Received:16May2023 Accepted:25May2023 DOI:10.1111/srt.13382
Dermalfibroblastcell-derivedexosomesforatopicdermatitis: In-vitrotest
DearEditor,
Theexosome,akindofextracellularvesiclewithadiameterinthe rangeof50–200nm,hasbeenextensivelyinvestigatedintreatingvariousskinissues,suchasskinagingmitigationandcutaneouswound healingstimulation,andatopicdermatitis(AD).1–3 Comparedtostem celltherapy,advantageousproperties,suchasalonghalf-life,small size,favorablepenetration,andlowimmunogenicity,makeexosomes morefavorableandbeneficial.4 Althoughexosomeshavebeenextensivelyinvestigatedfortheirapplicationinskindamagerepair,theonly sourcesshowntobeeffectivearethosederivedfrommesenchymal stemcells(MSCs).5 However,exosomeisolationfromMSCsaccompaniesmultipledrawbacks,suchasthetimeandeffortrequiredfor MSCcultureandexpansion,theriskofcontaminationwithredblood cellsduringtheisolationprocess,neovascularizationpotential,diminishedcellviability,andtheextremeinvasivenessofremovingMSCs frombonemarrow.6 Therefore,findinganalternativecellsourcefor exosomeisolationhasbecomeimperativefortheeffectiveapplicationofexosomesintherepairofdysfunctionalskin.Dermalfibroblasts (DFs)arethepredominantcelltypeinthedermisandareresponsible forregulatingtheextracellularmatrix(ECM)whileplayingimportant rolesinthemaintenanceofthenormalstructureandfunctionofthe skin.Accordingtothepreviousstudy,DFsareconsideredausefulcell lineinwhichtostudyexosome-relatedtreatmentsbecausethesecells areeasilycultivated.Inthisarticle,wesuggestDFsfortheextraction ofexosomesbecause,incontrastwithMSCs,fibroblastsmoreclosely resembletheskinandmaybeobtainedfromtheskinusinglessinvasive techniques.7
ThisstudythusaimedtodetermineifDF-derivedexosomesarea promisingcandidateforskindamagerepair.Toinvestigatetheeffects ofDFexosomesonskinpermeabilitybarrierprotection,weevaluatedtheexpressionlevelsofskinpermeabilitybarriermaintenance biomarkersinkeratinocytestreatedwith1-chloro-2,4-dinitrobenzene (DNCB).
Weharvestedexosomesfromconditionedmediaoffibroblastcells (Gibco,GrandIsland,NY,USA)usingdifferentialultracentrifugation. Whenthefibroblastcellsreached ∼95%confluencyina150-mmpetri dish,theyweretreatedwithcycloheximide(5–100 µg/ml)togenerateastress-inducedcondition.Post-treatment-conditionedmedia KwanghoYooandNikitaThapacontributedequallytothiswork.
werecollectedandsubjectedtodifferentialcentrifugation.Lastly, pelletscontainingharvestedexosomeswereseparatedandcollected fromtheultracentrifugedsupernatant,resuspendedinphosphatebufferedsaline,andstoredat 80◦ Cforfurtherexamination.Light microscopyrevealedthattheculturedfibroblastsexhibitedatypicalspindle/fibroblast-likestructurewhileadheringtotheculture vesselsurface(Figure 1A).Thecharacterizationoftheharvestedexosomesfromthefibroblastcellculture-conditionedmediawasdone bynanoparticletrackinganalysis(NTA).NTAwasusedtodetermine theparticlenumberandsizeoftheisolatedexosomes.ZetaView (Analytik,Cambridge,UK)wasusedtodeterminethevesicleconcentrationandsizedistribution.Themeanparticlesizesoftheisolated fibroblastexosomeswere215.4 ± 116.1nm,withaconcentrationof 2.33×1010 ± 2.22×109 particles/ml.(Figure 1B)
Thecytotoxicityoftheisolatedexosomeswasleastatconcentrationsof1×104 ,1×105 ,and1×106 particles/mlafter24and48h oftreatment.However,whentheexosomeconcentrationwas1×107 particles/ml,wedetectedconsiderablecytotoxicityafter24hoftreatment.Interestingly,thecytotoxiceffectsontheHaCaTcellswere furtherincreasedafter48hoftreatmentwiththeharvestedexosomes. (Figure 2)
Exosometreatmentincreasedtheproteinlevelsofskinpermeability barrierbiomarkers,comparedtothecontrolinHaCaTcells.However, theoptimalconcentrationatwhichthisepidermalbarrierproteinwas expressedwas1×104 particles/ml(Figure 3A).Theexpressionlevelof theepidermalbarrierproteinswassignificantlyreducedintheDNCBtreatedHaCaTcellscomparedtothecontrol.Intriguingly,exosome treatmentat1×104 particles/mlincreasedtheproteinexpressionlevel ofallskinpermeabilitybarrierbiomarkerproteinsincomparisonto otherconcentrations(Figure 3B).InthecaseofFLG,allthreeexosomeconcentrations(1×104 ,1×105 ,and1×107 particles/ml)were showntosubstantiallyrecoverthediminishedproteinexpressionlevel. However,inthecaseofIVL,LOR,andHAS1,onlythe1×104 particles/mlconcentrationeffectivelyandgreatlyrestoredthediminished epidermalmarkerproteinlevelcomparedtotheremainingexosome concentrations.Thisdataledustoconcludethat1×104 particles/ml isthemostoptimum,safe,andeffectiveexosomeconcentrationtobe usedclinicallyfortherepairofdamagedskincells.
Recently,theeffectsofexosomesonvariousskindiseaseshave beenextensivelystudied.Thekeybenefitsofexosomesaretheirhigh
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FIGURE1 Characterizationofdermalfibroblastcellsandisolatedexosomes.(A)Themorphologyoffibroblastcellsobservedunderlight microscopy.(B)Thesizedistributionofisolatedexosomesdetectedthroughnanoparticletrackinganalysis.
FIGURE2 Humanskinkeratinocytes(HaCaT)werepurchasedfromAddexBio(SanDiego,CA,USA).HaCaTcellswereculturedinDMEM, alongwith10%fetalbovineserum(FBS)and1%antibiotic-antimycotic(AA)solutionina37◦ C,5%CO2 incubator.Every48to72h,freshDMEM wasusedtoreplenishthecellculturemedia.Thecytotoxicityofthefibroblast-derivedexosomeswasassessedusing 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazoliumbromide(MTT;Sigma-Aldrich,St.Louis,MO,USA).HaCaTcellsweredistributedinto 96-wellplates(1×104 cells/well)andincubatedfor24hundercellcultureconditions(37◦ C,5%CO2 ).Then,themediumwasreplacedwitha supplement-freemedium,anddifferentexosomeconcentrationsof1×104 ,1×105 ,1×106 ,and1×107 particles/mlwereaddedtothecellsand incubatedfor24and48h.Post-exosomeaddition,thecellsweretreatedwith5mg/mlMTTsolution(Sigma-Aldrich,St.Louis,MO,USA;M5655) andincubatedina37◦ Cincubatorfor4h.Afterincubation,thesupernatantwasremoved,andtheformazanformedbyMTTreductionwaslysed byaddingdimethylsulfoxide(DMSO;MilliporeSigma,Billerica,MA,USA,1.02952).Theabsorbancewasthenmeasuredat540nmusinga spectrophotometer(MolecularDevices,SanJose,CA,USA).
stability,non-immunerejection,anddirectstimulationoftargetcells. Especially,themiRNAscontainedintheexosomesareengagedinthe regulationofvariouscellularresponsesthroughbindingtothe3′untranslatedregions.Therefore,asingleingredient(exosomes)can contributetomultipletherapeuticeffects.Sofar,multipleendogenous exosometypes,suchasthosefromadipose-derivedstemcellsand mesenchymalstemcells,havecrucialrolesinaging,wounds,andAD.7,8
HumanDFs(HDFs)constitutethemostpredominantmesenchymalcelltypeinconnectivetissue,wherecollagenandelasticfibers oftheECMaredepositedandareresponsibleforthemaintenance ofthenormalstructureandfunctionsoftheskin.Asaresult,very fewstudieshaveinvestigatedthereparativeeffectsoffibroblastderivedexosomes,whichstimulatethemigrationandproliferationof collagenandthesynthesisoffibronectinandcollagenI.9 Inaddition,
FIGURE3 Toevaluatetheeffectofdermalfibroblast(DF)-derivedexosomesontheexpressionofbiomarkersassociatedwithskinbarrier functions,FLG,IVL,LOR,andHAS1expressioninhumanskinkeratinocytes(HaCaT)cellswasassessedthroughimmunoblotting.(A)Protein expressionanalysisofhumankeratinocytes(HaCaTcells)treatedwithfibroblast-derivedexosomes.(B)Proteinexpressionassociatedwith fibroblast-derivedexosometreatmentof1-chloro-2,4-dinitrobenzene(DNCB)-treatedhumankeratinocytes.
exosomesderivedfromautologousDFswerefoundtomoreefficiently amelioratesenescence-relatedtissuedamageandpromotecutaneous woundhealing,whichindicatesthatHDF-exosomescanbepotential candidatesforprotectingandrepairingskindamage.Furthermore, exosomesisolatedfromconditionedmediaduringthree-dimensional dermalfibroblastculturewereshowntostimulateECMsynthesisand secretionfromdermalfibroblasts,aswellascellularmigrationandproliferation,inpartthroughinterleukin-6signaling.Theseresultsimply thatDF-derivedexosomesparticipateinnumerousstagesofdermal ECMformation,thuscontributingtoskindamagerepair,andillustratingtheirpotentialmechanism.10 However,nostudyhasinvestigated themechanismusedbyDFexosomesonskinpermeabilitybarrierprotection.Thisarticleattemptstoevaluatethetherapeuticsignificance ofDF-derivedexosomesonskinepidermalmarkerproteins.Hence,we evaluatedtheexpressionlevelsofskinpermeabilitybarriermaintenancebiomarkersinkeratinocytestreatedwithDNCB,askinirritant, andinducerofdermatitis,includingAD.
Thoughexosomecytotoxicityhasbeenthesubjectofsomeinvestigations,itisnowbelievedthatexosomesgeneratedfromfibroblasts donotexhibitconsiderablecytotoxicity.Thisisprobablybecauseexosomesarenaturallysecretedcell-derivedvesiclesthatareengagedin regularphysiologicalfunctions.4 However,itisstillessentialtoemphasizethatthesafetyandefficiencyoffibroblast-derivedexosomesin clinicalsettingsrequirefurtherinvestigation.Inourstudy,thecytotoxicityoftheharvestedexosomesfromDFsusingtheMTTassaywas evaluatedbeforedeterminingtheirrepairefficacy.Theoptimalconcentrationwasidentifiedfromfourdifferentexosomeconcentrations (1×104 ,1×105 ,1×106 ,and1×107 particles/ml).Ourdatashowedthat DF-derivedexosomesatconcentrationsof1×104 ,1×105 ,or1×106 particles/mldisplayedtheleastcytotoxicityafter24and48hof treatment(Figure 2).
DownregulatingtheexpressionofIVL,LOR,FLG,andHAS1 isthehallmarkfeatureofADskinlesionsandisassociatedwith epidermalbarrierdysfunction.8 Thisstudyrevealsthepositiveeffects
ofDF-derivedexosomesonskincellswhereexosometreatmentof keratinocytesstimulatestheexpressionoftheepidermalmarker proteinsFLG,LOR,HAS1,andIVL.Inthiswork,weestablishedanAD modelofkeratinocytestreatedwith5 µMDNCBtoinvestigatethe skin-repairingeffectivenessoffibroblast-derivedexosomesonDNCBtreatedskin.Ourfindingshowedthefibroblast-derivedexosomes restoredtheDNCB-inducedreductioninFLG,LOR,IVL,andHAS1 expressionlevels(Figure 3).
Notably,inthisstudy,theoptimalexosomeconcentrationwasdetermined,whichshowedthemaximumefficacyinexpressingbiomarkersrelatedtotheintegrityandinflammationofthepermeability barrierinHaCaTcells.Allthreeexosomeconcentrations(1×104 , 1×105 ,and1×107 particles/ml)haveshownsignificanteffectson proteinexpression,butthe1×104 particles/mlconcentrationwas depictedastheoptimum.Similarly,regardingtheADmodelofkeratinocytes,the1×104 particles/mlconcentrationhasbeenshownto beoptimumforrepairingdamagedskin,whichmaybeattributed toseveralfactors.Apossiblehypothesisisthattheexosomesmay becomeexceedinglyconcentratedathigheramountsandmaynot beadequatelyabsorbedbytherecipientcells,leadingtodecreased efficacy.
Inconclusion,fibroblast-derivedexosomesataconcentrationof 1×104 particles/mlweretheoptimumtoshowefficaciousnessin increasingskinepidermalbarrierproteins,thusincreasingtherecoveryrateofskindamagewithoutcausingcytotoxiceffectsinthisstudy. Basedonthesefindings,weconcludethatonlyfibroblast-derived exosomesatspecificconcentrationscanbeofclinicalusefortheir prospectiveapplicationintreatingskinbarrierdysfunctiondiseases likeAD.Toourknowledge,thiswasthefirststudyofitskindtoinvestigatehowDF-derivedexosomesrecoverskinbarrierfunctions.This studymightthusserveasafirststeptowardthedevelopmentof fibroblast-derived,exosome-basedtreatmentsforADclinically.Additionally,thisstudyonlyusedinvitroexperimentstoillustratethe effectsofDF-derivedexosomes;hence,weplantoconductfurther
experimentsandanimalstudiestodelineatetheexactmechanismsof actionunderlyingtheefficacyofDF-derivedexosomes.
ACKNOWLEDGMENTS
Theauthorshavenothingtoreport.
CONFLICTOFINTERESTSTATEMENT
Theauthorsdeclarenoconflictofinterest.
FUNDINGINFORMATION
None.
DATAAVAILABILITYSTATEMENT
Contactthecorrespondingauthorfordataavailability.
ETHICSSTATEMENT
Notapplicable.
KwanghoYoo1
NikitaThapa2 JongjinLee3 YounaJang4 JungOkLee4
JaeyoungKim2
1 DepartmentofDermatology,Chung-AngUniversityGwangmyeong Hospital,Chung-AngUniversityCollegeofMedicine,Gyeonggi-do,Republic ofKorea
2 CK-Exogene,Inc.,NewDrugDevelopmentCenter,Seoul,RepublicofKorea
3 DepartmentofRegenerativemedicine,DaesungHospital,Seoul,Republic ofKorea
4 DepartmentofDermatology,Chung-AngUniversity,CollegeofMedicine, SeoulCampus,Seoul,RepublicofKorea
Correspondence JaeyoungKim,CK-Exogene,Inc.,NewDrugDevelopmentCenter, #803,10,Gwangnaru-ro8-gil,Seongdong-gu,Seoul,04799,Republic ofKorea.
Email: caput@naver.com
ORCID
KwanghoYoo https://orcid.org/0000-0002-0137-6849
NikitaThapa https://orcid.org/0000-0001-6460-9673
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