoarthritis[9],pulmonary ﬁbrosis,spinal cordinjury,myocardialdamage,kneecartilageinjury,dentalpulp regeneration,andorgantransplantation[10].Anincreasingnumber

1CordBloodBankCentre,GuangzhouWomenandChildren’sMedicalCentre,GuangzhouMedicalUniversity,Guangzhou,China. 2DepartmentofAlliedHealthSciencesFacultyof Science,TunkuAbdulRahmanUniversity,Ipoh,Malaysia. 3StateKeyLaboratoryofPharmaceuticalBiotechnology,theUniversityofHongKong,HongKongSAR,China. 4DepartmentofMedicine,theUniversityofHongKong,HongKongSAR,China. 5DepartmentofStemCell&RegenerativeMedicine,StateKeyLaboratoryofTrauma,Burnand CombinedInjury,DapingHospital,ArmyMedicalUniversity,Chongqing400042,China. 6DepartmentofChemicalBiology,TheMOEKeyLaboratoryofSpectrochemicalAnalysis& Instrumentation,CollegeofChemistryandChemicalEngineering,XiamenUniversity,Xiamen,Fujian361005,China. 7XiamenCardiovascularHospitalofXiamenUniversity,School ofMedicine,XiamenUniversity,Xiamen,China. 8HKUMedLaboratoryofCellularTherapeutics,theUniversityofHongKong,HongKongSAR,China. 9DepartmentofSurgery, ShenzhenHongKongUniversityHospital,Shenzhen518053,China. 10Theseauthorscontributedequally:MengKou,LiHuang. ✉email:qzlian@hku.hk EditedbyDrYufangShi

Received:14February2022Revised:8June2022Accepted:22June2022

ofstudieshasrevealedthatthepowerfultherapeuticeffectsof MSCsareduetoparacrine-likesecretionofcytokines(growth factorsandchemokines)[11, 12]andextracellularvesicles(EVs)as wellastheirinvolvementincellularcommunication[13–16]. ApplicationofMSCsascelltherapyisbasedonregulatingthe inflammatoryresponseandparticipatingintissuerepairand regeneration[17].ThetherapeuticeffectofMSCsismainly attributedtotheirimmunomodulatoryfunctionregulatedbythe inflammatoryenvironment[18].Whenstimulatedbyinflammatory factors,MSCsproducealargenumberofimmunomodulatory factors,cellchemokines,andgrowthfactors,therebyregulating thetissueimmunemicroenvironmentandpromotingtissue regeneration[19].ThereisaccumulatingevidencethatEVs derivedfromMSCspreservethetherapeuticactionoftheparent MSCsandtheiruseavoidsthesafetyconcernsassociatedwithlive celltherapy[20, 21].Therefore,useofMSC-EVstoreplaceMSCsas cell-freetherapymaybethefocusoffutureclinicaltreatments [20].WereviewrecentstudiesoftheroleofMSC-EVsin immunomodulationandregeneration,focusingontheirmolecular mechanismsinthetreatmentofosteoarthritis,spinalcordinjury, skininjury,andliver,kidney,andlung fibrosis.

EXTRACELLULARVESICLESCOMPOSITION

Extracellularvesicles(EVs)existinbody ﬂuids,arereleasedbycells, andhaveamembranestructure[22].Theycanbedividedintofour subgroupsaccordingtotheirdiameter:exosomes(30–150nm), microvesicles(100–1000nm),apoptoticbodies(50–5000nm,generatedduringcellapoptosis)[23, 24],andoncosomes(1–10 μm),newly discoveredandobservedincancercells[25].EVsencapsulatemany bioactivemolecules(proteins,lipids,nucleicacids,andorganelles) [26–28]thatcanbedeliveredtotargetcells.Largeamountsofdata suggestthatexosomesandmicrovesiclesarevitalmediatorsofEVs innumerousphysiological(pathological)processes[29](Fig. 1).

Exosomes

Exosomesaremicroscopicvesicleswithadensityof1.11–1.19g/mL. Theyhaveatypical “disk-like” structureand flatsphericalshapewhen seenunderanelectronmicroscope[24].Manykindsofcellsinvarious body fluidsandcellsupernatantscansecreteexosomesunder normalandpathologicalconditions.Exosomeswere firstdiscovered in1983insheepreticulocytesandwerenamed “Exosomes” by Johnstonein1987[30].Thesetinyvesiclescontainspecificproteins, lipids,andnucleicacidsthatcanbetransmittedandserveas signalingmoleculestoalterthefunctionofothercells[31, 32].

Duringtheformationofexosomes,theextracellularcomponentsandcellmembraneproteinsarewrappedbythe invaginatedplasmamembranetoformearlyendosomes.These canexchangematerialswithintracellularorganellesanddevelop intolateendosomes,eventuallyformingintracellularmultivesicularbodies(MVBs)[33, 34].MVBscontainmanyintraluminal vesicles(ILVs)[35].Theymaybedegradedandreleasedintothe cytoplasmbyfusionwithautophagosomesorlysosomes,or releasedintoextracellularvesiclesbyfusionwithplasma membrane,includingILVs,resultinginexosomeformation[34]. Exosome-mediatedintercellularcommunicationisachievedby directmembranefusion,receptor-mediatedendocytosis,phagocytosis,caveolae,andmicropinocytosis[36–38].

Proteinsinvolvedinexosomebiogenesis(suchastransportand fusion)includeRabGTPases[39–41],ESCRT(endosomalsorting complexrequiredfortransport)[42],annexin,lipidraftproteins,and fourtransmembraneproteins(CD63,CD81,andCD9)[43, 44].In addition,theyalsocontainbiosyntheticantibodies(AlixandTSG101) involvedinMVBs[45, 46],cholesterol,ceramide,phosphoglyceride thatprovidesstructuralstability,andimmune-relatedmoleculeMHC-II thatisinvolvedinantigenbindingandpresentation.Exosomesalso carryfunctionalmRNAsandmiRNAsthatcanbetransferredbetween cells[47].Exosomesreleasedbytumors containsingle-strandedDNA,

genomicDNA,cDNA,andatransposableelement[48, 49].Itisclear thatexosomeshavemanyfunctionsasbiomarkersofdisease.

Microvesicles

Microvesiclesarealsoknownasmicroparticles.BiogenesisofMVs differstothatofexosomessincetheyarereleasedfromoutward buddingand ﬁssionofplasmamembranewhenthecellis stimulatedorapoptotic[50].Nonetheless,theysharecharacteristicsofhighbiocompatibility,andlowimmunogenicityand targetingandcanbeusedasdrugcarriers[51].Studieshave shownthattheuseoftumorcell-derivedMVstodeliver chemotherapydrugsproducesinbettercancertreatmentresults withfewsideeffectsoradversereactions[52, 53].

MSC-DERIVEDEXTRACELLULARVESICLES

AlthoughMSCsderivefromavarietyofsources,theycanallbe adherentincultureanddifferentiatedintoavarietyofcelltypes withspecificsurfacemarkers[54].Withtheneedforclinical treatmentwithMSCs,theMesenchymalandTissueStemCell CommitteeoftheInternationalSocietyforCellularTherapy(ISCT) hasproposedminimumcriteriaforidentificationofhumanMSCs: (1)Culturedunderstandardconditionstheymustadhereto plasticsubstrates;(2)On flowcytometry,thepositiverateof CD105,CD73andCD90expressioninMSCsurfacemarkersshould reach95%,andnegativeexpressionrateCD45,CD34,CD14or CD11b,CD79aorCD19orHLA-DR(humanleukocyteantigen-DR) (≤2%positive);(3)Afterinductionbystandardmethodsinvitro, MSCsmustbeabletoinducedifferentiationintoosteoblasts, chondrocytesandadipocytes[55].Nonetheless,furtherresearch hasrevealedthatthesestandardsdonotfullydefineMSCs[56]. ThereisaccumulatingevidencethatheterogeneousMSCshave multiplecellsubpopulationswithcharacteristicsurfacemarkers [57, 58],butthedefinitionofsurfacemarkersandbiological functionsofthesesubpopulationsrequiresongoingexploration.

MSCsareeasytoresuscitateandproliferateinvitro,enabling themtobemass-producedforclinicalapplication[18].Inrecent years,theyhavebeenthemoststudiedstemcelltypeforclinical application,andhaveplayedaneffectivetherapeuticroleingraftversus-hostdisease(GVHD)[7],kidneyinjury[59],tissueandorgan transplantation,immunetolerance[60],nerveinjury,rheumatic disease,andliverdisease.Atpresent,MSCshaveattractedmuch attentioninthecontextoftheCOVID-19pandemic[61].Lengetal. demonstratedthatinanMSCtreatmentgroup,patientswith COVID-19infectionwerecuredortheirconditionsigniﬁcantly improvedasaresultofregulationofincreasedinterleukin10(IL 10)expression,inhibitionofoveractivatedimmuneTcellsandNK cells,andasigniﬁcantlyreducedTNF-α level[62].

Despitetheiradvantages,thereareaspectsofMSCtherapythat warrantconsideration.First,theproliferationabilityofMSCsis graduallyweakenedandaccompaniedbyacertaindegreeof differentiationandevenagingwithincreasingpassagesduring invitroculture.Thisimpactstheirregulatoryandtherapeutic ability[56, 63].Second,intheinvivoenvironment,heredityfactors andtheself-renewalabilityofMSCscannotbecontrolledwith consequentpotentialfortumorigenicity[64].Inaddition,although MSCshaveastrongregenerativeregulatorypotential,itis uncertainwhethertheycantargetorremainatthedamaged sitefollowingintravenousinjection[65].Thereissomeevidence thatonlyasmallnumberofMSCsreachthetargetsiteduetothe hostbody’sscavengingcapacity[66, 67].Althoughin-situinjection canpartiallysolvetheseproblems,thereremainproblemswith celldifferentiationandaging,andtheclinicaleffectsarenot optimistic[68].MSCshavealsobeenfoundtocauseandpromote thegrowthofvarioustypesofcancer[69].Inaddition,thereare theusualassociatedrisksofcelltherapysuchasviralinfectionand immunerejectionaswellasproblemswithstorageand transportation[70].

Fig.1 Thedevelopmentandmaintypesofextracellularvesicles.A Exosomesarederivedfromtheendosomalpathway. B Compositionof exosomes.

ThediscoverythatmosttherapeuticeffectsofMSCsdependon theirparacrineactionandthatEVscanreplacetheirparentcells offersexcitingprospectsforresearchers[21].EVsoffergreat advantages[71]:theyarenotself-replicatingandlargelyavoidthe riskoftumorigenicity[72];comparedwithcelltherapy,EVsare safer;asnanoparticlestheyhavebothbiocompatibilityandlow immunogenicity,enablingthemtocross-protectivebarrierssuch astheblood-brainbarrier[73];theycanbecontinuouslysecreted byimmortalizedcellstoobtainasufﬁcientnumber[74];EVs protecttheirinternalbiomolecularactivityviatheirlipidmembranestructure,canbepreservedforaprolongedperiodat-80°C, andarenotsubjecttodeactivation,evenafterrepeatedfreezing andthawing[75, 76];andtheyhaveanencapsulationcapability, canloadspeciﬁcdrugsandtransportthemtotargetcells[77].

Notably,MSC-EVsexpressEVsurfacemarkersCD63,CD9and CD81,aswellasmesenchymalstemcellsurfacemarkersCD44, CD73,andCD90[78].Inaddition,proteinscontainedinthe extracellularvesiclessecretedbyMSCsareaspeciﬁcprotein subclassthatdeterminestheiruniquebiologicalfunctions[36].At thesametime,theencapsulatedmRNAandmiRNAinMSC-EVs formthemolecularbasisfortheirfunction[79].Accordingly,MSCEVstransmitinformationandcommunicatewithtargetcells throughinternalsubstances,thuschangingtheactivityand functionoftargetcells[80].

Withtheiruniqueadvantages,MSC-EVsplayanimportantrole inimmuneregulationandregeneration.Studiesofthepromotion ofregenerationthroughimmuneregulationaredescribedin

detailbelow.Meanwhile,inthetreatmentofautoimmune diseases,Wuetal.foundthatBM-MSC-derivedEVstargeted inhibitionofthecyclinI-activatedATM/ATR/p53signalingpathwaybyupregulationofmiR-34a,therebyinhibitingRA fibroblastlikesynoviocytes(RA-FLSs)andsignificantlyamelioratingRA inflammationinvivo[81].Anotherstudyontheregulationof type-Iautoimmunediabetesmellitus(T1DM)showedthatADMSC-derivedexosomesamelioratedT1DMsymptomsbyupregulatingtheexpressionofregulatoryTcells,interleukin4(IL4),IL10 andtransforminggrowthfactor-beta(TGF-β)anddown-regulating IL17andinterferon-gamma(IFN-γ)[82].Additionalstudiesof autoimmunediseaseregulationhavebeensummarizedelsewhere [83].RecentlyMSC-EVshavealsobeenappliedinclinicalpractice. Nassaretal.areintheprocessofevaluatingtheeffectofhuman UC-MSC-derivedEVsonislet β cellsinpatientswithT1DM(trial NCT02138331).Recentclinicaltrialshavebeenconductedto evaluatethesafetyandefficacyofMSC-EVsinpatientswitha varietyofdiseasesbasedontheirpotentialforimmuneregulation andregeneration(Table 1).

APPLICATIONOFMSC-EVSINIMMUNEREGULATIONAND REGENERATION

ThetherapeuticpotentialofMSC-EVshasbeenreportedin immuneregulationandtissueregenerationbasedonEVmediatedcellularcommunicationbetweenMSCsandseveral targetcells,includingmacrophages,microglia,chondrocytes,

Table1. SummaryofregisteredclinicaltrialsbasedonMSC-EVswithpotentialforimmuneregulationandregeneration.

RegisterNo.TitlePhaseConditionInterventionURL

https://ClinicalTrials.gov/show/ NCT05127122

https://ClinicalTrials.gov/show/ NCT04493242

https://ClinicalTrials.gov/show/ NCT05078385

https://ClinicalTrials.gov/show/ NCT05130983

https://ClinicalTrials.gov/show/ NCT04657458

https://ClinicalTrials.gov/show/ NCT05125562

https://ClinicalTrials.gov/show/ NCT04327635

https://ClinicalTrials.gov/show/ NCT05116761

https://ClinicalTrials.gov/show/ NCT05176366

https://ClinicalTrials.gov/show/ NCT04173650

https://ClinicalTrials.gov/show/ NCT05215288

https://ClinicalTrials.gov/show/ NCT04223622

https://ClinicalTrials.gov/show/ NCT04270006

I/IIARDSBMSC-EVs; IV

NCT05127122BoneMarrowMesenchymalStemCell-DerivedExtracellularVesicles InfusionTreatmentforARDS

IICOVID-19ARDSBMSC-EVs; IV

IBurnwoundsBMSC-EVs;apply towound

ICrohn ’ sDiseaseBMSC-EVs; IV

BMSC-EVs; IV

CriticallyillCOVID- 19ARDS

open- label

BMSC-EVs; IV

NCT04493242ExtracellularVesicleInfusionTreatmentforCOVID-19 AssociatedARDS

NCT05078385SafetyofMesenchymalStemCellExtracellularVesicles(BMSC-EVs) fortheTreatmentofBurnWounds

NCT05130983APhaseIStudyofExoFlo,anexVivoCulture-expandedAdult AllogeneicBoneMarrowMesenchymalStemCell-Derived ExtracellularVesicleIsolateProduct,fortheTreatmentofMedically RefractoryCrohn ’ sDisease

NCT04657458ExpandedAccessProtocolonBoneMarrowMesenchymalStemCell- DerivedExtracellularVesicleInfusionTreatmentforPatientsWith COVID-19AssociatedARDS

IIMild-to-Moderate COVID-19

NCT05125562BoneMarrowMesenchymalStemCell-DerivedExtracellularVesicles InfusionTreatmentforMild-to-ModerateCOVID-19:APhaseII ClinicalTrial

IAMIEVs; Intracoronaryinfusion

NCT04327635SafetyEvaluationofIntracoronaryInfusionofExtracellularVesiclesin PatientsWithAMI

I/IICOVID-19BMSC-EVs; IV

NCT05116761ExoFlo ™ InfusionforPost-AcuteCOVID-19andChronicPost-COVID- 19Syndrome

IUlcerativeColitisBMSC-EVs; IV

NCT05176366StudyofExoFlofortheTreatmentofMedicallyRefractoryUlcerative Colitis

NCT04173650MSCEVsinDystrophicEpidermolysisBullosaI/IIDEBBMSC-EVs;apply towound

ISolidOrganTransplant Rejection BMSC-EVs; IV

NCT05215288IntermediateSizeExpandedAccessfortheUseofExoFlointhe TreatmentofAbdominalSolidOrganTransplantPatientsWhoAreat RiskofWorseningAllograftFunctionWithConventional ImmunosuppressiveTherapyAlone

NCT04223622EffectsofASCSecretomeonHumanOsteochondralExplantsopen- label OAASCsecretome; * IV

IPeriodontitisASC-EVs

NCT04270006EvaluationofAdipose-DerivedStemCellsExo.inTreatmentof Periodontitis

AMI acutemyocardialinfarction, ARDS acuterespiratorydistresssyndrome, ASC adipose-derivedstemcell, BMSC bonemesenchymalstemcell, COVID-19 coronavirusdisease2019, DEB dystrophicepidermolysis bullosa, EVs extracellularvesicles, IV intravenousadministration, OA osteoarthritis. * ASCsecretome,eithercompleteconditionedmediumorEVs.

articularchondrocytes,endothelialcells, fibroblasts,pericytes, neuralstemcells(NSC),neurons,hepaticstellatecells,and podocytes.Inthispaper,wediscussthemolecularmechanisms ofMSC-EVsintissuerepairandanti-fibrosis,inwhichseveral clustersofmiRNAandtheirdownstreampathwayshavebeen revealedtoplayimportantrolesinosteoarthritis,spinalcord injury,skininjury,liver fibrosis,kidney fibrosis,andlung fibrosis (Tables 2–7).

Supporttissuerepair

Osteoarthritis.Osteoarthritis(OA)istheprincipalformofjoint diseasewithunclearpathogenesis,presentingwithpainand stiffness,andinsomecases,disability[84].Recently,MSC-EVshave beenproventohavebothregenerativeandimmunoregulatory beneﬁtsinOA(Table 2).

SeveralstudieshavereportedthathBMSC-EVsplayasignificant roleinthetreatmentofOAbyinhibitingsomepro-inflammatory pathwaysandfactors,andenhancingtheproliferationand migrationofchondrocytes.Vonketal.determinedthatMSC-EVs blockedNFκBsignalingbyinhibitingphosphorylationofIκBα, therebydown-regulatingTNF-α-inducedCOX2expression,and interleukinsandcollagenaseactivity.Additionally,MSC-EVsupregulatedtheexpressionofSOX9andWNT7A,andpromotedthe productionofproteoglycanandtypeIIcollagenininvitrostudies [85].Lietal.concludedthathBMSC-EVspromotedOA-chondrocyte (OA-CH)proliferationandmigrationandreducedapoptosisvia downregulationofMMP13,ALPL,IL-1β-activatedpro-inflammatory Erk1/2,PI3K/Akt,p38,TAK1,andNF-κBsignalingpathwaysand increasedgeneexpressionofPRG4,BCL2,andACAN(aggrecan) [86].Inaddition,inOA-likechondrocytes,MSC-EVsinducedthe expressionoftypeIIcollagenandaggrecan(chondrocytemarkers), whileinhibitingMMP-13andADAMTS5(catabolic)andiNOS (inflammatorymarkers).InaCIOAmodel,treatedmicealso exhibitedreducedcartilageandbonedegeneration[87].Inan OAmodel,RuizshowedthattheeffectofMSC-EVswasduetothe presenceofTGFBImRNAandprotein[88].Analogously,inthe samemodel,BMSC-EVspromotedtheconversionofRAW264.7 fromM1toM2,reducedtheexpressionofproinflammatory cytokinesIL-1β,TNF-α,andIL-6,andenhancedtheexpressionofIL10,chondrogenicgenes,collagenIIandSOX9[89].Interestingly, Wooetal.revealedintheirmonosodiumiodoacetate(MIA)ratand thesurgicaldestabilizationofthemedialmeniscus(DMM)mouse modelthatMSC-EVscouldamelioratecartilagedegenerationby increasingtypeIIcollagensynthesisanddecreasingMMP-1,MMP3,MMP-13andADAMTS-5expressioninthepresenceofIL-1β [90]. RecentstudieshavealsoexaminedtheeffectofmiRNAsinMSCEVs.Insynovial-derivedMSC-EVs(SMSC-EVs),Taoetal.overexpressedmiR-140-5ptoblockWnt5aandWnt5btoactivateYAP viatheWntsignalingpathwayandsignificantlyreduceextracellularmatrix(ECM)secretion[91].Wangetal.foundthat exosomesderivedfrommiR155-5p–overexpressingSMSCs(SMSC155-5p-Exos)promotedECMsecretionbytargetingRunx2,which enhancedcartilageregenerationandamelioratedOA[92].Likewise,SMSC-EVshighlyexpressedmiR-31andrelievedOAviathe KDM2A/E2F1/PTTG1axis[93].Ofinterest,hypoxiaincreasedthe expressionofmiR-216a-3pinHIF-1α-inducedBMSC-EVsand promoteddown-regulationofJAK2,promotingproliferation, migration,andreducedapoptosisofchondrocytesviainhibition oftheJAK2/STAT3signalingpathway[94].Acombinationofthese miRNAsandMSC-EVsmayserveasapotentialtherapyforOA.In contrast,severalstudieshaveshownthatmiRNAscauseside effectsinOA.Intra-articularinjectionofantagomir-miR-100-5p dramaticallyattenuatedtheinfrapatellarfatpad(IPFP)MSC-EV (MSCIPFP-EVs)-mediatedprotectiveeffectonarticularcartilage invivo[95].MiR-29b-3ptargetsFoxO3 geneandenhances chondrocytedestruction.lncRNAH19fromumbilicalcordMSCEVscouldcompetitivelybindtomiR-29b-3ptoattenuateits inhibitionofthetargetgeneFoxO3 [96].

Spinalcordinjury.Spinalcordinjury(SCI)arisesfollowing damagetoitsstructureandfunctionbyvariouspathogenic factors,withconsequentspinalcorddysfunctionincludingthatof movement,sensation,andreflexes[97].Duetothelimited regenerativeabilityofnervecomponents,MSC-EVshavebeen recentlyviewedasapromisingclinicaltreatmentforSCI(Table 3). AratmodelofSCIhascommonlybeenappliedtoevaluate treatmentwithMSC-EVs.Theyhavebeenfoundtobeableto regulateimmunityandrestorefunctionthroughavarietyof pathways.First,Huangetal.studiedtheadministrationofhBMSCExosinananimalmodel,anddemonstratedthatinhibitionof apoptosisprotein(Bax)andpro-inflammatoryfactors(TNFα andIL 1β),andpromotionofanti-apoptoticprotein(Bcl-2),antiinflammatoryprotein(IL10)andangiogenesis,couldimprove motorfunction[98].Interestingly,thereducedpericytemigration mediatedbyBMSC-EVscorrelatedwithinhibitionoftheNF-KB P65signalingpathwaywithconsequentweakeningofthebloodspinalcordbarrier(BSCB)[99].Inaddition,Zhouetal.showedthat treatmentwithBMSC-Exossuppressedtheexpressionofcaspase1 andIL1β byreducingpyroptosis,andenhancedneuronal regenerationtoamelioratemotorabilityinratswithspinalcord injury[100].Hanetal.foundthatTGF-β inBMSC-EVsenhanced theexpressionofSmad6,inhibitedtheexcessivedifferentiationof neuralstemcells(NSCs)intoastrocytes,andpromotedregenerationofneurons[101].Consecutively,Nakazakietal.proposedthat BMSC-EVsshouldbeadministeredover3daystoup-regulate transforminggrowthfactor-β (TGF-β),TGF-β receptor,andrelative proteinsoftightjunction[102].Moreintriguingly,Zhouetal. providedevidencethatexosomessecretedbyhPMSCsincreased theactivationofproliferatingendogenousnervestem/progenitor cellsinvivo,whilepromotingNSCproliferationandupregulating MEK,ERK,andCREBphosphorylationlevelsinvitro,resultingin functionalrecovery[103].

MiRNAshavealwaysbeenpotentbiologicaleffectorsofMSCEVs,andwithoutexception,theyplayastrongroleinimmune regulationandregenerationinspinalcordinjury.Jiaetal. confirmedthatoverexpressionofmiR-381inMSC-EVscould promoteSCIrepairbyup-regulatingRashomologousA(RhoA)/ RHOkinaseactivityanddown-regulatingBRD4expressionand DRGcellapoptosisbyWNT5A[104].Lietal.observedthatmiR-133 carriedbyMSC-Exoscoulddirectlytargetanddown-regulatethe expressionofRhoA,andalsopromoteexpressionofERK1/2STAT3 andCREBsignalingpathwayproteinsrelatedtoneuronalsurvival andaxonregeneration,thusrescuingneuronapoptosisand promotingaxonregeneration[105].Ofinterest,whenmiR-17-92, miR-26a,andmiR-216a-5pwereenrichedinBMSC-Exos,they respectivelyinducedactivationofmTOR/PI3K/Akt,PTEN/Akt /mTOR,andtheTLR4/NF-κB/PI3K/Aktsignalingpathwaycascade, withconsequentpromotionofaxonalregenerationandnerve functionrepairafterSCI[106–108].Inaddition,miRNA-22 encapsulatedinBMSC-EVspromotesneurogenesisandinflammationsuppressionbydownregulatingtheexpressionofinflammatorycytokinesandGSDMD,andblockingthepyroptosisof microgliaafterSCI[109].OverexpressionofmiR-199a-3p/145-5pin exosomessecretedbyhumanumbilicalcord-derivedMSCshas beenshowntoactivatetheNGF/TrkAsignalingpathwayaffecting TrkAubiquitination,andimprovelocomotorfunctioninratswith SCI[110].

Skininjury.Skininjuryisquitecommon.Skinregenerationis typicallyaccompaniedbyfouroverlappingprocesses:inﬂammation,angiogenesis,newtissueformation,andremodeling [111–113](Table 4).

Thereisrecentevidencethathuman-derivedMSC-Exos effectivelybeneﬁtskindamageandacceleratewoundhealing bymodulatingrelatedsignalingpathways.Intriguingly,Zhouetal. adoptedacombinationtherapy,applyinghADSC-Exosbothlocally andintravenouslytoaccelerateskinwoundhealing.

Table2. Summaryofstudiesontheroleofextracellularvesiclesinosteoarthritis.

AnimalmodelMolecularmechanismActioneffectRef

EVssourceTargetcellsor tissues

[ 85 ]

Promotetheproductionofproteoglycan,typeII collagen,andchondrocytesregeneration

DownregulateTNFα -inducedexpressionofCOX2,ILs andcollagenaseactivity

[ 86 ]

Promotecellproliferationandmigrationand reduceapoptosis.

DownregulateIL-1ß-activatedpro-in ﬂ ammatoryErk1/2, PI3K/Akt,p38,TAK1,andNFκ Bsignalingpathways

[ 87 ]

CIOAInhibitMMP-13,ADAMTS5andiNOSReinducetheexpressionoftypeIIcollagen, aggrecan,andprotectedmicefrom jointdamage

BMSC-EVsChondrocytes –

88 ]

Increasechondrocyteproliferation[

OATGFBIinhibitcartilageandbonedegradation,andlimit calci ﬁ cationandosteophyteformation

hBMSC-EVsChondrocyte –

89 ]

InhibitOAprogression[

[ 90 ]

[ 91 ]

[ 92 ]

Promotetheproliferationandmigrationof humanOAchondrocytes,andprotected cartilagefromdegeneration

Enhanceproliferation,migrationof chondrocytes,andpreventOA

[ 93 ]

Alleviatecartilagedamageandin ﬂ ammationin kneejoints

Promoteproliferation,migrationandreduce apoptosis

MurineBMSCs-EVsOA-like chondrocytes

hBMSC-EVsOA-like chondrocytes

BMSC-ExosMacrophagesOAPromotetheconversionofRAW264.7fromM1toM2, reducetheexpressionofIL-1 β ,TNFα andIL-6,and enhanceIL-10,chondrogenicgenes,collagenIIandsox9

hASC-EVsChondrocytesMIA,DMMIncreasetypecollagensynthesisanddecreaseMMP-1, MMP-3,MMP-13,andADAMTS-5expressioninthe presenceofIL-1 β

OAHighly-expressmiR-140-5pblockedECMsecretion decreaseviaRalA

OAHighly-expressedmiR-155-5ppromotedECMsecretion viaRunx2

EncapsulatemiR-31ameliorateskneeOAviathe KDM2A/E2F1/PTTG1axis.

SMSC-EVsArticular chondrocytes

SMSC-ExosArticular chondrocytes

SMSC-EVsKneeOAHumankneeOA patients

[ 95 ]

[ 96 ]

Protectarticularcartilagefromdamageand ameliorategaitabnormalityinOAmiceby maintainingcartilagehomeostasis

BMSC-EVsChondrocyteOAHypoxiaincreasedtheexpressionofmiR-216a-3pand promoteddown-regulationofJAK2

[ 94 ] infrapatellarfatpad

ChondrocyteOAMiR100-5p-regulateinhibitionofmTOR-autophagy pathway

MSCs-Exos

UMSC-ExosChondrocyteOAExosomalH19againstmiR-29b-3ptoupregulateFoxO3Promotechondrocytemigration,matrix

secretion,apoptosissuppression,aswellas senescencesuppression

BMSC bonemesenchymalstemcell, CIOA collagenase-inducedosteoarthritis, DMM destabilizationofthemedialmeniscus, ECM extracellularmatrix, EVs extracellularvesicles, Exos exosomes, hASC human adipose-derivedstemcell, MIA monosodiumiodoacetate(inducedosteoarthritis), OA osteoarthritis, OA-CH osteoarthritis-chondrocyte, SMSC synovialmesenchymalstemcell, UMSC umbilicalcordmesenchymal stemcell.

Table3. Summaryofstudiesontheroleofextracellularvesiclesinspinalcordinjury.

EVssourceTargetcellsortissuesAnimalmodelMolecularmechanismActioneffectRef

[ 98 ]

hBMSC-ExosEndothelialSCIInhibitBaxandTNF α andIL1 β ,andBcl2,IL10andangiogenesisAttenuatethelesionsizeandimproved functionalrecoveryafterSCI

BMSC-EVsPericyteSCIInhibitNF-KBP65signalingpathwayAmeliorateblood-spinalcordbarrier[ 99 ]

]

[ 100 ]

Amelioratethemotorabilityofspinal cordinjuryrats

BMSC-ExosPericyteSCISuppresstheexpressionofcaspase1andIL1 β byreducing pyroptosis

BMSC-EVsNSCsSCITGFβ enhancedtheexpressionofSmad6Promotetheregenerationofneurons[ 101 ]

Improvelocomotorrecovery[ 102 ]

BMSC-EVsM2macrophageSCIUp-regulateTGFβ ,TGFβ receptorandrelativeproteinsoftight junction

Promotespinalcordfunctionalrecovery[

SCIPromoteNSCsproliferationandupregulateMEK,ERK,andCREB phosphorylationlevels

hPMSC-ExosEndogenousneuralstem/ progenitorcells

PromoteSCIrepair[ 104 ]

MSC-EVsDRGcellsSCIOverexpressmiR-381up-regulatesRhoA/RHOkinaseactivityand down-regulateBRD4expressionandDRGcellapoptosisbyinhibiting theBRD4/WNT5Aaxis

[ 105 ]

[ 106 ]

[ 107 ]

Improvetherecoveryofhindlimb locomotorfunctionfollowingSCI

Enhanceneuro-functionalrecovery ofstroke

Promoteaxonalregenerationand neurogenesisandattenuateglia scarringinSCI

[ 108 ]

Promotefunctionalbehavioralrecovery afterSCI

NervefunctionrepairafterSCI[ 109 ]

PromotelocomotorfunctioninSCIrats[ 110 ]

MSC-ExosNeuronsSCIMiR-133btargetdown-regulatestheexpressionofRhoA,and promotesERK1/2STAT3andCREBsignalingpathway

BMSC-ExosNeuronsMCAOMiR-17-92inducesactivationofmTOR/PI3K/Aktsignalingpathway cascade

BMSC-ExosNeuronsSCIMiR-26ainducesactivationofPTEN/Akt/mTORsignalingpathway cascade

BMSC-ExosMicrogliaSCIHypoxicexosomalmiR-216a-5pmodulatemicroglialpolarizationby TLR4/NFκ B/PI3K/AKTsignalingcascades

BMSC-EVsMicrogliaSCIMiRNA-22downregulatestheexpressionofin ﬂ ammatorycytokines andGSDMD

hUC-MSC-ExosNeuronsSCIMiR-199a-3p/145-5paffectedTrkAubiquitinationandpromotedthe NGF/TrkAsignalingpathway

BMSC bonemesenchymalstemcell, DRG dorsalrootganglia EVs extracellularvesicles, Exos exosomes, hPMSC humanplacentalmesenchymalstemcell, MCAO middlecerebralarteryocclusion, MSC mesenchymal stemcell, NSCs neuralstemcells, SCI spinalcordinjury, UC-MSC umbilicalcordmesenchymalstemcell.

Mechanistically,hADSC-ExosachievedthiseffectbydownregulatingTNF-α,IL-6,CD14,CD19,CD68,andC-caspase3,and up-regulatingVEGF,CD31,Ki67,PCNA, ﬁlaggrin,loricrinandAQP3 [114].Jiangetal.demonstratedthathBMSC-ExossuppressedTGFβ1,Smad2,Smad3,andSmad4bytargetingtheTGF-β/Smad signalingpathway,butincreasedtheexpressionofTGF-β3and Smad7,thusimprovingscarformationandpromotingwound healing[115].Remarkably,fetaldermalmesenchymalstemcellderivedexosomes(FDMSC-Exos)havebeenshowntoactivate adultdermal ﬁbroblast(ADFs)topromotecellproliferation, migrationandsecretionbytargetingJagged1ligandinthe Notchsignalingpathway,andultimatelyacceleratewound healing[116].

Similareffectshavealsobeenobservedforhuman-derived MSC-ExoscarryingmiRNAs.Ofinterest,Heetal.showedthat hBMMSCsandjawbonemarrowMSCs(JMMSCs)couldinduce macrophagestowardM2polarizationandpromotewound healing.Themechanismsuggestedthatexosomessecretedby donorsmayregulatethepolarizationofmacrophagesbycarrying miR-223targetingPknox1.Nonetheless,researcherscannot confirmwhetherothermiRNAsorfactorscarriedbythese exosomesareinvolvedintheinductionofM2polarization,and furtherstudiesareneeded[117].Likewise,Wuetal.utilizedBMSCExostreatedwith50µg/mLFe3O4 nanoparticlesand100mTSMF toformafunctionalexosome(mag-BMSC-Exos).Notably,miR-215pwasoverexpressedinmag-BMSC-Exosandpromotedangiogenesisinvivoandinvitrotoaccelerateskinwoundhealingby targetingSPRY2toactivatethePI3K/AKTandERK1/2signaling pathways[118].Additionally,Chengetal.foundthathUCMSCsEVsarehighlyenrichedwithmiR-27bandpromotetheexpression ofJUNBandIRE1α bytargetingtheItchyE3ubiquitin-protein ligase(ITCH),therebyacceleratingcutaneouswoundhealing[119]. Inaddition,hUMSC-ExoscanbeenrichedwithasetofmicroRNAs (miR-21,-23A,-125b,and-145)toattenuateexcessmyofibroblast formationandscarringviarepressionoftheTGF-β2/SMAD2 pathways[120].AnotherstudyshowedthathADSC-Exosderived miR-19bregulatetheTGF-β pathwaybytargetingCCL1[121].Li etal.verifiedthathADSC-Exosdown-regulatedtheexpressionof Col1,Col3, α-SMA,IL-17RA,andP-SMad2/P-SMad3,andupregulatedthelevelofSIP1bysuppressingmultiplicationand migrationofhypertrophicscar-derived fibroblasts(HSFs).In addition,miR-192-5pwashighlyenrichedinADSC-EXOand reducedthelevelofpro-fibrosisprotein,improvedhypertrophic scar fibrosis,andacceleratedwoundhealingviatargeted inhibitionofIL-17RAexpression[122].Alongsidethis,overexpressionofmiR-486-5PinhADSC-EVsenhancedthemigrationof humanskin fibroblasts(HSFs)andtheangiogenicactivityof humanmicrovascularendothelialcells(HMECs)bytargetingSp5 andmotivatingCCND2expression,therebypromotingwound healing[123].Interestingly,Gaoetal.foundthatoverexpressionof Mir-135ainhAMSC-Exossignificantlydown-regulatedLATS2, therebyincreasingcellmigrationandpromotingwoundhealing [124].

Anti-ﬁbrosis

Liver fibrosis.Liver fibrosisisapathophysiologicalprocessand referstotheabnormalproliferationofintrahepaticconnective tissueduetovariouspathogenicfactors[125].Recently,useof MSC-EVshasbeenconsideredanewtherapeuticapproachto repairliver fibrosis(Table 5).Rongetal.showedthathumanbone MSC-EVsinhibitedexpressionofWnt/β-cateninpathwaycomponents, α-SMA,andtypeIcollagen,therebypreventingstellatecell activationandincreasinghepatocyteregeneration.Invivo injectionofhBMSC-Exoshasbeenshowntoeffectivelyalleviate CCL4-inducedliver fibrosisinratsandrestoreliverfunction[126]. Likewise,usingaCCL4-inducedliver fibrosisanimalmodel,Ohara etal.provedthatEVsfromamnion-derivedMSCs(AMSC-EVs) couldsignificantlyreducethenumberofKupffercells(KCs),mRNA

expressionofinflammatoryfactors,activationofhepaticstellate cells(HSC),andthelipopolysaccharide(LPS)/toll-likereceptor4 (TLR4)signalingpathway,therebyreducinginflammationand fibrosis[127].

Theanti-fibroticeffectofmiRNAsinMSC-EVshasbecomea focusofresearchintoCCL4-inducedliver fibrosisinrats.MiRNA181-5poverexpressioninADSC-EVshasbeenshowntodownregulatetranscription3(STAT3)andBcl-2andactivatedautophagyinHST-T6cells,alongsideasignificantdecreaseincollagenI, vimentin,a-SMA,and fibronectininliver[128].Similarly,high expressionofmiR-122inADSC-EVsmodulatedtheexpressionof targetgenessuchasinsulin-likegrowthfactorreceptor1(IGF1R) cyclinG(CCNG1),andproline-4-hydroxylaseA1(P4HA1),thereby moreeffectivelyblockingtheproliferationofHSCsandcollagen maturation[129].Interestingly,Kimetal.reportedthatmiR-486-5p washighlyexpressedinT-MSC-EVsthatcouldtargetthehedgehogreceptor,smoothened(Smo),andinhibithedgehogsignaling, therebyattenuatetheactivationofHSCsandliver fibrosis[130].

Kidney fibrosis.Renal fibrosisisagradualpathophysiological processduringwhichkidneyfunctionprogressesfromhealthyto injured,thentodamagewithanultimatelossoffunction[131]. Increasingly,MSC-EVshavebeenstudiedinthetreatmentofrenal fibrosisusingvariousmodels(Table 6).

Jietal.determinedthathUC-MSC-ExosrepressedYesassociatedprotein(YAP)throughcaseinkinase1δ (CK1δ)andE3 ubiquitinligase β-TRCPinaratmodelofunilateralureteral obstruction(UUO),thusamelioratingrenal fibrosis[132].Similar effectsinaUUOmodelwereconfirmedinLiu’sstudy.They revealedthathUC-MSC-Exosattenuatedrenal fibrosisbyinhibitingtheROS-mediatedp38MAPK/ERKsignalingpathway[133]. Likewise,Shietal.showedthatmilkfatglobule–epidermalgrowth factor–factor8(MFG-E8)wasincludedinBMSC-EVs,and amelioratedrenal fibrosisbyblockingtheRhoA/ROCKpathway inaUUOmodel[134].Ofinterest,inaUUOmousemodel,BMSCExosloadedmiR-34c-5pinhibitedcorefucosylation(CF)bycd81EGFRcomplex,therebyimprovingrenalinterstitial fibrosis(RIF) [135].Correspondingly,recentstudiesalsosuggestthatexosomes fromADSCsamelioratethedevelopmentofDNviamiRNAs.Jin etal.usedmiRNA-215-5ptoinhibitZEB2andimproveddiabetic nephropathy(DN)symptoms.Theyalsorevealedthatupregulated expressionofmiR-486couldsuppresstheSmad1/mTORsignaling pathwayinpodocytes[136, 137].MV-miR-451afromhUMSCs repressedcellcycleinhibitorP15andP19expressionbytargeting their3′-UTRsites,therebydecreasing α-SMAandincreasing e-cadherinexpression.Thisresultedinepithelial-mesenchymal transformation(EMT)reversalandimprovedDNsymptoms[138]. InanotherstudyofameliorationofDN,BMSC-Exossignificantly enhancedtheexpressionofLC3andBeclin-1,anddecreasedthe levelofmTORand fibroticmarkersinastreptozotocin-inducedrat modelofdiabetesmellitus[139].Interestingly,Grangeetal. reportedthatrenal fibrosisandtheexpressionofcollagenIwere significantlyamelioratedviamultipleinjectionsofHLSCs(human liverstem-likecells)andMSC-EVsinNOD/SCID/IL2Rγ KO(NSG) mice.Additionally,relatedgenes(Serpina1a,FASligand,CCL3, TIMP1,MMP3,collagenI,andSNAI1)weresignificantlydownregulated,therebyattenuatingDNsymptoms[140].

Lung fibrosis.Pulmonary fibrosisisaterminalchangeinlung diseasecharacterizedby fibroblastproliferationandaccumulation ofalargeamountofextracellularmatrixaccompaniedby inflammatoryinjuryanddestructionoftissue.Normalalveolar tissueisdamagedandabnormalrepairleadstostructural abnormalities[141, 142].Theetiologyinthevastmajorityof patientswithpulmonary fibrosisisunknown[143].Idiopathic pulmonary fibrosis(IPF)manifestsmainlywithpulmonary fibrotic lesionsandisaseriousinterstitiallungdiseasethatcanleadto progressivelossoflungfunction.IPFhasahighermortalitythan

Table4. Summaryofstudiesontheroleofextracellularvesiclesinskininjury.

AnimalmodelMolecularmechanismActioneffectRef

Accelerateskinwoundhealing[ 114 ]

Down-regulateTNFα ,IL-6,CD14,CD19,CD68,and C-caspase3,up-regulateVEGF,CD31,Ki67,PCNA, ﬁ laggrin,loricrinandAQP3

[ 115 ]

Improvescarformationandpromote woundhealing

TargetonTGFβ /Smadsignalingpathway,butincreased theexpressionofTGFβ 3andSmad7

[ 116 ]

InhibitMMP-13,ADAMTS5andiNOSReinducetheexpressionoftypeIIcollagen, aggrecan,andprotectedmicefrom jointdamage

[ 117 ]

Full-thicknessskin defectmodel

EVssourceTargetcellsor tissues

hADSC-Exos –

hBMSC-ExosHaCaTcells andHSFs Full-thicknessskinwounds injurymodelinrats

FDMSC-ExosADFsFull-thicknessdermal woundinjurymodel

hBMSC-Exosand JMMSC-Exos MacrophagesSkinWound-HealingBycarryingmiR-223targetingPknox1InducedmacrophagestowardM2 polarizationandpromotewoundhealing

Accelerateskinwoundhealing[ 118 ]

mag-BMSC-ExosHUVECsandHSFsRatSkinWoundModelHighly-expressmiR-21-5pandtargetSPRY2toactivating PI3K/AKTandERK1/2signalingpathways

119 ]

Acceleratecutaneouswoundhealing[

Highly-expressmiR-27bpandpromotetheexpression ofJUNBandIRE1 α bytargetingtheItchyE3ubiquitinproteinligase(ITCH)

[ 120 ]

Attenuateexcessmyo ﬁ broblastformation andanti-scarring

121 ]

Promotethehealingofskinwounds[

Highly-expressmicroRNAs(miR-21,-23A,-125band -145)repressedtheTGFβ 2/SMAD2pathway

Highly-expressmiR-19bregulatedTGFβ pathwayby targetingCCL1

[ 122 ]

[ 123 ]

[ 124 ]

Reducethelevelofproﬁ brosisprotein, improvehypertrophicscar ﬁ brosisand acceleratewoundhealing

Down-regulatetheexpressionofCol1,Col3, α -SMA,IL- 17RA,andP-SMad2/P-SMad3,andup-regulatethelevel ofSIP1,whileoverexpressionmiR-192-5ptarget inhibitionofIL-17RAexpression

Promoteproliferation,migrationand reduceapoptosis

OverexpressionmiR-486-5pinhibitSp5andelevatethe CCND2expression

Increasecellmigrationandpromote woundhealing

DownregulationofLATS2afteroverexpressionofmiR- 135a

hUCMSCs-EVsHaCaTcells andHSFs Cutaneouswound mousemodel

hUCMSC-ExosMyo ﬁ broblastFull-thicknessskindefect mousemodel

Woundhealingofskin- injuredmice

hADSC-ExosHaCaTcells andHSFs

hADSC-ExosHSFsFull-thicknessskindefects inthebacksofrats

hADSC-EVsHSFsandHMECs –

hAMSC-ExosFibroblastsFull-thicknessskindefects inthebacksofrats

EVs extracellularvesicles, Exos exosomes, FDMSC fetaldermalmesenchymalstemcell, hADSC humanadipose-derivedstemcell, hAMSC humanamnionmesenchymalstemcell, hBMSC humanbonemesenchymal stemcell, HMEC humanmicrovascularendothelialcell, HSF Humanskin ﬁ broblast, hUCMSC humanumbilicalcordmesenchymalstemcell, JMMSC jawbonemarrowMSC.

[ 126 ]

Effectivelyalleviateliver ﬁ brosis,andenhance liverfunctionality,hepatocyteregeneration

InhibitedtheexpressionofWnt/ β -cateninpathway, α -SMA,andCollagenI

127 ]

Improveliverin ﬂ ammationand ﬁ brosis[

[ 128 ]

[ 129 ]

hBMSC-ExosHepaticstellatecellsCCl4-inducedliver ﬁ brosis

Effectiveanti-liver ﬁ broticandattenuate liverinjury

AMSC-EVsHepaticstellatecellsNASH,liver ﬁ brosisDecreasethenumberofKCsandthemRNA expressionlevelsofTNFα ,IL1β ,IL6,TGFβ ,LPS, andTLR4

Down-regulateSTAT3andBcl-2andactivated autophagy

ADSC-ExosHST-T6cells * Inducedliverinjury byCCl4

AMSC-ExosHepaticstellatecellsCCl4-inducedliver fi brosis miR-122Enhancethetherapeuticef fi cacyofAMSCsinthe treatmentofliver fi brosis

MiR-486inactivateshedgehogsignalingAttenuateHSCactivationandliver ﬁ brosis[ 130 ]

CCl4-inducedliver ﬁ brosis

hTMSC-EVsHumanprimaryhepatic stellatecells

ADSC adipose-derivedmesenchymalstemcell, AMSC amnion-derivedmesenchymalstemcell, BMSC bonemesenchymalstemcell, CCl4 carbontetrachloride, EVs extracellularvesicles,Exosexosomes, HSC hepatic stellatecell, NASH nonalcoholicsteatohepatitis, TMSC tonsil-derivedmesenchymalstemcell.

mosttumorsandisconsideredatumor-likedisease[142]. Recently,MSC-EVshavebecomeaneffectivetreatmentfor pulmonary ﬁbrosis(Table 7).

BMSC-Exosexerttheirtherapeuticeffectthroughimmunomodulation.Inamousemodel,BMSC-Exoshavebeenshownto significantlyamelioratehyperoxia(HYRX)-inducedbronchopulmonarydysplasia(BPD),alveolar fibrosis,andpulmonaryvascular remodelingbysuppressingM1macrophageproductionand enhancingM2macrophagegeneration[144].Likewise,BMSCExoshavebeenshowntosignificantlyreverse fibrosisina bleomycin-inducedpulmonary fibrosismodelbyregulatingtotal lungimbalanceofMΦ phenotype[145].Inaddition,theWnt5a/ BMPsignalingpathwayregulatedbyUC-MSC-Exoscanenhance Wnt5a,Wnt11,BMPR2,BMP4,andBMP9expression,anddownregulatethatof β-catenin,CyclinD1andTGF-β1.Inamonocrotaline(MCT)-inducedratmodelofpulmonaryhypertension(PH), MSC-Exoswereshowntosignificantlyamelioratepulmonary vascularremodelingandpulmonary fibrosis[146].Ofinterest, Chaubeyetal.showedthatUC-MSC-Exosplayedatherapeutic roleinimprovingpulmonaryinflammation,pulmonarysimplification,pulmonaryhypertension,andrightventricularhypertrophy throughimmunomodulatoryglycoproteinTSG-6inaneonatal BPDmousemodel[147].

Additionally,MSC-EVscanreverselunginjuryandpulmonary fibrosisbyexpressinginfluentialmiRNAs.Wanetal.determined thathighexpressionofmiR-29b-3pbyBMSC-EVsamelioratedIPF byFZD6[148].Zhouetal.foundthatmiR-186enrichedbyBMSCEVsrepressedtheexpressionofSOX4andDickkopf-1(Dkk1), therebyeffectivelyinhibiting fibroblastdevelopmentandattenuatingIPF[149].Inaddition,Lei’sstudyrevealedthathPMSC-EVs couldcarrymiR-214-3panddownregulateATM/P53/P21signaling, thusrelievingradiation-inducedlunginflammationand fibrosis [150].InBLM-inducedlung fibrosisandamousemodelofalveolar epithelialcelldamage,exosomessecretedfromMenSCs(MenSCsExos)havebeenshowntoamelioratepulmonary fibrosisby transferringmiRNALet-7tosuppressreactiveoxygenspecies (ROS),mitochondrialDNA(mtDNA)damage,andactivationof NLRP3inflammasome[151].Similarly,Xiaoetal.usedanotherLPSinducedAcuteLungInjury(ALI)mousemodelanddemonstrated thatMSC-ExosrepressedNF-κBandhedgehogpathwaysby transportingmiR-23a-3pandmiR-182-5p,therebyimprovinglung injuryand fibrosis[152].

CHALLENGESANDAPPLICATIONOFMSC-EVSASAN ADVANCEDTHERAPY

*HST-T6,mousehepaticstellatecellline.

AlthoughMSC-EV-basedtherapyholdsgreatpromiseasanovel “cell-free” therapeuticproduct,thereremainmanychallengesto overcomepriortotheirclinicalapplication.Atpresent,several limitationsrestricttheclinicaltranslationofMSC-EVsincludingthe discrepanciesinthecomponentsofEVsfromvarioussourcesand thelackofstandardoperationprocessesforlargescaleproduction, bothofwhichlargelydependonqualitycontrolofthesourcesof EVs.Itisplausibletoovercomethesehurdlesbyintroducinga strategytocontrolthequalityofMSCsfromtheoriginalsource ofEVs.

ThequalityofMSC-derivedEVsfromdifferentgroupsand batchesisheterogeneous MSCsaremostcommonlyderivedfrombonemarrow,fat, umbilicalcordandothertissues,butmaintainingconsistent qualityofMSCsandtheirEVsfromdifferentsourcesandacross batchesisdifﬁcult.Thisseverelyrestrictsthequalitycontroland managementofMSCsandtheirEVsasdrugs,andincreasesthe problemofdrugresistance[153].Thisresultsinlimited reproducibilityoffunctionalmeasurementsinvitroandinvivo [154].

UUOInhibitROS-mediatedp38MAPK/ERKsignaling pathway Attenuaterenal

]

hUC-MSC-ExosRenaltubular epithelialcells

BMSC-EVsHK-2cellsUUOInhibitRhoA/ROCKpathwayAttenuaterenal

AmeliorateRIF[ 135 ]

UUOMiR-34c-5pinhibitsthecorefucosylationofmultiple proteins

[ 136 ]

ImprovepodocytedysfunctionandDN symptoms

MiR-215-5pshuttlestopodocyte,andinhibitsthe transcriptionofZEB2

137 ]

AmeliorateDNsymptom[

[ 138 ]

Decreasethemorphologicandfunctional injuryofkidney

139 ]

AttenuateDNsymptom[

[ 140 ]

BMSC-EVsPericytes;Fibroblasts; Macrophages

ADSCs-ExosPodocyte

ADSCs-ExosPodocyteSpontaneousdiabetesmiceEnhancetheexpressionofmiR-486,inhibitof Smad1/mTORsignalingpathway

DiabetesandhyperuricemiamiceMiR-451adecreases α -SMAandincreasese-cadherin expressionbytargeting3 ′ -UTRsitesofP15andP19

EnhancetheexpressionofLC3,Beclin-1anddecrease thelevelofmTORand ﬁ broticmarker

hUC-MSC-EVsHK-2cells *

BMSC-EVsRenaltissueStreptozotocin-induceddiabetes mellitusrat

brosisandtheexpression ofcollagenI,attenuateDNsymptom

Amelioraterenal ﬁ

KO(NSG)miceDownregulateSerpina1a,FASligand,CCL3,TIMP1, MMP3,collagenIandSNAI1

hBMSC-EVsGlomerulusNOD/SCID/IL2R γ

ADSC adipose-derivedmesenchymalstemcell, BMSC bonemesenchymalstemcell, DN diabeticnephropathy, EVs extracellularvesicles,Exosexosomes, RIF renalinterstitial ﬁ brosis, UC-MSC umbilicalcord mesenchymalstemcell, UUO unilateralureteralobstruction. * HK-2,humanproximaltubularepithelialcellline.

Intheangiogenesisstudy,BMSC-,ADSC-,andUCBMSC-derived EVswerecomparedandfoundtoreducemyocardialapoptosis, facilitateangiogenesis,andimprovecardiovascularfunction. Notably,EVsfromADSCsstimulatedcardioprotectionfactors VEGF,bFGF,andHGF[155].Inaddition,BMSC-derivedEVs appearedtohaveagreaterangiogenicpotentialthanADSCderivedEVswhencomparedintwoindependentischemicmodel studies,withanapproximately4-foldincreaseinendothelialcell numberscomparedwithcontrols,anda1.5-foldchangeinthe latter[156, 157].Nonetheless,anotherstudyshowedthatEVsfrom endometrialmesenchymalstemcellsresultedinagreaterlevelof angiogenesisthanEVsfromBMSCSorADMSCs[158].

Instudiesofosteogenesisstudies,intwoseparateratskull defectstudies,BMSC-EVtreatmentincreasedbonevolumefourfoldrelativetothecontrolgroup[159],whileADSC-EVincreased bonevolumebyabout1.33times[160].Inotherstudies,BMSCandADSC-derivedEVsacceleratedchondrocyteproliferation, migration,andosteogenicdifferentiation[161, 162].

ComparisonoftheimmunomodulatorydifferencesofMSCderivedEVsfromdifferentsourcesrevealedthatBMSC-EVsand ADSC-EVscouldinduceM2polarizationofmacrophagesinvivo andinvitro[163, 164].Interestingly,inaseparateexperiment, Wangetal.showedthatBMSC-EVspromptedasigniﬁcant(3.2fold)increaseintheexpressionofCD206ofM2-polarization markerinanacutelunginjurymousemodel[163].Nonetheless Liuetal.reportedthattheM2polarizationabilityofADSC-EVs increasedonlybyafactorof1.5inamousemodel[165].

TheproliferationcapacityofMSCsextractedfromadult tissueswaslimited,andaffectedthelargescaleproductionof EVs

TodevelopMSC-EVsintocommerciallyadvancedtherapeutic products(ATPs),qualityassurance(QA)isrequiredoftheoriginal material,includingparentalgroupsorcellsusedinthe manufactureofMSCs.Thereremainmanydifficultiesinmass productionofEVsfromadulttissuesforclinicaltrialssince proprietaryMSCshavealimitednumberofpassagetimes,age easily,andcomeatahigh financialcost.Inaddition,their heterogenicitymakestraditionalcellcultureinefficientintermsof timeandcost.

MSCsderivedfrompluripotentstemcellsovercomethe problemsofmassproductionofMSC-EVsandquality heterogeneity

TheoriginalsourceMSCsrequiresgood,consistent,and controllablequality,withastrongabilitytoproliferateandto secretelargenumbersofEVs.Toachievethis,weestablishedan inductionsystemofMSCsusingpluripotentstemcellsto overcometheproblemsofmassproductionofMSC-EVsand variationinquality.WesuccessfullyinducedMSCsfrompluripotentstemcells(PSC)[166–170].ComparedwithMSCsextracted fromtraditionalsources,ourMSCswerederivedfromthesame parentPSCs,consequentlyovercomingtheproblemofEV heterogeneitywhenMSCsfromavarietyofsourcesareused. Recently,GMP-gradeMSCsderivedfromhumanPSCs(hPSC)have beenusedinclinicaltrialsforrefractorygraft-versus-hostdisease (GVHD)[171].ThetherapeuticpotentialofMSC-EVshasbeen showninpreclinicalstudiesofbothacuteGVHD(aGVHD) [172–174]andchronicGVHD(cGVHD)[175]models.The preliminarybenefitsofhPMSC-EVshavebeenreportedina patientwithcutaneouscGVHD.Thestiffeninganddrynessofskin wereimprovedsignificantlyafterintravenousinjectionofhPMSCEVs[176].Basedonthepreliminaryefficacyandsafetyprofiles,a phase1studyhasbeenlaunchedtoevaluatethesafetyand efficacyofBM-MSC-derivedEVsinpatientswithacuteorchronic rejectionfollowingabdominalsolidorgantransplantation (NCT05215288,Table 1).ItisplausiblethathPSC-MSC-derived EVswillpromotetheclinicaltranslationofMSC-EVsowingtothe

ﬁ brosis.

Table7. Summaryofstudiesontheroleofextracellularvesiclesinlung

EVssourceTargetcellsortissuesAnimalmodelMolecularmechanismActioneffectRef

[ 144 ]

Improvelungfunction,decrease ﬁ brosisandpulmonary vascularremodeling,andamelioratepulmonaryhypertension.

BMSC-ExosLungmacrophageHyperoxia-inducedBPDSuppressM1macrophageproductionandenhanceM2 macrophagegeneration

145 ]

ﬁ brosis[

RegulatetotallungimbalanceofmacrophagephenotypePreventorreverselung

hBMSC-ExosLungmacrophageBleomycin-inducedpulmonary ﬁ brosis

146 ]

RegulateWnt5a/BMPsignalingpathwayAttenuatepulmonaryvascularremodelingandlung ﬁ brosis[

UC-MSC-ExosPAECandPASMCMonocrotaline-inducedrat modelofPH

UC-MSC-ExosLungtissueBPDImmunomodulatoryglycoproteinTSG-6Improvepulmonaryin ﬂ ammation,pulmonarysimpli ﬁ cation, pulmonaryhypertension,andrightventricularhypertrophy [ 147 ]

148 ]

BMSC-EVsIPFpulmonarytissueIPFMiR ‐29b ‐3pAmeliorateIPF[

150 ]

AmeliorateIPF[

BMSC-EVsLung ﬁ broblastPFMiR-186suppressedSOX4andDKK1expression,blocked ﬁ broblastactivation

MiR-214-3pdownregulateATM/P53/P21signalingRelieveradiation-inducedlungin ﬂ ammationand ﬁ brosis[

149 ] hPMSC-EVsLung ﬁ broblastWholethoraxirradiation mousemodel

151 ]

Remitpulmonary ﬁ brosis[

MenSCs-ExosRecipientalveolarepithelialcellsBLMMiRNALet-7suppressesROS,mtDNAdamage,and NLRP3in ﬂ ammasomeactivation

152 ]

ReversedtheLPS-inducedlunginjuryand ﬁ brosis[

MSC-ExosMLE-12cells * LPS-inducedALITransmitmiR-23a-3pandmiR-182-5ptoinhibitNFκ Band hedgehogpathways

ALI acutelunginjury, BLM bleomycin, BMSC bonemesenchymalstemcell, BPD bronchopulmonarydysplasia; EVs extracellularvesicles, Exos exosomes, hPMSC humanplacenta-derivedmesenchymalstemcell, IPF idiopathicpulmonary ﬁ brosis, LPS lipopolysaccharide, PAEC pulmonaryarteryendothelialcell, PASMC pulmonaryvascularsmoothmusclecell, PF pulmonary ﬁ brosis, PH pulmonaryhypertension, MenSCs menstrualblood-derivedstemcell, UC-MSC umbilicalcordmesenchymalstemcell.

qualitycontrolandlargescaleproductiveadvantagesofhPSCMSCscomparedwithtraditionalMSC.hPSC-MSCshavemore passages(morethan30generations),strongampliﬁcationability, canwithstandsenescence[166, 167, 170],andhavestrong secretionability(includingcytokinesandexosomes)[168] comparedwiththetraditionalMSCs.Nonetheless,thepassage timesoftraditionalMSCsaregenerallylessthan10generations, andtheproliferationanddifferentiationabilitiesofMSCsare reducedafternumerouspassagesinculture,andaffectsthe secretionofextracellularvesicles.Therefore,ourhPSC-MSCshave greatadvantagesforlarge-scaleproductionandcostcontrolof EVs.MassproductionofMSCsandtheirEVsisnowpossibleusing bioreactorsandmicrocarrierstomaximizeMSCgrowthandEV releaseperunitsurfacearea.Weevaluatedmesenchymalstem cellsfromdifferentsourcesandfoundthatPSC-MSCshadthe highestEVproduction.TooptimizeEVproduction,weacquired hPSC-MSCsinascalablecellfactory-basedcultureandwereable toovercomethemajorobstaclesduringtransformationofMSCEVsintoATPs.

CONCLUSIONSANDFUTUREPERSPECTIVE

Extracellularvesiclesderivedfrommesenchymalstemcellsplaya criticalroleinthedevelopmentofimmuneregulationand regeneration.TheseEVsmimictheeffectsofstemcellsand performpowerfulfunctionsbymodulatingimmunepathways, promotingeffectorcellmigrationandproliferation,andreducing apoptosis.Todate,15clinicaltrialshavebeenregisteredin ClinicalTrial.gov,butnonehasbeencompleted.AlthoughEVs comparedwithMSCcelltherapyincitealowerimmuneresponse andhaveahighersafetyprofile,thereremainchallengestotheir clinicalapplication[56].Inaddition,thesuccessfulapplicationof EVsdependsonlowcostformassproduction,aswellasimproved separationefficiencyandmoreaccuratecharacterizationmethods. ThisreviewhasdiscussedthetherapeuticeffectsofEVsbasedon thefunctionofMSCsortheintroductionofspecificmolecules (suchasmiRNAsandlncRNAs).Asworkcontinues,researchersare activelydevelopingengineeredEVsthataremoreeffectiveand capableoftargeting,throughloadingofbioactivemoleculesand surfacemodification.Ofinterest,Fengetal.developed ε-polylysine-polyethylene-distearylphosphatidylethanolamine (PPD)tomodifyMSC-EVsandinverttheirsurfacecharge.Asa result,thestericandelectrostatichindranceofcartilagematrix werealleviated,andtheefficiencyofMSC-EVsinthetreatmentof OAwasimproved[177].Thesetreatmentstrategieshaveachieved promisingresultsattheinitialstageandprovideexcitingnew avenuesforregenerativemedicinetherapy.

DATAAVAILABILITY

Allrelevantdataareincludedinthismanuscript.

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ACKNOWLEDGEMENTS

Forthecollaborationandgeneralsupport,wewouldliketothankourcolleagues fromthecordbloodbankcentre,aswellasallcollaborationpartners.Graphswere assembledusingdynamicBioRenderassets(icons,lines,shapes,and/ortext).

AUTHORCONTRIBUTIONS

KMcollectedtheliteratureandwrotethemanuscript.HLcontributedtothe revisionsofthemanuscriptandtablesfor importantintellectualcontent.YJ,CZ, CS,LJ,GL,ZX,andZXcontributedtotheliteraturesummary.XX,YX,WY,ZJ,TH, andXAcontributedtoreviewandlanguageediting.LQconceptualizedthe manuscriptandcontributedtofundingacquisition.Allauthorsreadandgave ﬁ nal approvalforpublication.

FUNDING

ThisstudyisinpartsupportedbyStart-upGrantforStemCellRegenerativeMedicine (GuangzhouWomenandChildren’sMedicalCentre,GrantNo:5001-4001010),and ShenzhenScienceandTechnologyProgram(JCYJ20210324114606019).

COMPETINGINTERESTS

Theauthorsdeclarenocompetinginterests.

ADDITIONALINFORMATION

Supplementaryinformation Theonlineversioncontainssupplementarymaterial availableat https://doi.org/10.1038/s41419-022-05034-x

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