Repair and regeneration of the respiratory system

2021-11-29 来源：欧得旅游网

CellStemCell

Review

RepairandRegenerationoftheRespiratorySystem:Complexity,Plasticity,

andMechanismsofLungStemCellFunction

BrigidL.M.Hogan,1,*ChristinaE.Barkauskas,2HaroldA.Chapman,3JonathanA.Epstein,4RajanJain,4ConnieC.W.Hsia,5LauraNiklason,6ElizabethCalle,AndrewLe,ScottH.Randell,7JasonRock,8MelindaSnitow,9MatthewKrummel,3BarryR.Stripp,10ThiennuVu,3EricS.White,11JeffreyA.Whitsett,12andEdwardE.Morrisey13,*

1Department2Division

ofCellBiology,DukeMedicine,Durham,NC27705,USA

ofPulmonary,AllergyandCriticalCareMedicine,DukeMedicine,Durham,NC27705,USA

3DivisionofPulmonaryandCriticalCare,DepartmentofMedicine,UniversityofCalifornia,SanFrancisco,SanFrancisco,CA94143,USA4DepartmentofCellandDevelopmentalBiology,UniversityofPennsylvania,Philadelphia,PA19104,USA5DepartmentofInternalMedicine,UniversityofTexas,SouthwesternMedicalCenter,Dallas,TX75390,USA6DepartmentsofAnesthesiologyandBiomedicalEngineering,YaleUniversity,NewHaven,CT06520,USA

7DepartmentofCellBiologyandPhysiology,TheUniversityofNorthCarolinaSchoolofMedicine,ChapelHill,NC27599,USA8DepartmentofAnatomy,UniversityofCalifornia,SanFrancisco,SanFrancisco,CA94143,USA9PerlemanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA19104,USA

10RegenerativeMedicineInstitute,Cedars-SinaiMedicalCenter,LosAngeles,CA90048,USA11DepartmentofInternalMedicine,UniversityofMichigan,AnnArbor,MI48109,USA

12SectionofNeonatology,PerinatalandPulmonaryBiology,DepartmentofPediatrics,CincinnatiChildren’sHospitalCenter,UniversityofCincinnatiCollegeofMedicine,Cincinnati,OH45229,USA

13DepartmentsofMedicineandCellandDevelopmentalBiology,UniversityofPennsylvania,Philadelphia,PA19104,USA*Correspondence:brigid.hogan@duke.edu(B.L.M.H.),emorrise@mail.med.upenn.edu(E.E.M.)http://dx.doi.org/10.1016/j.stem.2014.07.012

Respiratorydiseaseisthethirdleadingcauseofdeathintheindustrializedworld.Consequently,thetrachea,lungs,andcardiopulmonaryvasculaturehavebeenthefocusofextensiveinvestigations.Recentstudieshaveprovidednewinformationaboutthemechanismsdrivinglungdevelopmentanddifferentiation.How-ever,thereisstillmuchtolearnabouttheabilityoftheadultrespiratorysystemtoundergorepairandtoreplacecellslostinresponsetoinjuryanddisease.ThisReviewhighlightsthemultiplestem/progenitorpop-ulationsindifferentregionsoftheadultlung,theplasticityoftheirbehaviorininjurymodels,andmolecularpathwaysthatsupporthomeostasisandrepair.

Introduction

Thereparativebehaviorofadulttissuesfallsalonganinjury-responsespectrum.Atoneendofthescalearetissuessuchastheepidermis,intestine,andhematopoieticsystemwithaconstitutivelyhighrateofcellturnoverandawell-delineatedstem/progenitorcellhierarchy.Attheotherendareorgansliketheheartandbrainthatcontainfewstemcellsandcannotrepairefﬁciently,resultinginscarringafterinjury.Inbetweenthesetwoextremesaretissuessuchasthelung,liver,andpancreasthathavealowsteady-statecellturnoveryetcanrespondrobustlyafterinjurytoreplacedamagedcells.Thisremarkablecapacityhaspromptedstudiesintothemechanismsthatmediateinduc-iblerepair,aswellasstrategiestoharnessthemtherapeutically.ThisReview,writtenbymembersoftheNIH-fundedLungRepairandRegenerationConsortium(LRRC;http://www.lungrepair.org/)hasthreegoals:ﬁrst,toprovideanoverviewofthestem/progenitorcellsthatbuildtherespiratorysystemandtheirde-scendantsthatrepairtheadultorgan;second,tosurveysomeofthemolecularpathwaysregulatinglungstem/progenitorpop-ulations;andthird,tohighlightrecentdiscoveriesinlungregen-erationbiology,includingbioengineeringofthelung.

Stem/ProgenitorPopulationsinLungDevelopment

Themammalianrespiratorysystemconsistsofatree-likear-rangementofbranchedairwaytubesconnectedtoasingletra-cheaandterminatinginmillionsofdelicateandhighlyvascu-larizedgas-exchangeunitsknownasalveoli(Figure1).Theepitheliumliningthewholesystemiscontinuousandinitiallyarisesfromasmallregionofanteriorventralforegutendoderm,markedbythetranscriptionfactorNkx2-1.Bythetimetheorganismature,theepitheliumdifferssigniﬁcantlyalongtheproximal-distalaxis,bothincellularcompositionandstructuralorganiza-tionand,relatedtothis,instemcellcompositionandstrategiesforrepair.Mostofthelungmesenchymelikewisearisesfromasmallpopulationofmesodermcellsthatwillgenerateairwayandvascularsmoothmuscle,cartilage,myoﬁbroblasts,lipoﬁ-broblasts,andpericytes.Thedevelopmentandpatterningoflungendodermandmesodermhasbeenthetopicofseveralcomprehensivereviews(CardosoandWhitsett,2008;HerrigesandMorrisey,2014;MorriseyandHogan,2010;OrnitzandYin,2012;Shietal.,2009),andonlyrecenthighlightsarediscussedhere.

Fromthepointofviewofregenerativebiology,therearemul-tiplereasonswhystudyinglungdevelopmentisimportant.Forexample,somepretermbabiesarebornatthestageoflungdevelopmentwhenprogenitorsofalveolarstemcellsarebeinglaiddown(Blackwelletal.,2011).Perinatalinfectionsandinﬂam-mationthatdisruptalveologenesisandcausebronchopulmonarydysplasia(BPD)maythereforehavelong-termconsequencesthatmightbeavoidedifweknewmoreaboutunderlying

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Figure1.AnatomyoftheAdultHumanandMouseLungandExamplesofHumanLungPathology

Upperpanels:regionalepithelialhistologyinhumanandmouse.Leftpanel:thehumantrachea,bronchi,andbronchioles>1mmindiameterarelinedbyapseudostratiﬁedepitheliumwithbasal,multiciliated,andsecretorycells.Mucousgobletcellspredomi-nateinthelargerairways,andClubcellspredomi-nateinthesmallerairways.Individualneuroen-docrinecellsandneuroendocrinebodies(NEBs)arescatteredinthelargerairwaysandincreasedistally.Cartilage,smoothmuscle,andstromalcellsareassociatedwithintralobarairwaysdowntothesmallbronchioles.Thesimplecuboidalepitheliumliningtheterminalbronchiolesleadingintotheal-veoliispoorlycharacterized.ThealveoliarelinedbysquamousAEC1sandcuboidalAEC2s.Rightpanel:inthemouse,onlythetracheaandmainstembronchihavecartilageandapseudostratiﬁedmu-cociliaryepitheliumwithBCs.ThesmallerbronchiandbronchiolesarelinedbyasimpleepitheliumwithmulticiliatedandClubcellsandfewerneuro-endocrinecellsandNEBs.Theinsetillustratesamouselungtothesamescaleasthehumanlungintheleftpanel.Lowerpanels:normalandpathologichumanlung.(AandB)Imagesofthealveolarregionina2-month-oldinfantandanormaladultillustratethatalveolarnumberincreasespostnatallythroughsecondaryalveolarseptalcrestformation.(C)Inpulmonaryemphysema,septaldestructionandlossofalveolarcellsresultsinalveolarenlargement.(D)Inpulmonaryﬁbrosis,theterminalbronchiolesarepluggedwithmucus,alveolarepithelialmorphologyisabnormal,andalveolararchitectureisdrama-ticallyalteredbyﬁbroblasticdepositionofextra-cellularmatrix.(EandE0)Bronchiolitisobliteranssyndromeshowingmassiveinﬁltrationofimmunecells,severedisruptionofthesmallairwayepithe-lium,andthickeningoftheunderlyingsmoothmuscleandstroma(boxedregionmagniﬁedinE0).(F)Normalpseudostratiﬁedmucociliarybronchialepitheliumfromalungtransplantdonor.(GandH)Gobletsecretorycellhyperplasiaandsquamousmetaplasia,respectively,inchronicobstructivelungdisease.(A–E)and(F–H),respectively,arethesamemagniﬁcation.Scalebarin(A)is400mm.

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mechanisms.Moredetailedinformationaboutthemolecularidentityofdifferentcelltypesandtheirlineagespeciﬁcationcanalsoinformstrategiesforgeneratinglungcellsexvivofrompluripotentstemcells(PSCs)andprovidenewtoolstomarkandfollowthebehaviorofstem/progenitorcellsinmodelsofhu-manlungdisease.

BranchingMorphogenesisandProximal-DistalPatterningoftheEpitheliumOccurEarlyinLungDevelopment

Perhapsthebest-studiedphaseinlungdevelopmenttodateistheprocessofbranchingmorphogenesis,bywhichthetwopri-marylungbudsthatarisearoundembryonicday(E)9.5inthemouseand4–5weeksgestationinthehumangiverisetotheairwaytree.Thebudsarecomposedofasimpleendodermalepitheliumsurroundedbymesodermandavascularplexus.Thesetissuesareencasedinathinlayerofmesotheliumthatmakesatransientearlycontributiontomesenchymallineages(Dixitetal.,2013).Thebudsextendandbranchinapatternthatisinitiallyverystereotypicbutbecomeslesssoasdevelop-mentproceeds(Metzgeretal.,2008;MorriseyandHogan,2010;Shortetal.,2013).Alllungendodermalcellsinitiallyexpress

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Nkx2-1andthismarkerpersistsintotheadult.However,astheprimarybudsextendandbranch,distinctpatternsofgeneexpressionemergeintheendodermofthestalksversusthebuds.Thisproximal-distaldifferenceisexempliﬁedbytheexpressiondomainsofSox2andSox9,twotranscriptionfactorsrequiredforearlylungdevelopment.Sox2expressionisconﬁnedtotheproximalstalks,whileSox9isdynamicallyex-pressedinthemorehighlyproliferativecuboidalcellsofthedistalbuds(Changetal.,2013;Rockichetal.,2013).Manyothergenesaredifferentiallyexpressedinthetips,includingId2,whichen-codesabHLHtranscriptionfactor(Alanisetal.,2014;Herrigesetal.,2012;Rawlinsetal.,2009a).Originallineagelabelingex-perimentssuggestedamodelinwhichtheId2+tipcellsareapopulationofmultipotentprogenitors(Rawlinsetal.,2009a).Earlyindevelopmentthetipcellsgeneratedaughtersthattrans-locateintothestalksandgiverisetoSox2+precursorsofallthecelltypesintheintrapulmonarybronchiandbronchioles.Theseprogenyincludeneuroendocrinecells,multiciliatedcells,anddome-shapedsecretorycells(originallycalledClaracellsbutnowknownasClubcells).EvidencesuggeststhatNotchsignalingplaysakeyroleinthepatterningandspeciﬁcation

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Review

Figure2.PrenatalandPostnatalAlveologenesisintheMouse

Upperpanel:schematicofthecanalicularandsaccularstagesindicatingthatprecursorsofalveolarepithelialcellsarelaiddownbeforebirth.(Imageofwhole-mountE16lungimmunostainedforE-cadherinwasprovidedbyRossMetzger.)Adistalcanaliculartubuleadjacenttotheoutermesotheliumisshowningreaterdetail(boxes).Evidencesupportsamodelinwhichthepopulationofdistaltipcells(thatareSox9+/Id2+)isbipoten-tial,expressesmarkersofbothmatureAEC2andAEC1cells,andgivesrisetoAEC2s(Sftpc+,orange)andAEC1s(Pdpn+,green,andAGER+,yellow)throughaseriesofintermediatepro-genitors(Changetal.,2013;Desaietal.,2014;Rawlinsetal.,2009a;Treutleinetal.,2014).Duringthesaccularstagethedistaltubulesbegintobudintomultiplesacsthatareinclosecontactwithvascularendothelialcells.AfewSox9+/Id2+pu-tativebipotentialprogenitorsremainatthisstage.Lowerpanel:schematicrepresentationofpost-natalchangesinlungarchitecture.Thenewbornlunghasonlyprimarysepta.AroundP4secondarysepta(asterisks)developfromcrestsoftissuecontainingcapillaryandstromalcellsthatmigrateinfromthewallsandsubdividealveoli.Thestromalpopulationisincompletelydeﬁnedbutincludespericytes,ﬁbroblasts,lipoﬁbroblasts,andmyoﬁ-broblasts.Thelatterarethoughttobethemainproducersoftheelastindepositedatthetipofeachseptumandinthewalls,forminganinte-gratedﬁbroelasticnetwork.Inset(redrawnfromSiriannietal.,2003)showsaschematicoftheputative‘‘niche’’ofanAEC2stemcell.Thisin-cludesAEC1s,lipoﬁbroblasts,endothelialcells,pericytes,andextracellularmatrix(dashedlinerepresentsbasallamina,anddotsarecollagen,elastin,andmanyothercomponents).

ofthesedifferentcelltypes(Morimotoetal.,2012;Tsaoetal.,2009).LaterindevelopmenttheId2+tipcellsgiverisetoalve-olarcelltypes.MorerecentlineagetracingstudieswithaShh-

CreERallelesupporttheideathattipcellsaremultipotentprogenitors,al-thoughwhetheractivityofthiscredriverisrestrictedtothedistaltipatthisstageofdevelopmentwasnotdirectlytested(Desaietal.,2014).FuturestudiesusingSox9-CreERandotherprogenitorcell-speciﬁcdriversshouldfurtherreﬁneourunderstandingofthemultipotencyofdistaltipcells.

Alveologenesis:ACriticalStageinPreparingtheLungforGasExchange

AroundE15.0,branchingmorphogenesisslowsandimportantchangestakeplaceinthedistalepithelium(Figure2).Thetubesbecomenarroweror‘‘canalicular’’andmorecloselyassociatedwiththesur-roundingvasculature.Cellsinthedistalpartofthetubulesbegintoexpressgenescharacteristicofthetwoepithelialcelltypesthatmakeupthematurealveoli,

namelythetype1cells(AEC1s,whichexpressHopx,podoplanin(Pdpn,alsoknownasT1alpha),andAGER)andtype2cells(AEC2s,whichexpressproteinsassociatedwithhighlevelsof

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surfactantproductionandsecretion,suchasSftpA-C)(Treutleinetal.,2014).Subsequently,thetubulesenterthe‘‘saccular’’phasethatinvolvesthe‘‘budding’’oftinyperipheralsacssepa-ratedbyprimarysepta.Postnatally,thesesacsarefurthersub-dividedbysecondarysepta(Figure2).LineagetracingsuggeststhepresenceofbipotentialcellsinthetipsofthedistaltubulesthatcangiverisetobothAEC1andAEC2cells(Desaietal.,2014).Therehasbeenprogressindeﬁningmorepreciselythein-termediateprogenitorsinthelineagesbetweenbipotentialcellsandmatureAEC1andAEC2cells,andsingle-cellRNA-seqanal-ysishasemergedasapowerfultoolinthisendeavor(Changetal.,2013;Desaietal.,2014;Treutleinetal.,2014).Amajorquestionforthefutureisthenatureofthesignalsthatcontroltheonsetofthecanalicularandsaccularstages.Rolesforsig-nalsfromtheadjacentmesoderm,includingglucocorticoids,ret-inoicacid,Fgf,parathyroidhormone,andinsulin-likegrowthfactor1,havebeenproposed(Alanisetal.,2014;Changetal.,2013;ElAghaetal.,2014;Epaudetal.,2012;Moreno-Barriusoetal.,2006;Ramirezetal.,2000).Signalsfromthevasculaturemayplayimportantrolestoo,giventhecloseassociationbe-tweenendotheliumandendodermatthistime(Figure2).

Developmentofthealveolidoesnotstopatbirth;theirnumberandsurfaceareaincreasesdramaticallypostnatallyandcon-tinuesforweeks(mice)andmonths(humans)withtheformationofnewsecondarysepta(Figures1Band2).Thesecondaryseptaarisefromridgesonthewallofthealveolarsacsandthecapil-larieswithinthemundergoremodelingbymechanismsthatarepoorlyunderstood.Thecapillarymeshworkalsoincreasesinsizebyaprocessknownasintussusceptivemicrovasculargrowth(Burrietal.,2004).Onlyaverythinlayerofmatrix,asharedbasallamina,eventuallyseparatesAEC1sandthecapil-laryendothelialcells.Stromalcellsalsomoveintotheseptaanddifferentiateintopericytes,lipoﬁbroblasts,myoﬁbroblasts,andotherpoorlydeﬁnedlineages.Lipoﬁbroblastsappeartoasso-ciatecloselywithAEC2swhilemyoﬁbroblastslaydownelastinﬁbersatthetipoftheseptaandinthestalks.Theseﬁbersformanintegratednetworkthroughoutthealveolarregion,providingaﬂexibleandelasticscaffoldthatiscriticalformain-taininglungfunctionandforkeepingthesmallterminalairwaysandalveolarductsopen(Weibel,2013).Mechanicalforcesandphysicalstressareemergingaskeyregulatorsofalveolardevel-opment,maintenance,repair,andregrowth(reviewedinHsiaetal.,2004;WirtzandDobbs,2000).

EmbryonicMesodermContributestoLungDevelopmentandPromotesEpithelialDifferentiation

Themesodermalcomponentofthelungprimordiumplaysmul-tiplerolesinlungdevelopment(reviewedinHerrigesandMorri-sey,2014).ThecellsaroundthedistaltipsfunctionasacriticalsignalingpopulationproducingFgf10andothersignalingmole-culesthatdriveoutgrowthofthedistalbudsandbranchingmorphogenesis.Thisearlymesodermalsocontainsprogenitorsforspeciﬁccellpopulationsoftheadultlung.Amajorfocusofrecentstudiesistounderstandhowthesemesodermalderiva-tivescontributetoadultlungfunctionandtoregenerationandrepair.Recentcelllineagetracingexperimentshavedemon-stratedthattheearlymesodermharborsacardiopulmonarymesodermprogenitor(CPP)thatexpressesWnt2/Gli1/Isl1andcangeneratebothcardiacandlungmesodermalderivatives,includingcardiomyocytes,endocardium,pulmonaryvascular

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andairwaysmoothmuscle,andpulmonaryPdgfrb+pericyte-likecells(Pengetal.,2013).Asdevelopmentproceeds,CPPslosetheirabilitytocontributetothevariouslungmesodermalderivativesandbytheendofgestationcanonlymakePdgfrb+pericyte-likecellsandnotsmoothmuscleorendothelium.Thissamestudyalsoshowedthatthebulkofthevascularendothelialplexusinthealveolarregionisderivedfrompreexistingembry-onicendothelium,likelythroughanangiogenicprocess(Pengetal.,2013).SinceWnt2+andGli1+cellspersistintheadultlung,animportantquestioniswhetherthesecellsplayaroleinregenerationafterinjury.Gli1isadownstreameffectorofHedge-hogsignalingandcomponentsofthispathwayhavebeenas-sociatedinGWASstudieswithchronicobstructivepulmonarydisease(COPD)(Pillaietal.,2009;Wangetal.,2013a;Wilketal.,2009).Therefore,theHedgehogpathwayandGli1+cellsmayplayaroleinadultlunghomeostasisandinjuryresponsebycontrollingmesodermalexpansion.

LineagetracingstudieshavealsoshownthattheFgf10-pro-ducingcellsofthedistallunggiverisetomultiplemesodermalcomponentsintheadult,includingairwaysmoothmuscleandalveolarlipoﬁbroblasts(ElAghaetal.,2014).ThereisevidencethatFgf10isreactivatedinparabronchialsmoothmuscleafterinjury,includingexposuretonaphthalene,andmaypromotethereparativeprocess(Volckaertetal.,2011).

PreviousworkhasestablishedarequirementforPdgfra-ex-pressingcellsinthedistalembryoniclungforthedevelopment

ofalveolarmyoﬁbroblasts(Bostro

¨metal.,1996).RecentstudieshaveindicatedthatPdgfraisdynamicallyexpressedinalveolarmyoﬁboblastsduringregrowthafterpneumonectomy(PNX)(Chenetal.,2012b).Pdgfraalsomarksapopulationoflipoﬁbro-blast-likecellsthatarespatiallyassociatedwithAEC2cellsandlikelyformpartofthealveolarstemcellniche(Figure2)(Barkaus-kasetal.,2013).Studiessuchasthesehavebeguntoaddresssomeoftheoutstandingquestionsabouttheorigin,heterogene-ity,andfunctionoflungmesodermalcellsandhowtheyinteractwiththevariousepithelialcomponentstoestablishandmaintainnormallungfunctionandcontributetorepair.

ApplyingDevelopmentalPathwaystoDeriveLungProgenitorsfromPSCs

OneareaofresearchwheredevelopmentalstudiesarehavingabigimpactisinthederivationoflungcellsfromPSCs.SeveralgroupshavedemonstratedthatstepwiseapplicationofsignalingfactorsinamannerthatmimicsthesequenceofeventsduringearlyanteriorforegutandlungdevelopmentiscriticalforderivinglungendodermprogenitorsfrommouseandhumanPSCs.KeypathwaysincludethosedownstreamofActivin/Nodal,Wnts,Bmps,andFgfs(Ghaedietal.,2013;Huangetal.,2014;Longmireetal.,2012;Mouetal.,2012;Wongetal.,2012).PSC-derivedlungendodermcanbeusedforbasicstudiesofdiseasessuchascysticﬁbrosisandpo-tentiallyforcell-basedtherapies.Thisareaofresearchwillundoubtedlycontinuetobeanimportantfocusinlungregen-erationresearch.

RegionalEpithelialStem/ProgenitorCellPopulationsMediatingAdultLungHomeostasisandRegenerationThreeinterrelatedconceptshaveemergedfromrecentstudiesonadultlungreparativecellbiology.Theﬁrstisthat,dependingonthecompositionandorganizationoftherespiratory

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epithelium,distinctregionsofthelungcontaindifferentpopula-tionsofepithelialcellsthatfunctionasadultstemcells,asdeﬁnedbytheirabilitytoundergolongtermself-renewalandgiverisetodifferentcelltypesduringhomeostaticturnoverorcellreplacementafterinjury.Thesecondisthatlungcellsexhib-itingstem/progenitoractivityarenotnecessarilyundifferen-tiated.WhileTrp63+basalcells(BCs)inthepseudostratiﬁedmucociliaryepitheliumaremorphologicallysimple,AEC2cellsinthealveoliandClubcellsinmousebronchioles,bothofwhichfunctionaslong-termstemcells,alsoexpressgenesassociatedwithspecializedfunctions,suchassurfactantproteinsynthesisandsecretionandglycoproteinproduction,respectively.Thethirdconceptisthatinresponsetotissuedamage,epithelialcellsthatexpressmarkersofonedifferentiatedcelltypecan,undercertaincircumstances,changetheirphenotypeandgiverisetocellsthateithertransientlyorstablyexpressmarkerscharacter-isticofanothercelltype.Insomecaseslineagetracingexperi-mentshaveshownthatthisphenotypicswitchingorplasticityinvolvesaprocessof‘‘dedifferentiation’’toalessspecialized,multipotentandproliferativeintermediate,followedbyrediffer-entiation.Inothercasestheprecisestepsinvolvedhavenotyetbeenidentiﬁedanditremainspossiblethatthephenotypicswitchis‘‘direct’’anddoesnotinvolveaspeciﬁcundifferentiatedandproliferativeintermediate.Phenotypicplasticityisnotuniquetothelunganddedifferentiationortransdifferentiationappar-entlyoccurquitefrequentlyinresponsetoadverseeventsinvarioustissues(BlanpainandFuchs,2014;Fuhrmannetal.,2014).Importantly,theprocesscanbestronglyinﬂuencedbytheparticularkindofdamagesustained,whetheritisacuteorchronic,andwhetheritinvolvesinﬂammationorimmunemodulation.

Modernstudiesofstemcellbehavior,includinginvestigationofplasticityinthephysiologicalcontextofanadulttissueunder-goingrepair,areheavilydependentonthetechniqueoflineagetracing(BlanpainandSimons,2013).Inthefollowingsectionswesummarizesomeoftherecentﬁndingsaboutepithelialstem/progenitorcelllineagesindifferentregionsoftheadultlung,focusingonstudiesbasedonrigorouslineagelabelingstra-tegies.

StemandProgenitorCellsofthePseudostratiﬁed

MucociliaryAirwayEpitheliumContributetoRepairandRegeneration

Mostoftheairwaysofthehumanlung,downtoabout1.0–1.5mMindiameter,arelinedbyapseudostratiﬁedmucociliaryepitheliumcontainingmulticiliated,secretory,tuft,andneuroen-docrinecells,aswellasapopulationofBCsthataretightlyattachedtothebasallaminathroughhemidesmosomescontain-inga6b4integrin.TheheightoftheluminalcellsandpropertiessuchastheproportionofgobletanddifferentsecretorycellsandthedensityofBCsvaryalongtheproximal-distalaxis.Un-derlyingtheepitheliumarebloodvessels,smoothmuscle,carti-lage,stromalﬁbroblasts,andnerves.AsshowninFigure1andschematicallyinFigure3,thenormalorganizationofthemuco-ciliaryepitheliumisdisruptedincommonpathologicalcondi-tions.Inthecaseofmucushyperplasiatherearemanymoregobletcellsthannormal,whileinsquamousmetaplasiatherearemultiplelayersofBCsthatgiverisetokeratinizedsquamouscells.BothmucushyperplasiaandsquamousmetaplasiaareseenintheconditionknownasCOPD.

Asimilarbasicorganizationofpseudostratﬁedmucociliaryepitheliumandunderlyingmesenchymeisfoundinthemousetrachea(whichisabout1.5mMindiameter),extralobarbronchi,andtheﬁrsttwoorthreegenerationsofbranchesalongtheintra-lobarmainaxialpathway.Theseregionsthereforeprovideanexperimentalplatformformodelinghumanairways(Rocketal.,2010)(Figure3).TheBCsofthemouseproximalairwayscharacteristicallyexpressTrp63,Ngfr,Pdpn,thealpha1-3galepitopethatbindsGSIB4lectin,andcytokeratin5(Krt5)(Pauletal.,2014;Rocketal.,2009;Tataetal.,2013).Atsteadystateabout20%ofBCsalsoexpressKrt14(Coleetal.,2010).Thiscy-tokeratinisalsoconstitutivelyexpressedinBCsliningtheductsofsubmucosalglands(thatarenotconsideredfurtherhere)andisupregulatedinmostKrt5+BCsduringrepair(Hongetal.,2004;Wansleebenetal.,2014).ThenumberandproportionofKrt5+BCsdeclinewithageinthemousetrachea,whichoccursalongwithothersigniﬁcantchangesinepithelialarchitectureandcompositionofstromalimmunecells(Wansleebenetal.,2014).TheconsensusfrominvivolineagetracingexperimentsisthatBCsarestemcellsthatself-renewoverthelongtermandgiverisetociliatedandsecretoryluminalcellsduringpost-natalgrowth,homeostasis,andepithelialrepairafterlossofluminalcells(Figure3).Differentexperimentalmodelshavebeendevelopedtoinducethislossinthemouse.Forexample,exposingmicetotoxicgases(e.g.SO2,orchlorine)ordetergentkillsallproximalluminalcells,whilesystemicnaphthalenekillsClubcellsinthetracheaaswellasthebronchioles.TherearemanyquestionsaboutBCsthathavenotyetbeenfullyanswered.Forexample,wedonotknowwhetherallTrp63+/Krt5+BCsaremultipotent,withthesamereparativecapacity,orwhethertherearesubsetsthatarequiescent(forexampleKrt5+cellsversusKrt5+/Krt14+cells)orhaveintrinsicallydifferentpotentials.Thesequestionsawaitquantitative,single-cellclonalanalysisunderbothsteady-stateanddifferentrepar-ativeconditions.Inaddition,wedonotknowtheprecisemech-anismsbywhichBCsgiverisetociliatedandsecretorycellsandwhatenvironmentalsignalsaffectthefatedecision.Onemodel,basedonstudiesoftheintestinalstemcellsoftheDrosophilalarvalmidgut(LucchettaandOhlstein,2012;OhlsteinandSpra-dling,2007),holdsthatBCsgiverisetoTrp63À/Krt5+/Krt8+earlyprogenitorsor‘‘intermediate’’cellsthatcanproliferateandgiverisetoeithersecretoryorciliatedcellsdependingonlocalsignals,includingthelevelofintracellularNotchsignaling(Gusehetal.,2009;Rocketal.,2011b).Recentstudiesinthehu-manlunghavedescribedapowerfulnoninvasivelineagetracingmethodologyforaddressingthepotentialofindividualairwayprogenitorcellstoself-renewanddifferentiate(Teixeiraetal.,2013).TheresultsareconsistentwithamodelinwhichBCsareapopulationofmutipotentprogenitorsthatself-renewand/ordifferentiatestochasticallytomaintaintissuehomeostasis.However,theydonotruleouttheexistenceofaquiescentstemcellpoolthatisonlyactivatedafterinjury.

Onequestionofpracticalrelevanceforregenerativethera-piesinthehumanlungiswhetherBCsaretheonlycellthatcanefﬁcientlyrepairthepseudostratiﬁedepitheliumorwhetherdifferentiatedcellscanbecomeBCsundercertainconditions.Thisquestionhasbeenaddressedusingtherodenttracheaasamodel.PioneeringstudiesbyRandellandcolleaguesus-ingdenudedrattracheassuggestedthatGSIB4Àluminalcells

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Figure3.Homeostasis,Repair,andRemodelingofPseudostratiﬁedMucociliaryAirwayEpitheliumwithBCs

Solidlinesrepresenttransitionsorlineagesthataregenerallyaccepted,whiledottedlinesarespeculativelineagesorrelationships.Curvedarrowsrepresentself-renewal.Leftpanel:schematicrepresentationofpseudostratiﬁedmucociliaryepitheliumofthehumanlung.ThedensityofBCsandheightandcompositionofluminalcellsvariesalongthemainaxis.Afew‘‘intermediate’’cells,whichmayrepresentimmediateundifferentiatedprogenyofBCs,arepresent.Inmucushyperplasiathenumberofgobletcellsincreaseseitherbyproliferationofexistinggobletcellsorbydifferentiationofothersecretorycells.Insquamousmetaplasia,BCschangetheirbehaviorsothatthepoolofproliferativeKrt5+/Krt14+BCsexpandsandstratiﬁesandupperlayersdifferentiateintokeratinizedsquamouscells.Bothconditionsmaybereversible.Rightpanel:repairandremodelinginthemousetracheaandprimarybronchi.Sincefewgobletcellsarenormallypresentinmouseairways,theirincreaseinnumberinresponsetoimmunestimuliiscalledmetaplasiaratherthanhyperplasia.GeneticlineagetracinghasshownthatScgb1a1+cellsarethepredominantsourceofgobletcellsinresponsetoallergenexposure(Chenetal.,2009).IfluminalcellsarekilledbySO2,survivingBCsspreadandproliferate.TheygiverisetoapopulationofKrt8+progenitorsthatdifferentiateintociliatedandsecretorycells.Thereistransientinﬂuxofimmunecellsintotheunderlyingstroma.IfBCsarekilledgenetically,secretorycellslineage-labeledwithadriverfordifferentiatedcellproducts(Scgb1a1orAtpv1b)giverisetosomeoftheregeneratedBCsthatcontinuetofunctionasstemcells(Tataetal.,2013).InsetshowssomeofthenonepithelialcomponentsoftheBC‘‘niche.’’Inadditiontothebasallamina,theseincludeﬁbroblastsandvasculatureandimmunecells.Lowerpanel:summaryoflineagerelationshipsfromstudieswithbothmouseandhumanairways.InbothtissuesBCsareheterogeneousforexpressionofKrt14;whethertheseBCsareinterconvertableand/orwhetherKrt14+cellshaveahigherprobabilityofdifferentiationratherthanself-renewalisnotknown.Theexistenceofanintermediateprogenitorcellisinferredfromstudiesonrepair.ThiscellisKrt8+butmaytransientlyexpressKrt5/Krt14.BCsandtheirimmediatedaughtersgiverisetociliatedandsecretorycellsduringrepair(Rocketal.,2011b).Whethertheydirectlygiverisetoneuroendocrinecellsandgobletcellsorwhethergobletcellsonlyarisefromsecretorycellsisnotknown.SecretorycellsincludeScgb1a1+ClubcellsaswellasvariantClubcellsthatpredominantlyexpressotherproducts(seetext).Scgb1a1+cellscangiverisetociliatedcells.

werejustasabletoregeneratetheentireepitheliumaslectin+BCs(Liuetal.,1994).Subsequentlineagetracingstudiesshowedthatsecretorycellsexpressingsecretoglobin1a1(Scgb1a1;alsocalledCCSPorCC10)cangiverisetoKrt5+

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BCsafterSO2-inducedinjury,butthefrequencyofconversionwasverylow,probablybecausemostlyBCssurvivethisinjury(Rawlinsetal.,2009b).Toovercomethisproblem,RajagopalandcolleaguesusedastrategytospeciﬁcallykillKrt5+BCs

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Figure4.EpithelialCellsofMouse

BronchiolesandtheCellularResponsestoInjury

Schematicrepresentationofdifferentcelltypesinthemousebronchiolarepithelium.Allcellsarelikelyattachedtothebasallaminathrougha6b4integrin.ThefullheterogeneityofepithelialcelltypesisstillunderinvestigationandthepresenceofrareTrp63+cellsiscontroversial.Shownwithdashedarrowsareputativecells-of-originoftheKrt5+/Trp63+basal-likecellspresentin‘‘pods’’inthelungsofmiceafterinfectionwithH1N1in-ﬂuenzavirus.ThereissomeevidencethattheseKrt5+cellscontributetotheregenerationofdamagedalveoli,butmorelineagetracingdataarerequired.

inthemousetracheainvivo(Tataetal.,2013).UndertheseconditionstheyfoundthatdifferentiatedScgb1a1+orAtpv1b1+lineagelabeledsecretorycellscanundergodedif-ferentiationintoTrp63+/Krt5+BCs.TheseBCspersistoverthelongtermandbehavelikenormalKrt5+stemcells.Animportantquestionraisedbyallthesestudiesconcernsthemechanismsthatnormallyconstrainthepotentialplasticityofsecretorycells.DatafromthestudiesbyTataetal.usinginvitrocultureexperimentssuggestthatcontactwithBCspre-ventsluminalcellsfromdedifferentiation,buttheprecisemechanismsdrivingreprogrammingandsubsequentstemcellfunctionneedfurtherstudy.

Howbasalandluminalcellsrepopulateanairwaydenudedofepitheliumbyinjuryhasconsiderablerelevancetoproposalstobioengineerreplacementlungsorairwaysegmentsbyseedingcellsintodecellularizedlung‘‘scaffolds’’(seelatersection).Ev-idencefrominvivoinjury/repairmodelssuggeststhatifthebasallaminaisnotcompletelycoveredbyBCs,theunderlyingstromaproliferatesoutofcontrolandgivesrisetogranulationtissuecontainingﬁbroblastsandimmunecellsandblockstheairways(O’Korenetal.,2013).Thisﬁndingsuggeststhatthereisnormallyatightinterplaybetweentheairwayepitheliumandtheunderlyingstromathatkeepsﬁbrosisincheck.Identifyingthefactorsthatmediatethissignalingmaynotonlyinformstra-tegiesforbioengineeringreplacementpartsbutalsoprovideinsightsintorespiratorydisorders,suchasbronchiolitisobliter-ans,inwhichﬁbrosisrestrictssmallairwaysinthehumanlung(Figure1).

LungInjuryModelsRevealDifferentialRegenerative

CapacitiesofEpithelialCellsoftheMouseBronchioles

Thesmallintralobarairwaysofthemouselungarecalledbronchioles;theydonothavecartilagesoradedicatedsystemicbloodsupplyandaresurroundedbyairwaysmoothmuscleandﬁbroblasts(Figures1and4).TheepithelialliningisasimplecuboidalepitheliumcontainingFoxj1+multiciliatedcellsandneuroendo-crinecellsthatareusuallyclusteredintoneuroendocrinebodies(NEBs).Thebron-chiolesalsocontainaheterogeneous

populationofsecretorycellsthatarestillnotfullydeﬁned.Thebest-studiedareClubcellsthatinthematurestatehaveachar-acteristicdomedappearancewithvesiclescontainingthesecre-toglobinScgb1a1.TherehasbeenrecentprogressinidentifyingnewmarkersforClubcellsthatcanbeusedtofollowtheirdevel-opmentandfunctionalheterogeneity(Guhaetal.,2014).ClubcellsimmediatelyadjacenttoNEBsareresistanttonaphtha-lene-induceddepletionandcharacteristicallyexpresslowlevelsofScgb1a1buthighlevelsofScgb3a2anduroplakin3a(Upka3)(Guhaetal.,2012).

Usingstandardimmunohistochemicalanalysis,thebronchi-olesofthemouselungdonotappeartocontainBCs,butthepresenceofrareTrp63+cellshasnotbeenruledout.Thetransi-tionfrombronchiolestoalveolarsacsinthemouselungisknownasthebronchioalveolarductjunction(BADJ).Itcontainsafewcells(<1perBADJ)thatcoexpressScgb1a1andsurfactantpro-teinC(Sftpc)proteinsandareproposedasputativebronchioal-veolarstemcells(BASCs)(Kimetal.,2005)(Figures3and4).Itshouldbenotedthatthetransitionbetweenterminalbronchiolesandalveoliisquitedifferentinmouselungascomparedtohu-manlung(Figure1).

Turnoverofmousebronchiolarepitheliumisnormallyquitelow,butlineagetracingstudieshaveestablishedthatScgb1a1+cellsdoself-renewandgiverisetociliatedcellsoverthelongterm(Rawlinsetal.,2009b).Thus,theScgb1a1+populationmeetsthedeﬁnitionoflong-termstemcellsalthoughitdemon-stratesadifferentiatedphenotype.Moreover,asdiscussedhereandinthenextsection,thereisnowevidencethatthe

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Figure5.StemCellsoftheMouseAlveolarRegionandTheirRoleinResponsetoInjury

Schematicdiagramofnormalmousealveolarregionandchangeselicitedbyexposuretobleomycin.AtsteadystatethereislittlecellturnoverandAEC2sself-renewandgiverisetoAEC1swithlowfrequency.Bleomycindamagesmultiplealveolarcelltypesresultingintheexposureofdenundedbasallaminaandmatrix(dashedlines)andinﬂuxofimmunecells.Variousmesenchymalcellsproliferateandgiverisetomyoﬁbroblastsandlargeamountsofextra-cellularmatrix.Inthismodelevidencearguesagainstmyoﬁbroblastsbeingderivedfromepithelialcellsandtheﬁbrosisistransient(Rocketal.,2011a).BothAEC2sandScgb1a1+cellsintheBADJproliferateandgiverisetothemajorityoftheAEC2andAEC1cells(dottedcells)intheﬁbroticregions(Barkauskasetal.,2013).EpithelialcellsinthealveolarregionthatareSftpcÀ/b4integrin+mayalsobeasourceofreparativeAEC2s(Chapmanetal.,2011).Lowerpanel:schematicofdifferentcelltypesinvolvedinalveolarturnoverandrepairinthebleomycininjurymodel.Adetailedinventoryofepithelialmarkerscanbefoundfromsingle-cellRNA-seqstudies(Treutleinetal.,2014).

proliferationandphenotypeofScgb1a1+Clubcellscanchangeinresponsetocertaininjury/repairmodels(Figures4and5).SystemictreatmentofmicewithnaphthalenekillsClubcellsthatexpressthecytochromeCyp2f2.Ciliatedcellsspreadtocoverdenudedmatrixbutdonotproliferate,andtheepitheliumisrestoredbytheproliferationofnaphthalene-resistantClub

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cells,includingthoseadjacenttoNEBsandintheBADJ(Gian-grecoetal.,2002;Guhaetal.,2014).IthasbeensuggestedthattheNEBsprovideaspeciﬁcnicheforreparativeClubcells.However,deletionofneuroendocrinecellsdidnotaffecttheabil-ityofClubcellstoregenerateafternaphthaleneinjury(Songetal.,2012).LineagetracingstudiesusingaCgrp-CreERallelehaveprovidedevidencethatdifferentiatedneuroendocrinecellscanproliferateafternaphthalene-inducedlossofClubcellsandgiverisetolineagelabeledClubandciliatedcells.Thisconclu-sionistemperedbythefactthatthewash-outperiodbetweentamoxifenadministrationandnaphthalenetreatmentwasveryshort,sothatintheseexperimentsClubcellsupregulatingCgrpmighthavebeenlineagelabeled(Songetal.,2012).

Thedamagecausedbynaphthalenetreatmentisrelativelyspeciﬁcandappearstoberapidlyrepaired.However,muchmoreextensivedamageiscausedinthemouselungbyH1N1inﬂuenzaviralinfection(Kumaretal.,2011).Althoughthereisclinicalevidenceforrepairofthehumanlungafteracuteinﬂu-enzainfection(Toufenetal.,2011),precisemechanismsarestillpoorlyunderstood.Inthemouseitappearsthataquiteunex-pectedlevelofplasticitycanbeevokedinepithelialcellsinresponsetosignalsexpressedaftersuchviralinfection.Immu-nohistochemistryofmouselungafterinfectionshowsthepres-enceofepithelial‘‘pods’’containingKrt5+/Trp63+basal-likecellsbotharoundthebronchiolesandwithinthealveoliintheareasofgreatestdamage(Kumaretal.,2011)(Figure3).LineagetracingexperimentsusinganScgb1a1-CreERallelehavesug-gestedthatthepodsarisefromClubcells.However,intheseexperimentsthewash-outperiodbetweenadministrationoftamoxifenandviralinfectionwasagainquiteshort(7days).Consequently,anScgb1a1ÀcellactivatedbytheinjurymighthavebeenlabeledifittransientlyupregulatedScgb1a1earlydur-ingtherepairprocess(Zhengetal.,2014).Alternatively,thepodsmayarisefromasyetuncharacterizedprogenitorpopulationsinthebronchiolesand/orfromAEC2cellsinthealveoli(XianandMcKeon,2012).Krt5+podshavebeenidentiﬁedinmouselungsafterbleomycininjury(Zhengetal.,2014),butagain,theiroriginisnotyetresolved.Giventhehighrelevanceofinﬂuenzainfectiontohumanhealth,thisisanimportantareaofresearch.Morein-formationisneededabouttheextenttowhichtheKrt5+repara-tivecellsdoinfactcontributetoalveolarrepair,howthisisachieved,andwhetherthesamecelltypeortypesplayaroleinthehumanlung.

TheImportanceofInjuryModelsforStudyingStemandProgenitorCellsoftheAlveolarCompartment

ThemajorepithelialcelltypesofthegasexchangeregionarecuboidalAEC2s,specializedforsurfactantproteinproductionandsecretionandﬂat,highlyextendedAEC1s,specializedforgasexchange(Figure5).Cellturnoverisnormallyverylow,butnumerousstudieshaveshownchangesincellbehavior,includingproliferation,normaldifferentiation,andphenotypicplasticityinresponsetoavarietyofinjurymodels.Theseincludeexposuretonitricoxide,highlevelsofoxygen(hyperoxia),thechemotherapydrugbleomycin,cigarettesmoke,irradiation,andviralinfection.

Alveolarepithelialtype2cells(AEC2).Studiesmorethan40yearsagowithH3thymidinelabelingshowedthatAEC2sinadultmonkeysandratsproliferateinresponsetoinjurybyhyperoxiaandnitricoxideandgiverisetoAEC1s(Evansetal.,1975;

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Kaplanetal.,1969).Thiscapacityforself-renewalanddifferen-tiationofadultAEC2shasbeenconﬁrmedbyrecentinvivoge-neticlineagetracingstudiesusingCrerecombinasedrivenbygenesassociatedwithdifferentiatedfunctionssuchasSftpcandLyz2(LysM)(Barkauskasetal.,2013;Desaietal.,2014).Insteadystate,thereisrelativelylittleclonalexpansionofindividualAEC2sandverylittledifferentiationintoAEC1s.Afterinjuryofthealveolarregionbybleomycin(Rocketal.,2011a)andbyhyper-oxia(Desaietal.,2014),therateofdifferentiationoflineagelabeledAEC2sintoAEC1sismuchhigher.Tofollowthisregen-erativecapacityintheabsenceofconfoundingﬁbrosis,anewmodeloflunginjurywasdevelopedinwhichabout50%oftheAEC2cellsarespeciﬁcallykilledbyinducedexpressionofdiphtheriatoxin.Lineagetracing,coupledwithnewimagingtechniques,showedthatsurvivingAEC2cellsundergoclonalexpansion,withdaughtercellsdispersingamongneighboringal-veoli,mostlikelybyactivemigration(Barkauskasetal.,2013).TherateofdifferentiationintoAEC1sislow,presumablybecausethiscellpopulationisnotdamaged.

Theseobservationsraiseseveralinterestingquestions.Forexample,isthereheterogeneityintheAEC2populationordotheyallhavethesamecapacityforself-renewalanddifferentia-tion?Someevidenceforheterogeneitycomesfromthefactthatabout10%ofadultAEC2sarelineagelabeledinvivobyanScgb1a1-CreERknockinalleleandgiverisetoadenocarci-nomasafterKrasactivation(Xuetal.,2012).InadditionarecentreportsuggeststhatanSftpcÀ/integrinb4+celltypeexistsinthealveolithatcangenerateSftpc+AEC2andAEC1cellsafterinjury(Chapmanetal.,2011).AnotherimportantquestionisthenatureofthenicheinwhichAEC2sresideandwhetheritproducesspe-ciﬁcsurvivalandhomingsignalsafterlossofAEC2s.Toaddressthisquestion,assayshavebeendevelopedforclonalexpansionoflineagelabeledAEC2sin3Dorganoidculture.Inthisassay,AEC2sonlyproliferateinthepresenceofPdgfra+stromalcellsandtheyform‘‘alveolopheres’’containinglineagelabeledAEC1sexpressinganumberofmarkers,includingPdpn,Hopx,AGER,andAquaporin5(Barkauskasetal.,2013).Similarassaysarebeingusedtoidentifyfactorsmadebyothermeso-dermallineagessuchasendothelialcellsthattogetherwiththeﬁbroblastslikelycomprisethealveolarstemcellniche(Leeetal.,2014).Inturn,understandinghowAEC2sregulatethebehaviorofthenicheisalsoimportant,andrecentstudiessug-gestedaroleforBmp4andthrombospondininthereciprocalinteractionbetweenepithelialprogenitorsandendothelialcells(Leeetal.,2014).

ItishasbeenhypothesizedthatAEC2sthatarestressedduetocellulardamageoragingareunabletoundergoself-renewalandgenerationofAEC1sbutinsteadsignalﬁbroblasticprolifer-ation,oratleastfailtokeepstromalproliferationincheck.Inthehuman,stressmaybecausedbygeneticmutationsresultinginunfoldedsurfactantproteins.Inthediseasedyskeratosisconge-nita,environmentalstimulisuchassmokeordustcombinedwitharelativestemcelldeﬁciencyduetolackoftelomerasecompo-nentsresultsinpulmonaryﬁbrosis(reviewedinNobleetal.,2012).Identiﬁcationandcharacterizationofthesignalsgener-atedbyAEC2sthatkeepstromalpopulationsinaquiescentstatewillbeimportantforfuturedevelopmentoftherapiesforﬁ-broproliferativediseasessuchasidiopathicpulmonaryﬁbrosisandbronchiolitisobliterans.

Alveolarepithelialtype1cells(AEC1).PreviousstudieshavesuggestedthatAEC1snormallyhavealimitedproliferativeca-pacityinvivoandcanmodulatetheiridentitywhenculturedinvitro(Dantoetal.,1995;Evansetal.,1973).Inthefuture,noveltoolsincludinginducibleCrelinesmadeusinggenessuchasHopxandAGERexpressedinmatureAEC1s(Barkauskasetal.,2013;Takedaetal.,2013;Treutleinetal.,2014)mayallowtheabilityofthesecellstoproliferateandgiverisetoAEC2cellsinspeciﬁcinjurymodelstobeaddressedunambiguously.ThepotentialroleofchangesinAEC1s,AEC2s,andmesenchymalcellsinthedevelopmentofsmoking-andage-relatedemphy-sema,inwhichthereisadeclineinthenumberofAEC2sandsimpliﬁcationofalveoli(Figure1),isalsoanimportantavenueofinquiry.

RemodelingandRegrowthoftheAlveolarRegionafterPNXandBleomycinInjury

Uponreachingadulthood,cessationofribcageexpansioncoin-cideswiththecessationoflunggrowthinlargemammals.There-after,reinitiationoflunggrowth/regenerationrequireschangesinmechanicalsignalsandtheavailabilityofspaceforthelungtogrow.Thiscanbeprovidedexperimentallybytheprocessofuni-lateralPNX(Daneetal.,2013;Ravikumaretal.,2013,2014).Inthisprocedureinmice,thesingleleftlobeisremoved,leavingthefourrightlobesintact.Thisresultsintheregrowthor‘‘real-veolarization’’oftheremaininglungs.Newlobesarenotadded,butthereisadramaticincreaseinalveolarnumberthroughaddi-tionofnewseptaandarestorationinrespiratorycapacityintheremaininglungwithin2–3weeksforrodents.Newalveolartissueispreferentiallylaiddownattheperipheryofthelung(Fosteretal.,2002;MassaroandMassaro,1993),wheremechanicalstrainoftheseptaisaccentuatedduetoarelativelackofbron-chovascularsupport(Yilmazetal.,2011).EvidenceexistsforproliferationandchangesinthebehaviorofmultiplecelllineagesinthePNXmodel,includingbronchiolarcells,AEC2s,endothelialcells,andPdgfra+stromalcells(Chenetal.,2012b;Eisenhaueretal.,2013;Hoffmanetal.,2010;Hsia,2004;Thaneetal.,2014;Voswinckeletal.,2004).

EvidencepointstotheincreaseinproliferationafterPNXbeingregulatedbyacombinationofsignalingpathways,matrixcompositionandremodeling,especiallyrelatedtoelastin,andmechanicalforces.EarlystudiessuggestedthatEGFandFGFsignalingpromotepost-PNXalveolarregeneration(Kazaetal.,2000,2002),andthishasbeensupportedbylaterworkfocusingontheroleofpulmonarycapillaryendothelial(PCE)cellsinpro-ducingparacrinegrowthfactors(Dingetal.,2011).AfterPNX,thereisincreasedproliferationofPCEs,andtheiractivationofVEGFR-2andupregulationofFGFR1andMMP-14areneces-saryfortherestorationoflungmassandfunction.Despitetheseintriguingobservations,theprecisecellularmechanismsthatresultinthedramaticincreaseinalveolarnumberandlungca-pacityremainunclear.Detailedanatomicalstudies,includingscanningelectronmicroscopyofvascularcasts,suggestthatalveolarnumberincreasesbytheingrowthofcrestsoftissuecontainingcapillariesandstromalcellsfromthewallsofpreex-istingalveoli,aprocessthatmimicsnormalpostnataldevelop-ment(Figure2)(Ackermannetal.,2014).Elucidationofthemechanismsthatdriveregrowth,andhowtheyareattenuatedaslungcapacityisrestored,maysuggestpotentialtherapeutic

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interventionsforwhenrepairdoesnotoccurproperly.However,itwillﬁrstbeimportanttotranslateﬁndingswithsmallrodentmodelstolargeranimalmodelswithdifferentmechanicalandgrowthconditionsintheirlungs(Hsia,2004;Thaneetal.,2014).Aninjurymodelthatiswidelyusedinresearchisexposingmicetointratrachealbleomycin,achemotherapydrugthatcausesdamagetomultiplecelltypesinthealveoli,includingAEC1andendothelialcells.Oneoftheconsequencesofbleo-mycinexposureisatransientdisruptionofalveolararchitectureandﬁbrosis,togetherwiththeappearanceofmanymyoﬁbro-blastsandAEC2swithabnormalmorphology(Figure5).LineagetracingstudiesinthisinjurymodelhaveshowntwomainsourcesofreparativeAEC2sandAEC1s.SomeofthemarederivedfromSftpc+AEC2sinthealveoli,whileothersarederivedfromdif-ferentiatedScgb1a1+Clubcellsintheterminalbronchioles(Barkauskasetal.,2013;Chenetal.,2012a;Rocketal.,2011a;Tropeaetal.,2012).Dual-positiveScgb1a1+/Sftpc+cellsthatresideintheBADJ(calledBASCs)alsocontributetothisphenotypicconversion.However,knowingwhethertheyaremoreefﬁcientatthisthanneighboringClubcellswillrequirecell-speciﬁclineagetracingtools.Signiﬁcantly,ClubcellsdonotgiverisetoAEC2andAEC1cellsafterinjurytothealveolibyhy-peroxia.Thus,whileClubcellscanbeconsideredafacultativestemcellpopulationcapableofextensiveexpansionandpheno-typicﬂexibilityduringregeneration,thisdoesnotoccurinallinjury/repairmodels.

PathwaysthatPromoteLungRepairandRegenerationAmajorgoalintheﬁeldoflungrepairistodeﬁnethemolecularpathwaysthatregulatetheactivation,expansion,anddifferenti-ationofstemandprogenitorlineagesinresponsetoinjuryandrepair.Onestrategyistofocusoninteractionsbetweenepithelialandmesenchymalcellsinthereparativenichebecausecross-talkbetweenthesepopulationsisknowntobeanessentialpro-cessforproperdevelopmentofthelung.Arecentexampleofthisapproachisastudyshowingthatthrombospondin-1,expressedbylungendothelialcells,controlsthedifferentiationofasubpop-ulationofSca1+self-renewinglungepithelialcells(Leeetal.,2014).Bmp4activatedexpressionofthrombospondin-1inlungendothelialcells,whichinturnregulatedthedifferentiationofSca1+lungepithelialcells.However,thesourceofBmp4intheadultlunginvivowasnotdeterminedinthesestudies(Leeetal.,2014).

Othersignalingpathways,suchasWntandNotch,knowntoplayimportantrolesinstemcellself-renewalanddifferentiationalsoplaykeyrolesinlungrepairandregeneration.Wntsignalingisanessentialregulatorofearlylungendodermspeciﬁcationanddevelopmentandhasbeenimplicatedinregulatingregenerativeresponses.UsingWntreporterlines,severalgroupshavedemonstratedactivationofcanonicalWntsignalinginvariouscompartmentsinthelungundergoingactiveregrowthandregeneration(AlAlametal.,2011;Aumilleretal.,2013;Flozaketal.,2010;Hashimotoetal.,2012;Zhangetal.,2008).Intheairwayepithelium,Wntsignalingisactivateduponsecretorycelldepletionbynaphthalenetreatment.ActivationofWntsignalingthroughlossofthecriticaltranscriptionfactorGata6leadstoexpansionoftheputativeBASCpopulationafternaph-thaleneinjury(Zhangetal.,2008).However,postnataldeletionofb-catenin(Ctnnb1)inthismodeldidnotinhibitsecretorycell

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regeneration,suggestingthatcanonicalWntsignalingisnotessentialinthismodeloflungregeneration(Zemkeetal.,2009).Wntsignalingisalsoproﬁbrotic,andincreasedWntsignalinghasbeendemonstratedinidiopathicﬁbrosislesions(Chilosietal.,2003).LossofCtnnb1inpostnatalalveolarepitheliumleadstoincreasedﬁbrosisandalveolarepithelialcelldeath(Tan-joreetal.,2013).Moreover,blockingWntsignalingpharmaco-logicallycanreduceﬁbrosiscausedbybleomycintreatmentinmice,althoughwhetherthisisaneffectonalveolarepithelialormeseodermallineagesisunknown(Hendersonetal.,2010).Thus,theabilityofWntsignalingtopromoteproperrepairandregenerationafterinjuryiscontextdependentandchronicacti-vationcouldleadtoincreasedﬁbrosis.

TheroleforNotchsignalinginpromotingthesecretorycellphenotypeovertheciliatedcellphenotypeduringlungendo-dermdevelopment(Gusehetal.,2009;Tsaoetal.,2009)isreca-pitulatedintheresponseofBCsafterairwayinjury.NotchsignalingisessentialforBCself-renewalanddifferentiationofthesecellsintothesecretorycelllineageafterSO2-mediatedairwayepithelialinjury.IncreasedNotchactivationcanexpandthesecretorylineageattheexpenseoftheciliatedlineage(Rocketal.,2011a;Xingetal.,2012).Inrecentstudiesithasbeenfoundthatreactiveoxygenspecies(ROS)activateNotchsignalingbyactivatingNrf2(Pauletal.,2014).TheROS-NotchpathwayisimportantforBCself-renewalthroughregulationofcellproliferation,whichmaybecriticalformaintainingthecorrectnumberofBCsintheupperairwaysofmouseandhumanlungs.HistoneacetylationanddeacetylationactivitiesarealsoalteredinlungdiseasessuchasasthmaandCOPD.Asthmaticbronchialepitheliumdisplaysincreasedhistoneacetyltransfer-ase(HAT)activityanddecreasedhistonedeacetylase(HDAC)activity(Gunawardhanaetal.,2014).Corticosteroidtreatmentofasthmainducesacetylationandactivationofanti-inﬂamma-torygenesandrecruitmentofHDAC2complexestodeacetylateandsilenceproinﬂammatorygenes(Itoetal.,2002).COPDpa-tientbiopsiesshowacorrelationbetweendiseaseprogressionandlossofHDAC2expressionandactivity,butlowerHDACac-tivityisresistanttoanti-inﬂammatorysteroidtherapy(Itoetal.,2005).Inmice,geneticdeletionofHDAC1/2inthesecretoryepitheliumledtotheinductionoftumorsuppressorsRb1,p21/Cdkn1a,andp16/Ink4aafterinjury,whichresultedinreducedproliferationandapersistentlossofepithelialregener-ation(Wangetal.,2013b).Thislossinregenerationwaspersis-tent,indicatingthatHDAC1/2arerequiredforregenerationofse-cretoryepitheliumafternaphthalene-induceddepletioninthelung.HDACfunctionmayalsobeimportantforregulatingtheproperbalanceofAEC1andAEC2cellsduringdevelopmentandregeneration.Hopx,expressedbyAEC1sandbyalveolarprogenitors,recruitsHDAC2tonegativelyregulateAEC2-spe-ciﬁcgeneexpression(Yinetal.,2006).

HDACsarealsoknowntobindtononhistoneproteins.TheforkheadtranscriptionfactorsFoxp1/2/4areexpressedathighlevelsinthedevelopinglungepithelium(Luetal.,2002;Shuetal.,2007).TheinteractionbetweenFoxp1/2andHDACactivityplaysanimportantroleinlunginjuryandregeneration,asdemonstratedbytheresistanceofFoxp1/HDAC2doublehet-erozygousanimalstohyperoxicinjurymediatedbytheregula-tionofthecytoprotectivecytokineIL-6(Chokasetal.,2010).ThecombinedlossofFoxp1/4inthepostnatalScgb1a1+

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secretorylineageresultsinspontaneousdifferentiationofScgb1a1+cellsintoMuc5a/c+gobletcells(Lietal.,2012).Importantly,thisabnormalsecretoryepitheliumlackingFoxp1/4isunabletoregenerateafternaphthalene-inducedinjury,sug-gestingthatacquisitionofagoblet-likephenotypeimpairsairwaysecretorycellregeneration(Lietal.,2012).

DevelopmentalstudiesinthelunghaverevealedcriticalrolesformultiplespeciesofnoncodingRNAs,includingmiRNAsandlongnoncodingRNAs(lncRNAs),inepithelialbranchinganddifferentiation.TwomiRNAclustersplayimportantrolesinlungepithelialendodermproliferationanddifferentiation.miR17–92andmiR302–367arebothhighlyexpressedduringearlylungendodermdevelopmentbutaresigniﬁcantlydownre-gulatedorextinguishedbybirth(Tianetal.,2011;Venturaetal.,2008).SeveralofthemiRNAsinthesetwoclusterssharecom-monseedsequences.OverexpressionofthesemiRNAclustersresultsinincreasedlungepithelialproliferationbutinhibitionofdifferentiationwithincreasedexpressionofprogenitormarkers(Luetal.,2007;Tianetal.,2011).ThesedatasuggestthatthesemiRNAspromotetheearlylungprogenitorphenotypethatischaracterizedbyahighlyproliferative,undifferentiatedstate.SincemiRNAscanbeusedassmallmoleculetherapeu-tics,suchcapabilitiescouldbeharnessedtopromotelungregenerationviatherapeuticuseofmiRNAmimicsorantimiRs.ArecentstudyidentiﬁedhundredsoflncRNAsexpressedinthedevelopingandpostnatallung(Herrigesetal.,2014).Asigniﬁ-cantsubsetoftheselncRNAsislocatednearprotein-codinggenesincludingimportanttranscriptionfactorssuchasNkx2-1andFoxf1.OneoftheselncRNAs,calledNANCI,regulatesNkx2-1andisessentialforitsexpression,aswellasthatoftargetsofNkx2-1functionincludingSftpcandScgb1a1.GiventheimportantroleforNkx2-1inlungdevelopmentandpostnatalhomeostasis,lncRNAssuchasNANCIlikelyplayasimilarlyimportantroleinpostnatallunghomeostasisandrepair.

ImportanceoftheImmuneResponsetoLungInjuryRepair

Thelungcontainsanimportantpopulationofmesodermalcellsthatarisefromthehematopoeiticlineagebutresideforsigniﬁ-cantperiodswithinthelungparenchymaandwithinalveoli.Sig-niﬁcantevidenceisemergingforacomplexorchestrationbythesecellsduringrepairandregeneration.Recentlive-imagingapproacheshavehighlightedresidentpopulationsofneutrophilsandmonocytesatsitesofalveolarinjuryandhighlylocalizeddamageinresponsetoeitherischemiareperfusionorTLRsignaling(Kreiseletal.,2010;Looneyetal.,2011).Whileimme-diatedamageislikelyaproductofneutrophilextravasationandperhapsmyeloid-mediatedbreakdownofalveolarepithe-lium,theimmuneresponseisalsolikelyprotective.Forexample,alveolarCD11c+cells(macrophagesanddendriticcells)utilizeConnexin43(Cx43)-mediatedtightjunctionstocommunicatewiththeepitheliumandparticipateincalciumwavesthatpropa-gateacrossdamagedepithelium.InteractionsofsomeCD11ccellsinthiswayareapparentlyprotectivebecauseCD11c-drivendeletionofCx43resultedinaugmenteddamageinresponsetoTLRsignals(Westphalenetal.,2014).Itremainstobedeterminedwhethermyeloidcellswithamore‘‘M2’’pheno-typeassociatedwithTGFbandIL-10productionhavespeciﬁc

rolesininjury/repair,andindeedwhethermacrophagefunctioninthelungfallsunderthecontroloftheadaptiveimmuneresponse.EarlydatausingmicethatarepartiallydepletedforregulatoryTcellsindicatedthatthesecellsregulatedtheresolu-tionoflunginjury,butthemechanismandextentofthisremainsunclear(D’Alessioetal.,2009).

BioengineeringLungTissue

Thecurrentclinicalapproachforreplacementofdiseasedlungtissueisallogeneiclungtransplantation.However,thisproce-dureremainslimitedduetoarelativeshortageofdonororgans,immunologicrejectionbytherecipient,andcomplicationsduetointenseimmunosuppressionnecessarytoavoidrejection(Lauetal.,2004).IntheUnitedStates,5-yearpatientsurvivalafterlungtransplantationisapproximately50%,comparedtoabout70%forliver(Wangetal.,2014).

EngineeredTrachealReplacements

Engineeredairways(tracheaandbronchi)havebeeninuseforoveradecade,theﬁrstbeingdesignedtorepairatracheobron-chialanastomosisaftersurgeryformalignancy(Macchiarinietal.,2004).Sincethisinitialreport,fullyengineeredtrachealsegmentshavebeenmadeusingcadavericdonordecellularizedmatrices,uponwhichrecipient-derivedcellswereculturedtogenerateanautologousairwaygrafttorepairtracheobronchialmalacia(Macchiarinietal.,2008).Syntheticscaffoldshavealsobeenexploredforuseastrachealreplacements.Clinicaltrialsofsolidprostheseshaveincludedmaterialssuchasstainlesssteel(Cottonetal.,1952),steelcoil(Beattieetal.,1956),silicone(Nevilleetal.,1990),polyethylene(Clagettetal.,1952),Teﬂon(EkestromandCarlens,1959),andhydroxylapatite(Hiranoetal.,1989).However,mostsolidprostheseseventuallyfailtobecomewellintegratedwiththesurroundingtissueandcauseproblemswithinfection,dislodgement,migration,andobstruc-tionwithgranulationtissue(Grillo,2002).Whole-LungEngineering

Morerecently,whole-lungengineeringhasbeenattempted,albeitinanimalmodelsonly(Ottetal.,2010;Petersenetal.,2010).Theseapproachesmakeuseofwhole-lungdecellulariza-tionstrategies(Priceetal.,2010)followedbyreseedingairwaysandvesselswithprimaryepithelialandvascularendothelialcells,respectively,fromsyngeneicanimals.Afterculturewithinabioreactor,theorganswerereimplantedintosyngeneicrecipi-entsanddemonstratedefﬁcientgasexchange(Ottetal.,2010;Petersenetal.,2010).However,thelungsultimatelyfailafterseveralhourstodaysduetoacombinationofintravascularcoagulation(likelyduetoincompleteendothelializationofthede-cellularizedvasculature)anddefectsinbarrierfunctionleadingtoexudationofﬂuidintotheairways.

Morerecently,strategiesfordecellularizingwholelungsfrompigs,nonhumanprimates(Bonvillainetal.,2013),andhumans(Boothetal.,2012)havebeenestablishedthatpavethewayforrecellularizationstudies(Gilpinetal.,2013;Nicholsetal.,2013;Wagneretal.,2014).Althoughthesescaffoldsprovideanatomicaccesstotheairwayandvascularcompartments,onekeychallengewillbedeliveryofmesenchymalpopulationstotheinterstitiumandparticularlythedelicatesepta.Regionallyspeciﬁcmesenchymalpopulations,coupledwithdistinctextra-cellularmatrixcues(Hinenoyaetal.,2008;Sannes,1984),maysigniﬁcantlyenhancetheoutcomesofepithelialrepopulationof

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decellularizedmatrices.However,thecomplexityofhumanlungwillmakethisadauntingtaskformanyyearstocome.Conclusions

Muchprogresshasbeenmadeinrecentyearsindeﬁningthecelllineagesthatcontributetowardlungrepairandregeneration.Thefocusofmostofthisworkhasbeenontheepithelium,givenitsessentialfunctioninthelung.However,othercellularconstitu-ents,includingthelymphaticandvascularsystemsaswellastheimmunesystem,almostcertainlyplayimportantrolesinrepairandregeneration.Determiningtheircontributionisanimportantareaoffutureresearch.

Recentevidencesuggeststhatlungepitheliallineagesthatun-dergolong-termself-renewalanddifferentiationarethemselvesfunctionallydifferentiatedcells.ExamplesareScgb1a1+ClubcellsandSftpc+AEC2s.Moreover,theseandotherepithelialcellscanundergophenotypicswitchesinresponsetotissuedamageandtheresponsecanvaryaccordingtothetypeandseverityofinjury.Theseconceptshavehadanimportantimpactonourunderstandingofstem/progenitorbiology,notonlyinthelungbutinothertissuesaswell(BlanpainandFuchs,2014).Howthisplasticityisregulatedisanexcitingareaofinquiry.

Theidentiﬁcationofstem/progenitorlineagesandactivityintheadultlunghasproceededmuchfasterthanourunderstand-ingofthemolecularpathwaysthatregulatetheircellbehavior.Theconceptthatdevelopmentalpathwaysarereactivatedandplayimportantrolesinlungrepairandregenerationrequiresadditionaltesting,especiallyinmorephysiologicallyorclinicallyrelevantmodelssuchasinﬂuenzainjury.Thesestudieswillrequireadditionalinformationonhowstructuresareformedinthelung,inparticulartheprocessofalveologenesis,whichisstillpoorlyunderstood.Thisisatopicthatwillgreatlybeneﬁtfromadditionalresearchintothedevelopmentandmaturationofthelung.Importantly,theadventofnewexvivomodelsoflungstemcellactivitysuchastracheospheresandalveolospheresshouldallowfortestingoffactorsthatcanpromoteeitherstem/progenitorself-renewalordifferentiation.

Newtechniquessuchasafunctionalengraftmentassayarealsocriticalfortestingthetrueabilityofdifferentstem/progenitorlineagesthathavebeenandwillcontinuetobeidentiﬁedinthelung.Thiswillentailabetterunderstandingofthenichesthatlungstem/progenitorcellsresidein,whichwillincludedeﬁningtheroleofextracellularmatrixandanimprovedunderstandingofcell-cellsignalingmechanismsimportantforregionalcellbehaviorafterinjuryorindisease.Alloftheseoutstandingissueswillrequirearenewedcommitmenttobasicscienceaswellastheongoingpushtobetterunderstandhowanimalmodelscanbeusedtounderstandhumanlungdiseaseandregeneration.Muchhasbeengainedfromaninvestmentintobasiclungdevel-opmentandthereremainsmuchstilltobediscovered.Onlybycombiningthestrengthofsuchbasicstudiesalongwithexplora-tionofhowthehumanlungrespondstoinjuryandinsultcantheﬁelddevelopnewstrategiesandtherapiesforcombatinghumanlungdisease.

ACKNOWLEDGMENTS

Theauthorsapologizefortheomissionofanyreferencesduetothespacecon-straintsofthisReviewandwishtothankmembersoftheirlaboratoriesforinput

134CellStemCell15,August7,2014ª2014ElsevierInc.

Review

CellStemCell

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