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(MasterYodaStarWars,1977)

UniversityofAlberta EvaluationofMesenchymalStemCellBasedTherapies forInflammatoryLungDiseases by LaviniaIulianaIonescu AthesissubmittedtotheFacultyofGraduateStudiesandResearch inpartialfulfillmentoftherequirementsforthedegreeof DoctorofPhilosophy DepartmentofPhysiology LaviniaIulianaIonescu Fall2012 Edmonton,Alberta
PermissionisherebygrantedtotheUniversityofAlbertaLibrariestoreproducesingle copiesofthisthesisandtolendorsellsuchcopiesforprivate,scholarlyorscientificresearch purposesonly.Wherethethesisisconvertedto,orotherwisemadeavailableindigitalform, theUniversityofAlbertawilladvisepotentialusersofthethesisoftheseterms. Theauthorreservesallotherpublicationandotherrightsinassociationwiththecopyright inthethesisand,exceptashereinbeforeprovided,neitherthethesisnoranysubstantial portionthereofmaybeprintedorotherwisereproducedinanymaterialformwhatsoever withouttheauthor'spriorwrittenpermission.

Dedication TothosewhohavegivenmeallthechancestobewhoIam.Tomy grandfather,IonLeulescu,whohasdefinedmebeforeanythingelsecouldhave. ImisshimtothisdayandmystrongestwishistobewhohebelievedIwillbe. Tomymother,CarmenLeulescu,whoisquietlyencouragingeverythingabout me.Tomygrandmother,NiculinaLeulescu,whoisnotquietaboutthevalueof life.Tomyyoungerbrother,LeontinIonescu,forteachingmethejoyandtrust ofsharingbybeingfirsttoshare.Tothosewhosmiledwithme,tothosewho smiledforme,tothosewhoweretoostubborntoabandontheuncertainpath bymysidethroughoutmystruggles. ToGeorgiana,fortrustfullybeingthereforsolong.ToRaluca,forbeing therolemodelsisterbychoice.ToBev,forbeingagenuineembodimentofjoy. ToFarah,forherunabashedpatience.ToAnda,forbringinganimprobable friendshipalive.ToAhmed,forhavingthecouragetochallengemyfears.To Mamoona,forknowingmebeforeIdid.ToMira,forbeingwithinmyreach.To Ioana,forhertimelypresenceinmylife.ToMdlina,forhersoftspoken strength.ToVijay,forhispersistenceinbelievingIwouldunderstandwhoIam. Tobothmyhomelands. Thankyou.

ABSTRACT Recent discoveries in stem cell biology have generated enthusiasm about the possibility of harnessing stem cells for organ repair and regeneration.Theabilityofpluriandmultipotentstemcellstodifferentiate along various cellular lineages has placed them at the core of research that seeks to protect endogenous stem cell populations or to deliver exogenous stem cells to sites of organ injury. Lung diseases are a major health care concern and the prevalence of chronic lung diseases such as asthma, pulmonaryfibrosisandchronicobstructivepulmonarydiseaseisexpectedto continuetoriseoverthenextdecades.Meanwhile,improvedperinatalcare has allowed the survival of extremely premature infants that constitute a particularly vulnerable subpopulation because of their risk of developing bronchopulmonarydysplasia(BPD)withpotentiallifelongcomplications. Researchthataimstoevaluatethetherapeuticpotentialofexogenous stemcellsinlungdiseasesplacedaninitialemphasisontheengraftmentand differentiation of these cells in the lung. More recent studies demonstrate that multipotent stem cell populations, such as mesenchymal stromal cells (MSCs), exert paracrine activity that can modulate local inflammatory and immune responses in experimental lung disease models including asthma, acutelunginjury(ALI)andpulmonaryfibrosis.Thestudiespresentedhereby demonstrate that factors secreted by adult bone marrowderived MSCs can preventthedevelopmentofinflammatorylungdiseasesinmouse modelsof

asthmaandALIandprovidemechanisticinsightintotheantiinflammatory propertiesofMSCs. Theperspectiveofusingpluripotentstemcellsastherapeutic agents hasbeenrevivedbythelandmarkdiscoveryofinducedpluripotency,where embryonic stem cell (ESC)like cells can be generated by reprogramming terminallydifferentiatedsomaticcells.Whilethefieldofinducedpluripotent stem cell (iPSC) biology is still in its effervescent infancy, this finding may relievemanyESCsrelatedethicalconcernsandmayopenthewaytolarge scale production and evaluation of pluripotent stem cells for organ regeneration and repair. The additional study presented provides proofof conceptfortheutilityofiPSC. Together,thestudiespresentedherebyadvocatethepotentialofstem cellsasanovelclinicaloptionforthetreatmentofseverelungdiseases.

ACKNOWLEDGMENTS
My gratitude goes toward all those who made the work presented here

possible:Dr.BernardThbaud,mysupervisor;Dr.MarekDuszyk,whohasinitially cosupervised me when I was a starting Master of Science program student; Dr. Christopher I. Cheeseman and Dr. Harissios Vliagoftis, my Supervisory Committee members, who have thoughtfully contributed to designing my research path; our collaborators, particularly Dr. Zamaneh Kassiri; Dr. Lisa Cameron (University of Alberta); Dr. Kenneth Walsh and Dr. Tamar Aprahamian (Boston University), Dr. JamesEllis(UniversityofToronto);Dr.ValentinDu,whoseadviceacceleratedthe developmentofthemousemodelofasthma;manycurrentandformermembersof Dr.ThbaudsandDr.Vliagoftislaboratories:FarahEaton,BeverlyMorgan,Dr.Arul Vadivel,Dr.RajeshAlphonse,MelanieAbel,ThurayaMarshall,Dr.PaulWaszak,Dr. Narcy Arizmendi, Dr. Gaia Weissmann; staff members, especially Bronwyn Appleyard (Department of Pediatrics), Lynette Elder, Alana Eshpeter (Alberta DiabetesInstituteHistologyCore),DorothyKratochwilOtto(UniversityofAlberta flowcytometry facility), Dr. Nathan Bosvik and Dr. Greg Parks (Health Sciences Laboratory Animal Services), Drew Nahirney (Dr. Duszyks laboratory), Dr. Wilma SuarezPinzonfortheirworkandadvice. I would also like to thank the Department of Physiology (Dr. Keir Pearson,

Sharon Orescan, Barb Armstrong) and gratefully acknowledge the support I have receivedfromtheCIHRMFNStrategicTrainingProgram(PaulJacquier).

TABLEOFCONTENTS CHAPTER1INTRODUCTION.1 1.1.Overview..2 1.2.LungDevelopmentandRegeneration.......3 1.3.StemCells...8 1.4.ResidentLungStem/ProgenitorCells.....13 1.5.TherapeuticPotentialofExogenousStem/ProgenitorCells.15 1.5.1.CellReplacementbyMSCs........15 1.5.2.CellReplacementVersusParacrineActivityofMSCs.17 1.5.3.ESCsandInducedPluripotentStemCells(iPSCs)......19 1.5.4.StemCellsandCarcinogenesis.......20 1.6.BiotechnologyEngineeringLungTissue22 1.6.1.HumanExVivoLungProject(HELP)..22 1.6.2.ArtificialLungNovaLung......23 1.6.3.BioengineeredLungTissue....24 1.7.ClinicalStudies:Experience,Outcome,Limitations25 1.8.References......30 CHAPTER 2 AIRWAY DELIVERY OF SOLUBLE FACTORS FROM PLASTICADHERENT BONE MARROW CELLS PREVENTS MURINE ASTHMA....62

2.1.Abstract......63 2.2.Introduction....65 2.3.MaterialsandMethods.67 2.3.1.BMCsIsolationandCulture...67 2.3.2.LungFibroblastsIsolationandCulture..69 2.3.3.FluorescenceActivatedCellSorting.69 2.3.4.BMCsandLungFibroblastsCdMPreparation...70 2.3.5.AnimalModel..72 2.3.6.BronchoalveolarLavageFluid(BALF)analysis.73 2.3.7.LungFunctionTesting(LFT)...74 2.3.8.CytokineQuantification....75 2.3.9.LungHistologicalAnalysis.....76 2.3.10.StatisticalAnalysis....77 2.4.Results.78 2.4.1. BMCs Differentiation Along Mesenchymal Lineages and SurfaceMarkerProfile78 2.4.2. BMCs CdM Prevents Airway Inflammation in Experimental Asthma.....78 2.4.3. BMCs CdM Prevents AHR in Experimental

Asthma.79

2.4.4. BMCs CdM Improves Bronchial Responsiveness to Salbutamol and Prevents Chronic ASM Thickening and PeribronchialInflammation.......80 2.4.5.BMCsCdMAttenuatesThelper2(Th2)Lymphocytes CytokineResponse...81 2.4.6. APN Mediates Protective Effects of BMCs CdM on AHR...81 2.4.7. APN Mediates Protective Effects of BMCs CdM on AirwayRemodeling..82 2.4.8. BMCs CdM Induces Subsets of IL10Producing T RegulatoryCells(Tregs)andMacrophages..83 2.5.Discussion.84 2.6.References94 2.7.Figures.....108

CHAPTER 3 STEM CELL CONDITIONED MEDIUM PREVENTS ACUTE LUNGINJURYINMICE:INVIVOEVIDENCEFORSTEMCELLPARACRINE ACTION125 3.1.Abstract...126 3.2.Introduction..128 3.3.MaterialsandMethods......130

3.3.1. MSCs and Lung Fibroblasts Isolation, Culture and Characterization.....130 3.3.2.ConditionedMedium(CdM)Preparation...131 3.3.3.MurineLPSInducedALI...132 3.3.4.BALFAnalysisandAlveolarMacrophages(AM)Isolation..133 3.3.5.AssessmentofLungPermeability...134 3.3.6.LungHistologicalAnalysis...134 3.3.7.AMLPSExposureandAMPhenotypeCharacterization135 3.3.8.AntibodyArrayofMSCCdM.......137 3.3.9.StatisticalAnalysis....137 3.4.Results.....138 3.4.1.MSCsDisplayFunctionalCharacteristicsandSurfaceMarker PhenotypeofMurineMSCs..138 3.4.2. MSC CdM Decreased Lung Inflammation and Lung Vascular PermeabilityinLPSInducedLungInjury...138 3.4.3. MSC CdM Failed to Prevent LPSInduced Body Weight Loss.139 3.4.5.MSCCdMImprovedLPSInducedLungInjury139 3.4.6. MSC CdM Determined Alternative Activation of AMs FollowingLPSExposureInVitroandInVivo.139 3.4.7. MSC CdM Contains Soluble Factors That May Convey TherapeuticBenefit...140

3.5.Discussion.........142 3.6.References.....149 3.6.Figures.....161 CHAPTER4GENERALDISCUSSION..171 4.1.Overview....172 4.2.StudyLimitations..175 4.3. Conclusions and Future Perspectives on Lung Regenerative Therapies.177 4.4.References.....180 APPENDIX 1 INDUCED PLURIPOTENT STEM CELLS (IPSC) DIFFERENTIATE INTO ALVEOLAR EPITHELIAL CELLS IN VITRO AND PREVENTHYPEROXIAINDUCEDLUNGINJURYINVIVO...187

LISTOFTABLES Table11.Summaryofputativeendogenouslungstemcell populations29 Table21.Cytokineprofileintheacuteasthmamodel.107 Table22.Cytokineprofileinthechronicasthmamodel....108

LISTOFFIGURES Figure22.MesenchymallineagedifferentiationofBALB/cBMCs...108 Figure 23. Flowcytometric characterization of BALB/c BMCs surface markerprofile..109 Figure24.BALFanalysis....111 Figure25.InvasiveLFT...113 Figure26.Bronchodilatorresponsetosalbutamol..115 Figure27.Airwayremodelinginchronicasthma...116 Figure28.EffectsofAPNonAHR.118 Figure29.EffectsofAPNonchronicairwayremodelingparameters120 Figure210.FACSoflunganddraininglymphnodeslymphocytes..122 Figure211.FACSoflunglymphocytes.....123 Figure 212. FlowcytometricevaluationofCD45,Sca1,CD29expression byWTBMCs,CD45+BMCsandBMMSCs...124 Figure 31. Mesenchymal lineage differentiation of C57BL/6 BM MSCs.......................................................................................................................................162 Figure 32. Flowcytometric characterization of C57BL/6 BMMSCs surfacemarkerprofile...163 Figure33.BALFandlungpermeabilityanalyses...164

Figure 34. LPSinduced body weight loss and lung histological assessment....166 Figure35.AMpolarizationfollowinginvitroLPSexposure....168 Figure36.AMpolarizationfollowinginvivoLPSadministration.....169 Figure37.MSCsecretomeanalysis....170 Figure 38. Factors up or downregulated in MSC compared to LF secretome...171

LISTOFABBREVIATIONS ALI acutelunginjury airwayhyperresponsiveness alveolarmacrophage analysisofvariance allophycocyanin adiponectin acuterespiratorydistresssyndrome arginase1 alveolarepithelialtype2cells airwaysmoothmuscle alveolarepithelialtypeIcell alveolarepithelialtypeIIcell bronchoalveolarlavagefluid bronchoalveolarstemcell BlymphomaMoMLVinsertionregion1homolog plasticadherentbonemarrowderivedstromalcell bonemarrowderivedstromalcell bronchiolitisobliteransorganizingpneumonia bronchopulmonarydysplasia protooncogenec(tyrosineproteinkinase)Kit

AHR AM

ANOVA APC

APN ARDS Arg1 AT2 ASM AT1 AT2 BALF BASC Bmi1 BMC BMSC BOOP BPD ckit

CCL

chemokine(CCmotif)ligand Claracellsecretoryprotein(also:CC10,Scg1b1) conditionedmedium clusterofdifferentiation cysticfibrosistransmembraneregulator calcitoningenerelatedpeptide chitinase3like3(also:ECFL,Ym1) chronicobstructivepulmonarydisease chemokine(CXCmotif) chemokine(CXCmotif)ligand disabilityadjustedlifeyears DulbeccosmodifiedEaglemedium deoxyribonucleicacid Tlymphocytederived eosinophil chemotactic factor (also: CHI3L3,Ym1)

CCSP CdM CD

CFTR CGRP CHI3L3 COPD CXC

CXCL DALY DMEM DNA ECFL

ECM ECMO EDTA ELISA eNOS EPC EpCAM

extracellularmatrix extracorporealmembraneoxygenation ethylenediaminetetraaceticacid enzymelinkedimmunosorbentassay endothelialnitricoxidesynthase endothelialprogenitorcell epithelialcelladhesionmolecule

ESC FACS FBS FcRIIB Fib

embryonicstemcell fluorescenceactivatedcellsorting fetalbovineserum FcreceptorforimmunoglobulinG fibroblast oxygenfraction fluorescein foundininflammatoryzone1(also:RELM) forkheadboxp3 hematoxylinandeosin HumanExVivoLungProject hematopoieticstemcell isobutylmethylxanthine(1methyl3(2methylpropyl)7H purine2,6dione) interferongamma insulinlikegrowthfactor1 insulinlikegrowthfactorbindingprotein6 interleukin IL1receptorantagonist induciblenitricoxidesynthase inducedpluripotentstemcell intranasal

FiO2 FITC FIZZ1 Foxp3 H&E HELP HSC IBMX

IFN IGF1 IGFBP6 IL

IL1ra iNOS iPSC i.n.

i.p. i.t. i.v.

intraperitoneal intratracheal intravenous intravenousimmunoglobulin Janusactivatedkinase keratinocytegrowthfactor knockout lungfibroblast lungfunctiontesting lipopolysaccharideinducibleCXCchemokine lipopolysaccharide classically(canonically)activatedmacrophages alternativelyactivatedmacrophages mitogenactivatedproteinkinase monocyte chemotactic protein1 (also: CCL2, small induciblecytokineA2)

IVIG JAK KGF KO LF

LFT LIX

LPS M1 M2

MAPK MCP1

MCSF MDC

macrophage/monocytecolonystimulatingfactor macrophagederivedchemoattractant/chemokine

MDSCmyeloidderivedsuppressorcells MIP2macrophageinflammatoryprotein2 MIP1macrophageinflammatoryprotein1alpha MSC mesenchymalstemcells

NEB OVA PaO2 PBS PE

neuroendocrinebody ovalbumin partialarterialpressureofoxygen phosphatebufferedsaline phycoerythrin positiveendexpiratorypressure plateletfactor4(also:CXCL4) phosphoinositide3kinase probableleastsignificantdifference polymorphonuclearcell phenylmethylsulphonylfluoride pulmonaryneuroendocrinecell phosphataseandtensinhomolog penicillinstreptomycinfungizone(amphotericinB) regulated on activation, normal T cell expressed and secreted

PEEP PF4

PI3K PLSD PMN PMSF PNEC PTEN PSF

RANTES

RIPA

radioimmunoprecipitationassay

RPMI1640 RoswellParkMemorialInstitutemedium1640 qRTPCR quantitative reverse transcriptase polymerase chain reaction RELM Sca1 resistinlikemoleculealpha(also:FIZZ1) stemcellantigen1

SCF

stemcellfactor stromalderivedfactor1alpha standarderrorofthemean sidepopulationcells surfactantproteinC signaltransducerandactivatoroftranscription6 solubleTNFreceptorII thymusandactivationregulatedchemokine transforminggrowthfactorbeta totallungcapacity transforminggrowthfactorbeta Thelper1 Thelper2 interleukin10inducedandsecretingregulatoryTcell regulatoryTcell tumornecrosisfactoralpha vascularcelladhesionmolecule1 vascularendothelialgrowthfactorreceptor2 wildtype see:CHI3L3,ECFL

SDF1 SEM SP

SPC STAT6 sTNFRII TARC TGF TLC TGF Th1 Th2

Tr1 Treg TNF VCAM1 VEGFR2 WT

Ym1

CHAPTER1GENERALINTRODUCTION ThischapterwaswrittenbyLIIandeditedbyBT.Fragmentsofthis chapterhavebeenpublishedaspartof: ColtanL,ThbaudB.Chapter30:Lung.inRegenerativeMedicine,Steinhoff, Gustav(Ed.),1stEdition.,2011.ISBN9789048190744

1.1.

Overview Therespiratorysystemsupportsthevitalfunctionofbreathing.It

canbeviewedastheinterfacebetweentheoxygenrichenvironmentand thecarbondioxideproducinglivingorganism.Thefailureofthelungsto completetheirfunctionisimmediatelylifethreatening. From a functional and anatomical viewpoint, the respiratory system comprises two compartments: the conducting airways (nasal cavity, pharynx, larynx, trachea, bronchi and bronchioles) and the gas exchanging airways (respiratory bronchioles and the saccularalveolar compartment, where alveolar walls come in close contact with capillary walls in order to facilitate the exchange of oxygen and carbon dioxide). Lung injury can occur at any of these levels leading to impairment of breathing function, which can be reversible or irreversible. Obstructive respiratorydiseases,suchasasthmaandchronicbronchitis,arecausedby damage at the airway level, which limits airflow, whereas restrictive pulmonary diseases, such as lung fibrosis, acute respiratory distress syndrome (ARDS) and sarcoidosis are determined by inflammatory processesinthelunginterstitium,whichleadtoreducedlungcompliance with limitation of lung expansion. Although the advancements of biomedicalresearchoverthepastdecadeshavebroughtnoveltherapeutic approachesforrespiratorydisorders,manylungdiseases,suchaschronic

lung disease of prematurity (or bronchopulmonary dysplasia, BPD), chronicobstructivepulmonarydisease(COPD)andcysticfibrosisarestill lacking efficient treatments. According to the WHO World Health Report 2000,lungdiseasescontributetoatotalof17.4%ofdeathsand13.3%of disabilityadjusted life years (DALY) worldwide [180]. These facts highlight the absolute necessity to study the potential applicability of recentdevelopmentsinthefieldofregenerativemedicineastherapeutic optionsforlungdiseases. 1.2. Accordingtooneofitsmostrecentdefinitions,regenerativemedicine is an interdisciplinary field of research () focused on the repair, replacement or regeneration of cells, tissues, or organs to restore impairedfunctionresultingfromanycause().Itusesacombinationof convergingtechnologicalapproaches()[which]mayinclude()theuse of soluble molecules, gene therapy, stem and progenitor cell therapy, tissueengineering,andthereprogrammingofcellandtissuetypes.[32] Thisfieldhasevolveddramaticallyoverthepastcoupleofdecadesand evenmoresointherecentyears.Asearchforscientificpublicationsusing the keyword regenerative medicine on the United States Library of Medicine / National Institutes of Health database is returning 12,000 LungDevelopmentandRegeneration

results[158].Asforspecializedjournals,the17%increaseinthenumber ofarticlespublishedinCellTransplantationTheRegenerativeMedicine Journal over only one year (2008 compared to 2007) can serve as an eloquentexample[109]. Thefundamentalparadigmsinregenerativemedicineare: (i) insituorganregenerationfollowinginjurymayoccuraslongas theorganframeworkhasbeensufficientlypreserved; (ii) the regeneration principles would normally follow

evolutionaryprinciplesandwouldlikelyrecapitulateontogeny [26, 168]. This brings forth the need to understand organ development, as new developmental concepts may have immediateapplicabilityinregenerativemedicine. The intrauterine development of the lung has traditionally been subdivided in five overlapping stages, on the basis of gross histological features[24].Therespiratorysystemstartsformingasearlyasthethird week of gestation as an outpouching of the primitive forgut bifurcating intothetwomainstembronchi(embryonic stage).Duringthefollowing pseudoglandular stage, rudimentary bronchi divide by dichotomous branching; the resulting tubular structures are lined by columnar epitheliumsurroundedbymesenchymaltissue.The canalicular stageis characterized by the bifurcation of the last generations of distal bronchi. Inthisstagethereisalsocapillaryinvasionanddifferentiationoftheair

spaceepitheliumintoalveolartype2cells(AT2,responsibleforsurfactant production)andtype1cells(AT1,whichformthethinairbloodbarriers). Next, during the saccular stage, the peripheral air spaces expand in length and width, and, around 36 weeks of gestation these spaces form sacculesattheexpenseoftheinterveningmesenchyme. Alveolarization, thefinalstageoflungdevelopment,beginsintheneartermlungpriorto birth but primarily occurs postnatally, during the first 23 years of life, andmaycontinuebeyondchildhood,albeitataslowerrate. The alveolusisthedefinitivegasexchangingunitofthelung.Alveoli are, in part, formed by subdivision (septation) of the saccules. Septation involves budding of septal crests, which is followed by elongation of the septal walls to form individual alveoli. Septation increases the gas exchange surface area, without a proportionate increase of lung volume (i.e. alveoli have a larger surface/volume ratio than saccules). Microvascular maturation,thefinalimportantstepinlungdevelopment, follows and partly overlaps the alveolar stage. Initially, capillaries form doublelayersintheimmaturegasexchangeregion;duringthematuration step, these microvessels remodel to form a single capillary layer. The thickness of the alveolar wall decreases by about 20% and the distance between alveolar gas and capillary blood diminishes by about 25%. Morphometricstudiesshowthatfrombirthtoadulthoodthealveolarand

capillarysurfaceareasexpandabout20foldandthecapillaryvolume35 fold. Whilethehistologicalchangesarewelldescribed[76,87,190],much moreneedstobelearnedaboutthemechanismsthatregulatenormallung developmentinordertoharnesstheseprocessesfortherapeuticpurposes [168]. This is particularly relevant for the perinatal care of extremely premature infants who are born at the late canalicular stage, before the completion of the alveolar stage. The immaturity of the lungs, together with the ventilator support required places these infants at risk of developing BPD, which may lead to an irreversible arrest in alveolar developmentandimpairedlungfunctionbeyondchildhood[179]. Thelungsareparticularlyvulnerableorgansduetotheirroleasoneof theportsofentryforenvironmentaltoxinsandallergens.Thecomplexity of the lungs and their developmental programme, together with the current lack of efficient therapies that would prevent or repair lung damage, render the lung an especially challenging candidate for the current arsenal of regenerative medicine. Traditionally, respiratory diseases have been assigned to several pathophysiological categories; however, new insights into disease mechanisms may offer new approaches to old problems. One example is cystic fibrosis, which is caused by mutations in the gene encoding for the cystic fibrosis

transmembrane regulator (CFTR), an ion channel normally present in epithelialcells.Thismonogenicdiseasehasbeenclassicallyregardedasa purely electrophysiological disease due to the CFTR dysfunction; however, recent findings suggest that CFTR is also expressed in immune effector cells, such as macrophages [22], which opens new therapeutic perspectives that place more emphasis on the inflammatory aspects of cysticfibrosis. Aside from and also alike cystic fibrosis, numerous lung diseases are currentlylackingefficienttherapies:whileALI/ARDSorasthmahavean overwhelminginflammatorycomponent,othertypesofinjurymayhavea more obscure etiology (emphysema, COPD, pulmonary fibrosis). BPD impairslungdevelopment,yet,inrecentyears,advancesinperinatalcare have permitted the survival of extremely premature babies whose lungs areinearlierdevelopmentalstages;whereastheoldBPDhadastronger fibrotic component, the new BPD is an arrest in alveolarization with minimalinflammation[11].Theprolongedstalemateinfindingsolutions forpatientssufferingfromtheseincurablediseaseshasbroughtstemcell research at the core of RM today. The hallmark abilities of stem cells to selfrenew and differentiate along multiple cell lineages (stem cell plasticity) have rendered both tissueresident and circulating stem and progenitor cells extremely appealing for tissue regeneration purposes.

Together with advances in creating animal models of lung disease, the promise of stem cells has become a crucial research avenue in lung regenerativemedicine[80]. 1.3.StemCells Theconceptofaplasticcelltypethatcanrespondtothedemandsof itsmicroenvironmentbyacquiringtraitsspecifictoothercelltypeswas proposed by the German pathologist Julius Cohnheim in 1867 [27]. He theorizedthatthefibroblaststhatparticipateinwoundhealinghadabone marrow origin, a hypothesis that to this day has not yet been starkly resolved.Itwasnotuntilthedawnofthetwentiethcenturythatasimilar vision of the bone marrow as an origin and reservoir of blood cells was elaborated by the Russian scientist Alexander Maximow. He developed a noveltheoryofhematopoiesis,inwhichtheseelusivecellsthathenamed stem cells [92], had an overarching role. It is acknowledged that MaximowscommunicationduringascientificcongressinBerlinin1908 wasthefirstinstanceofthetermstemcells.Hisworkdescribedcomplex cellular interactions where the marrow stroma orchestrated the conditions of hematopoietic stem cell differentiation [46, 93]. The modernmilestoneofstemcellresearchwassetin1961withJ.E.Tilland E.A.McCullochsdescriptionofcoloniesconsistingofmultiplecelltypesin

thespleensofirradiatedmicethathadreceivedunirradiatedmarrowcells [153]. Before the end of that decade, the first allogeneic bone marrow transplantations[54]weretoprovidetheclinicalprooffortheexistence of hematopoietic stem cells (HSCs). These cells are the basis of the complete reconstitution of blood cells following transplantation of bone marrow into irradiated patients. To date, HSCs have been extensively characterized and are often employed as a model of stem cell hierarchy [145,173],whileresearchmethodsinitiallydevelopedtostudyHSCs,such as lineage tracing methods [45], have been enthusiastically adopted by otherfieldsofresearch. Theunprecedentedemergenceofresultsthatsuggestedacontinuous, organismwiderenewalofpostnataltissuesalsocomprisedthepioneering reportsofJosephAltmanandGopalDas,indicatingpostnatalneurogenesis inseveralanimalspecies:rats(1965)[7],Guineapigs(1967)[8]andcats (1971) [34]; however, the existence of a neural stem cell had to await more than a few years for the confirmation by Samuel Weiss group in 1992[125]. Meanwhile, evidence had started to gather with regards to the presence of a different type of stem cell harbored by the bone marrow, this time in the stromal compartment, which was, at the time, mainly regarded as a support for HSCs. These cells were the focus of A. Friedensteins 1976 report that named them colony forming units

fibroblast (CFUF) [47]; later, these cells were going to be assigned the termmesenchymalstemcells(MSCs)[4,115]. Stem cells are defined as cells that have clonogenic and selfrenewal potential and are able to differentiate along multiple cell lineages [173]. The size, shape and cellular compartments of the adult organs are determined by embryonic and fetal stem/progenitor cell behavior [121].Traditionally, stem cells are categorized based on their origin and differentiationpotentialintoembryonicandadult(postnatal)stemcells. Embryonic stem cells (ESCs), definitively established in culture in 1981[41,90],areisolatedfromtheinnermassofthetrophoblastandare pluripotent, i.e. able to differentiate along multiple cell lineages originating in any of the three germ layers: ectoderm, mesoderm or endoderm, whereas the differentiation potential of adult stem cells (multipotent or, for progenitor cells, oligo or unipotent) has been considered to be restricted to their original germ layer. However, recent studies are challenging this paradigm, as stem cells derived from bone marrow, classically considered to be partially committed to either the hematopoieticormesenchymallineages,havebeenshowntocrosslineage boundariesandtransdifferentiatealonglineagesderivedfromadifferent germlayer.

10

The discovery of ESCs determined another golden era of exponential discovery and reconceptualized biomedical research in its entiretybyallowingthegenerationofgeneticallyengineered(specifically knockout) mice [25, 40, 138] that are now widely used to model the effectsofanabundanceoffactors.Todate,ESCsarebettercharacterized thanadultstemcells.Yet,thelineagerelationshipbetweenembryonicand adultstem/progenitorcellshasnotbeenclearlydescribed. One of the defining features of stem cells is their ability to divide either symmetrically, generating two identical daughter cells or asymmetrically,givingrisetoanidenticaldaughterstemcellandamore specialized, lineagecommitted progenitor cell [121, 149] that lacks the selfrenewalabilitybutpossessesahigherproliferationratecomparedto itsparentstemcell. It becomes obvious that a tight regulatory control of the balance between symmetrical and asymmetrical division, as well as the proliferation rate of these cells, is critical for organ development and homeostasis.Forinstance,ithasbeenproposedthatateachstageoflung development the stem cells divide mostly in an asymmetrical fashion, leaving the specialized progenitor behind as the identical daughter cell movesdistallywiththebuddinglungtips[121]. Regenerativeapproachesmaythereforefollowseveraltherapeutic directions:

11

1) Targeting of endogenous local (resident) stem cell populationsprotecting/stimulatingthesecells(potential regenerativemechanismseffectors)asameanstopromote organ regeneration or, conversely, targeting cancer stem cells(e.g.lungcancerstemcells[6])inastemcelloriented therapeuticapproachtotreatcancer; 2) Cell replacement by exogenous stem cells, as stem cells may be able to regenerate damaged organs by differentiatingandengrafting invivo.Thisholdspromisefor degenerative diseases (e.g. multiple sclerosis, Parkinsons disease) or genetic diseases (cystic fibrosis, alpha1 antitrypsindeficiency); 3) Standardized stemcell based preparations (e.g.

conditioned medium) may eliminate the risks typically associated with heterologous stem cell transplantation (infectious agents carried over to the recipient [130], immune rejection, tumorigenesis [5, 155]) and even allow forautologoustherapy. 4) Regeneration/ reconstruction of damaged organs using stem cells as a source of terminally differentiated cells for tissueengineering.

12

1.4.ResidentLungStem/ProgenitorCells Atbirth,thenormallydevelopedlungiscomprisedofmorethan40 cell types that originate in both endoderm and mesoderm layers. In healthy adults, lung cellular homeostasis is viewed as a slow process compared to highly proliferating tissues such as the bone marrow, intestine or skin, which makes it more difficult to study lung resident stem/progenitor cells. However, it is widely accepted that

stem/progenitorcellscontributetomaintenanceoflungcellpopulations andthereisevidencethat (i) stem cell proliferation rate in the lung increases dramaticallyfollowinginjury; (ii) the type and amplitude of injury also determines the intensity,durationandtypeofcellularresponse[56]. Current approaches in lung regeneration include therapeutic approaches aimed at the protecting and/or exogenously administering both lungresident and circulating stem/progenitor cells. Local stem/progenitor cells divide to replace injured or postmitotic cells and require strict control over their proliferation rate. Traditionally, local endodermderived adult stem/progenitor cell populations have been considered to reside in welldelineated niches and categorized by lung region. Several cell populations have displayed progenitorlike behavior

13

following chemicalinduced lung injury in rodents, and the common feature generally employed to functionally define these cells has been their ability to incorporate [H3]thymidine into their DNA [30, 91, 139]. Lung cell populations that have been ascribed stem/progenitor cell functions [reviewed in 18, 84, 105, 119, 120, 122] are summarized in Table11. One particular category of cells, endothelial progenitor cells (EPCs),havebeentraditionallyconsideredtobecirculatingcells(foundin the bloodstream) that contribute to the homeostasis of the endothelium [64, 142, 186, 187], consistent with previous findings demonstrating the beneficialeffectofangiogenicgrowthfactorsinexperimentallungdisease models[79,151].ThereisevidencethatcirculatingEPCsmaycontribute tothemaintenanceofthelungparenchymainLPS[183],hyperoxia[15] and elastaseinduced lung injury [66, 67]. In patients, the number of circulatingEPCscorrelateswithsurvivalanddiseaseseverityinacutelung injury[23],severeCOPD[106]orrestrictivelungdiseases[42],idiopathic pulmonaryarterialhypertension[36,71,146]andpneumonia[184]. Recent findings have identified the presence of resident EPCs within the pulmonary microvascular endothelium with angiogenic capacity[9],highlightingthepotentialofnewtoolsinstemcellbiologyto identify resident lung progenitor cells. The significance of these cells in

14

healthanddiseaseaswellastheirtherapeuticpotentialiscurrentlybeing explored. 1.5.TherapeuticPotentialofExogenousStem/Progenitorcells 1.5.1.CellReplacementbyMSCs Beside local stem/progenitor cell populations, there is evidence that nonresident stem/progenitor cells contribute to lung repair following injury [1, 2, 56, 97, 100, 137, 144, 169172, 178]. Kotton et al. [77] and Krause et al. [78] showed that bonemarrowderived stem cells cangiverisetodaughtercellsintheairways.Thisability ofthecellsto engraft and differentiate has led to the hypothesis that they may reconstituteinjuredtissuesbyreplacingthedamagedcells.Thereisnowa large body of evidence in support of the hypothesis that bone marrow derived multipotent MSCs can differentiate into airway [166, 177] or alveolar[159]epithelialcells in vitro,engraftanddifferentiate in vivoand prevent lung injury in various disease models including bleomycin inducedlungfibrosis[102,103,129,131,191],lipopolysaccharide(LPS) induced ALI/ARDS [57, 181, 183, 184], oxygeninduced BPD [13, 159], radiation[1]andnaphthalene[135]inducedlunginjury,haemorrhhagic shock [110]. The ability of adult MSCs to differentiate into lung cells has

15

rendered them particularly important candidates for lung regeneration approaches; today, MSCs are the most widely used stem cells in regenerative medicine. Theestablishment ofa minimal set of criteria for defininghumanMSCs[37]createdaframeofreferenceforcomparisonof reportsfromdifferentgroups;however,anequivalentforrodentMSCsis still lacking. MSCs have proven therapeutic abilities in numerous organ injury models, including myocardial infarction [16], acute renal failure [61, 154], type 1 diabetes [43, 53, 162] and neurodegenerative diseases [70]. As of May, 2012, there are 238 clinical trials listed on clinicaltrials.gov,awebsitehostedbytheUnitedStatesNationalInstitutes ofHealth.AsofaruniqueclinicalsuccessemployedtheabilityofMSCsto differentiateintochondrocytesthatwereusedtorepopulateanacellular trachealacellularscaffold[86].Theengineeredstructurewassuccessfully transplantedasmainbronchusintoapatientwhoseownairwayhadbeen irreversibly damaged. Beyond their classically described mesenchymal lineage differentiation ability, the fact that MSCs can cross lineage boundariesanddifferentiateintolungepithelialcellscouldbeharnessed for diseases such as cystic fibrosis, in which the symptoms are mainly causedbymutationsinthegeneencodingfortheCFTR,achloridechannel typically expressed in the apical membrane of epithelial cells. The stem cells would be engineered to overexpress functional CFTR and act as a deliveryvehicletothedamagedtissues,includingthelung[22].Thesame

16

approachwouldbeapplicableforothermonogenicdiseasesthatseverely affect the lung such as alpha1 antitrypsin deficiency (which leads to irreversible emphysemalike lesions) or surfactant protein B deficiency (which results in fatal respiratory failure in newborns). Moreover, it has been suggested that stem cells could act as drug delivery vehicles [107] based on observed therapeutic effects in myocardial infarction [83] and cancer[73,85].ThepossibilityofisolatingMSCsfromavarietyofsources, includingthecordbloodandtheadiposetissue,makesautologoustherapy averypromisingclinicalapproachintheclosefuture. 1.5.2.CellReplacementVersusParacrineActivityofMSCs Despite the hope originally placed in cell replacementdriven studies,numerousreportsthatevaluatedthetherapeuticpotentialofstem celltransplantationinanimalmodelsoflungdiseasesharedonecommon feature: the degree of stem cell engraftment in the target organs was generally low and therefore alternate mechanisms needed to be considered in order to account for the observed therapeutic benefit. Moreover, MSCs have been shown effective in inflammatory diseases [136], such as graftversushost disease in humans [81] and rodent modelsofLPSinducedALI[57,95],fulminanthepaticfailure[108],sepsis [98]andasthma[20,55,99],wherethelocalcellengraftmentmaynotbe

17

the primary beneficial component. This has led to the current view that stem cells act through a paracrine mechanism by secreted factors [116]. Indeed,MSCssecreteantiapoptotic,angiogenic,andimmunomodulatory factors. Since the initial report indicating paracrinemediated protective effects of MSCs overexpressing the prosurvival gene Akt in the ischemic heart [52], this paracrine activity has now extensively been explored in vitro [57, 62, 159], showing cellprotective, proangiogenic and anti inflammatory properties. Ex vivo [82] and in vivo in oxygen [13], ventilator[31] and LPS [57] induced lung injury, MSCderived conditionedmediumconferredtherapeuticbenefit,evenwhencompared directly with wholecell therapy [13, 82]. The immunomodulatory, paracrine activity of MSCs may also have therapeutic potential in other inflammatory diseases [68, 116, 136]. Several factors found in the MSCs secretome, among which are interleukin10 [98], transforming growth factorbeta (TGF) [92], stanniocalcin1 [19] and keratinocyte growth factor (KGF) [82], have been proposed to mediate MSCs crosstalk with variouseffectorcelltypes,suchasmacrophages[98].Recentobservations also indicate cellprotective effects of MSCs on endogenous stem cells, such as bronchoalveolar stem cells (BASC) [157]. Identification of these MSCsecreted factors, along with clarification of their mechanisms of action,mayallowthedevelopmentofnewtreatments.

18

1.5.3.ESCsandInducedPluripotentStemCells(iPSCs) ESCs represent the most pluripotent stem cells but the clinical applicability of ESCbased clinical solution is hampered by the ethical controversy surrounding the need to isolate these cells from the early embryo. The recent landmark generation of ESClike, induced pluripotentstemcells(iPSC)usingviraldeliveryofpluripotencygenesto somaticcells[148]mayrelievemanyethicalconcernsrelatedtotheuseof ESCs for research andhas opened the way to largescale production and evaluationofpluripotentstemcellsforlungregenerationandrepair. When maintained in conditions that support the undifferentiated state,pluripotentstemcellsshowunlimitedproliferationpotential,which renders them ideal candidates for studies in developmental biology regeneration. ESCs can be directed to differentiate into definitive endoderm from which they may be further differentiated into lung cells using specific factors [128, 161]. Another method employed was the exposure of ESCs to microenvironments mimicking lung conditions (coculture with lung mesenchyme or lung cell extracts) [160]. Although thereareisolatedreportsindicatingtheattainmentoffullydifferentiated proximal airwaylike tissue [28], airway epithelium [133], distal lung progenitors[127]orevenpurepopulationsofAT2cells[165]fromESCs, most of the available literature indicates cellular heterogeneity of the

19

cultureswitharelativelylowyieldoflungcells. In vivoadministrationof ESCs or progenitor cells derived from ESCs or iPSCs has also generated inconclusive results so far, with limited and transient ESC expression in the lung [128, 174]. Further steps, such as stable differentiation and purificationofdesiredcellpopulationsneedtobetakeninordertoassess thepotentialofESCsandiPSCsforlungdiseases. 1.5.4.StemCellsandCarcinogenesis Thetermlungcancerencompassesseveraldifferentpathological

entities: squamous cell carcinomas, small cell carcinomas and adenocarcinomaswhichappearwithdifferentfrequencyindifferentareas ofthelung,suggestingthatlocallungenvironmentmayactuponcellfate. Thehypothesisoftumorinitiatingcells(cancerstemcells)couldexplain the relapse of certain tumors owing to the fact that these cells might be resistant to many conventional cancer therapies [111, 113, 182]. The identificationofputativeresidentstemcellsinlungtumors[75]leadsto the question whether the resident cells that survive pollutantinduced injurymayinfactbesuchacancerstemcell.Theexistenceofcancerstem cellsinthelungissupportedbyworkindicatingthatCD133+isamarker ofselfrenewingcellsthatsustaintumorpropagationinmice[39].These cellsareresistanttocisplatintreatment[17];however,theproportionof

20

cells expressing this marker lacks prognostic value [132]. Other work suggeststhatactivationofthe krasgene,whoseactivationwasshownto be directly linked to earlyonset lung cancer [69], upregulates the SP C+/CCSP+ (BASC) cells and leads to development of lung adenocarcinomas [75]. Similarly, deletion of phosphatase and tensin homolog (PTEN), phosphoinositide 3kinase (PI3K) or p38a mitogen activatedproteinkinase(MAPK)ledtoproliferationofSPC+/CCSP+cells simultaneous with the increase in susceptibility to develop lung neoplasms[185],whereasBmi1(BlymphomaMoMLVinsertionregion1 homolog)deletionhadoppositeeffects[38].Bmi1hasbeenshowntobe crucial for stem cell selfrenewal [189]. However, it has not yet been clearlydeterminedwhetherthereisalinkbetweentheCD133expressing and the dual SPC/CCSPexpressing cell population or whether either of these populations acts as an initiator or propagator of lung malignant tumors. Also, the cells in small cell carcinomas have been shown to express basal cell markers, whereas small cell carcinomas have been found to express markers reminiscent of PNECs [51], but the direct relationship between the putative stem/progenitor cells and the neoplastic cells, as well as the proposed contributions of bone marrow derived cells to cancer progression [48] has yet to be investigated. Although much work is still needed to identify and characterize cancer

21

stem cellsinitiating cells, the discovery opens therapeutic avenues for designingspecificcellulartargetsforthetreatmentofcancer[192]. 1.6.BiotechnologyEngineeringLungTissue Currently, lung transplantation is the only viable solution for incurable lung disease in patients under 65 years of age. These lung diseases include lung fibrosis, COPD, cystic fibrosis, primary pulmonary hypertension, sarcoidosis, lymphangioleiomyomatosis. However, the mortalityratefromthemomentthepotentialrecipientsareplacedonthe waiting list until they receive the transplant is currently around 30% [118]. Moreover, lung transplantation is not an option for patients with othermajoraccompanyinghealthproblems.Thishighlightsthenecessity to seek for alternative approaches, such as the development of the artificiallungorbioengineeredlungcomponents. 1.6.1.HumanExVivoLungProject(HELP) Currently, the supply of donor lungs does not match the demand

andoneofthefactsthatcontributetothisshortageisthatonlyabout20% of donor organs are considered acceptable for transplantation [118]. Improper oxygenation capacity (reflected by a PaO2 below 300 mm Hg

22

afteroxygenationwithaFiO2of100%for5minandPEEPgreaterthan5 cm H2O) leads to rejection of donor lungs. HELP involves the concept of reconditioning and transplantation of these otherwise rejected donor lungs.Lungsarereconditionedexvivobycontinuousperfusionwithalung evaluationpreservation solution (Steen solution) [175] mixed with erythrocytes for several hours, until the functional parameters reach acceptable values. After reconditioning, these lungs can be transplanted immediately or stored at 8C in ex vivo extracorporeal membrane oxygenation(ECMO)untiltransplantationcanbeperformed[63].Thefirst transplantoflungsharvestedfromadonorandreconditioned ex vivowas performed successfully in 2007 [141]. The impact of this promising strategyremainstobeevaluated. 1.6.2.ArtificialLungNovaLung Theartificiallungisarelativelynewmethod,similarinconceptto dialysisanddesignedtosupportrespiratoryfunctionwhilethepotential lungtransplantrecipientiswaitingforthedonorlungs[44].Thepatients blood flows into a device that removes carbon dioxide and enriches the blood in oxygen. As compared to conventional ECMO, the artificial lung eliminatestheneedforanextracorporealbloodpumpandcanbeusedfor extendedperiodsoftime(upto100days)[163]incentreswhereECMOis

23

not available. Other advantages of this system over ECMO are reduced anticoagulation and avoidance of longterm mechanical ventilation [150, 164]. 1.6.3.BioengineeredLungTissue The structural and functional complexity of the lung has so far restrictedthedevelopmentofbioengineeredlungtissue,whencompared totheprogressmadeinengineeringlesscomplexorgans,suchastheskin or the urinary bladder [12, 14]. A recent in silico model of the alveolar capillaryinterfacehasbeendevelopedemployingbiomaterialsandhuman alveolar epithelial cells at airliquid interface, along with human pulmonary microvascular endothelial cells [60]. This type of biomimetic microsystems could facilitate drug screening and toxicology studies by allowing highthroughput processing. On a larger scale, so far both ESCs and adult multipotent stem cells, as well as mixed cell populations containing progenitor cells or terminally differentiated cells such as fibroblasts or chondrocytes have been used with promising results to generate lung cell lineages or bioengineered lung components, including recellularization of a human tracheal scaffold with MSCsderived chondrocytes,followedbysurgicalimplantationasamainbronchus[86]. However, the lack of conclusive information with respect to the

24

tumorigenic potential of stem cells, especially ESCs, known for their karyotypic instability, together with the unanswered question regarding the local progenitor cells as potential cancer stem cells, demand careful safety evaluation of stem cellbased approaches. Also, the biomaterials used as scaffolds on which the lung tissue would be grown need to be evaluated with regards to their biocompatibility in terms of elasticity, adsorption kinetics, porosity and degradation kinetics [101]. So far, scaffoldscomposedofnaturalpolymerslikecollagen,Matrigel(amixture of basement membrane proteins and / or synthetic polymers, such as polyacrylamide have been used in attempts to engineer lung tissue [12]. Aside from constructed scaffolds, a recent breakthrough in lung bioengineering has been achieved by demonstrating that decellularized lungmatriceshavetheabilitytosupportrepopulationwithnewlyseeded epithelial and endothelial cells and, moreover, to sustain lung function followingtransplantationintoanimals[104,112,140]. 1.7.ClinicalStudies:Experience,Outcome,Limitations Severallimitationshavehamperedclinicaltrialsofstemcellbased therapies for lung diseases. There are certain risks to heterologous cell transplantation. The cells may carry infectious agents, which poses an even enhanced peril in the case of recipients who have developed graft

25

versushost disease [130]. Furthermore, there have been reports of bronchiolitis obliterans organizing pneumonia (BOOP) in patients who had undergone HSC transplantation [58]. Both heterologous and autologous transplantation bear the risk of tumor formation. ESCs and iPSCsdevelopteratomas in vivoandtherearealsoreportsindicatingthat transplantation of neural stem cells led to the development of tumors in the recipient brain [10]. MSCs, generally considered less prone to acquiring karyotypic abnormalities compared to ESCs, may also pose tumorigenicrisks[155].However,thesedangersmaybeovercome:recent findings indicating that stem cellsecreted factors exert therapeutic benefits may abrogate the need to deliver the cells themselves to the damagedtissues. Anotherlimitationistheinsufficientcharacterizationofstemcells in terms of both phenotype and function. For MSCs, minimal criteria for defininghumanMSCs,establishedbytheInternationalSocietyforCellular Therapy[37],havereducedsomeofthevariationswithregardstocellular compositionofMSCpopulationsisolatedaccordingtodifferentprotocols. Lung injury prevention obtained with MSCs in various animal models of lung disease, together with their ease of isolation and culture, as well as their immunomodulatory properties make these cells very promising candidatesforclinicaltrials.

26

Thusfar,MSCshavebeentransplantedinhumansaspartofwhole bone marrow transplantation for various disorders (including leukemia and genetic diseases of the immune system). Gendermismatched transplantation(maledonorbonemarrowtofemalerecipient)hasproven tobeausefultoolinassessingtheimpactofstemcelltransplantationon other organs than bone marrow. Donor male cells were identified in the lungsofrecipientsasepithelialandendothelialcells[147]andalsointhe liver[152],heart[35],brain[29,96]andkidney[114].Also,inthereverse case where males were recipients of sexmismatched organ transplants, theYchromosomeindicatingrecipientoriginwasidentifiedin avariable proportion of organspecific cells. With regards to lungs, the chimerism was present in bronchial epithelial cells, AT2 and seromucous glands [115]. Currently, one phase I clinical trial aimed at evaluating the tolerabilityandsafetyofprogenitorcellsforthetreatmentofpulmonary arterial hypertension (Pulmonary Hypertension: Assessment of Cell Therapy, PHACeT) is underway. Autologous endothelial progenitor cells areengineeredexvivotoexpressendothelialnitricoxidesynthase(eNOS), followed by injection of the cells via a pulmonary artery line. Previous pilotstudieshavesupportedthefeasibilityofthisapproachinidiopathic pulmonaryhypertension[167,193].

27

On the basis of initial reports of safety and efficacy following allogeneicadministrationofMSCstopatientswithCrohnsdiseaseorwith graftversushost disease, several trials studying the effect of MSCs in patients with lung diseases (COPD, BPD, idiopathic pulmonary fibrosis, emphysema)areongoingandprogramssuchastheProductionAssistance for Cellular Therapies (PACT) [124] in the United States have been initiatedtofacilitatethetranslationofcellbasedtherapiestotheclinical environment. Information on current clinical trials involving the use of stem cells or stem cellderived products is regularly updated on the United States National Institute of Healths website

www.ClinicalTrials.gov.

28

Celltype Basaland parabasal cells Location proximal airway epithelium submucosal glandular ductsand intercarti laginouszone bronchioalveo larjunction; proximityof neuroepithelia lbodies (NEBs)[49] Phenotype cytokeratin 5/14+ Lunginjury model polydocanol orSO2 induced[21] Remarks clonogeniccapacity, multilineagedifferentiation [134];repopulateairway epitheliumpostinjury[59]

TypeA (new, variant) Claracells

Claracell secretory protein (CCSP)+; nosecretory granules,no smoothER Sca1+/CD34 +/CD45/CD 31[74,75] SPC

naphthalene orozone induced

retainlabeledDNAprecursors [121];repopulateinjured airwayepitheliumwithboth mature,quiescentClaracells andciliatedepithelialcells

Bronchio alveolar stemcells (BASC) TypeII alveolar epithelial cells(AT2) Side population (SP)[146]

maycoexpressSPC

heterogeneo us population effluxthe DNAdye Hoechst[50] calcitonin generelated peptide (CGRP)

Pulmonary neuro endocrine cells (PNECs)

foundwith typeAClara cellsinNEBs associated regenerative foci[126].

naphthalene induced

proliferateandgenerateAT1 cellsfollowinginjury[4,123]. AT1differentiateintoAT2in vitro[33]:alternateprogenitor cellsdependingontypeoflung injury derivedfrombonemarrow, identifiedinlung[88]; differentiatealongendoderm andmesodermderivedlineages [89,143];endothelialpotential (newbornmice);decreasedin oxygeninducedarrested alveolargrowth[65] oxygensensing[188];ifboth naphthalenesensitiveand resistantClaracellsareablated airwayepitheliumdoesnot regeneratePNECsarenot airwayepithelialprogenitors [59] giverisetobronchialand alveolarepithelium[94] selfrenewing,clonogenic[72]

Lung epithelial progenitors Multipotent lung progenitors

EpCAM(hi)/ CD104+/ CD24(low) ckit+

Table11.Summaryofputativeendogenouslungstemcellpopulations.

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CHAPTER2AIRWAYDELIVERYOFSOLUBLEFACTORSFROM PLASTICADHERENTBONEMARROWCELLSPREVENTSMURINE ASTHMA Aversionofthischapterhasbeenpublished. IonescuLI,AlphonseRS,ArizmendiN,MorganB,AbelM,EatonF, DuszykM,VliagoftisH,AprahamianT,WalshK,ThebaudB.Airway deliveryofsolublefactorsfromplasticadherentbonemarrowcells preventsmurineasthma.AmJRespirCellMolBiol2012Feb; 46(2):20716.Epub2011Sep8.

LIIperformedthemajorityoftheexperiments.RSAperformedtheBMCs characterization.NA,BM,MAandFEprovidedtechnicalassistance.MDandHV aidedindesigningtheexperimentaloutlines.TAandKWprovidedtheadiponectin knockoutmice. ThemanuscriptwaswrittenbyLIIandeditedbyBT.

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2.1.Abstract Asthma affects an estimated 300 million people worldwide and accountsfor1in250deathsand15milliondisabilityadjustedlifeyears lost annually. Plasticadherent bone marrowderived cells (BMCs) administration holds therapeutic promise in regenerative medicine. However, given the low cell engraftment in target organs, including the lung,cellreplacementcannotsolelyaccountforthereportedtherapeutic benefits. This suggests that BMCs may act by secreting soluble factors. BMCsalsopossessantiinflammatoryandimmunomodulatoryproperties andmaythereforebebeneficialforasthma. Our objective was to investigate the therapeutic potential of BMCs secretedfactorsinmurineasthma. Inamodelofacuteandchronicasthma,intranasalinstillationofBMCs conditionedmedium(CdM)preventedairwayhyperresponsiveness(AHR) and inflammation. In the chronic asthma model, CdM prevented airway smooth muscle (ASM) thickening and peribronchial inflammation, while restoringbluntedsalbutamolinducedbronchodilation.CdMreducedlung levelsoftheTh2inflammatorycytokinesinterleukin(IL)4andIL13and increased levels of IL10. CdM upregulated an IL10induced and secreting subset of T regulatory lymphocytes and promoted IL10 expression by lung macrophages. Adiponectin (APN), an anti

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inflammatory adipokine found in CdM, prevented AHR, ASM thickening andperibronchialinflammation,whiletheeffectofCdMinwhichAPNwas neutralized or from APN knockout mice was attenuated compared to wildtypeCdM. Our study provides evidence that BMCsderived soluble factors preventmurineasthmaandsuggestsAPNasoneoftheprotectivefactors. FurtheridentificationofBMCsderivedfactorsmayholdpromisefornovel approachesinthetreatmentofasthma.

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2.2.

Introduction

Allergicdisorders,suchasanaphylaxis,hayfever,eczemaandasthma, nowafflictroughly2030%ofpeopleinthedevelopedworld[12].Asthma ischaracterizedbyairwayhyperresponsiveness(AHR),inflammationand remodelingwiththickeningoftheairwaysmoothmuscle(ASM)celllayer refractory to bronchodilator therapy [1, 23]. Despite the progress achieved in the past decades in the management of asthma, refractory asthmastillaccountsformorethan250,000deathsworldwideeveryyear [31]. Recentstudieshaveexploredthetherapeuticpotentialofstemcellsfor regenerativemedicineinmultipleclinicaldisordersincluding myocardial infarction, diabetes, sepsis, hepatic and acute renal failure [45]. Bone marrow (BM)derived cells also prevent various experimental lung diseases including pulmonary fibrosis [43, 47], acute lipopolysaccharide induced lung injury [14, 29, 37], and chronic oxygeninduced neonatal lungdisease[4,53].Overthepastdecadealone,numerousreviewshave summarized the increasing amount of evidence in support of the therapeutic benefits exerted by BMderived cells [22, 33, 47, 60, 61]. However, cell engraftment is consistently disproportionately low for cell replacement to account for the therapeutic benefit. An alternate hypothesisproposedformesenchymalstemcells(MSCs)isthereleaseof

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soluble factors with exertion of their therapeutic benefit through a paracrinemediatedmechanism[45]. In addition, BMderived cells display immunosuppressive and anti inflammatoryproperties[19,20,25,27,28,38],suggestingtheirpotential benefit in allergic and inflammatory disease such as asthma. Thus, we hypothesized that airway delivery of plastic adherent BMderived cells (BMCs) conditioned medium (CdM) prevents AHR, inflammation and remodelinginexperimentalmurineasthma.

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2.3.MaterialsandMethods 2.3.1.BMCsIsolationandCulture

BMCsfromadultwildtype(WT)BALB/cmice(810weeksCharles River Laboratories, Wilmington, MA) and adiponectin (APN) knockout (KO) mice (Dr. Kenneth Walsh, Boston University) were isolated and differentiated along mesenchymal lineages according to previously describedmethods[44]basedontheiradherencetotissuecultureplastic. Briefly, mice were anesthetized with sodium pentobarbital (65 mg/kg), the femurs and tibias were dissected, washed twice with phosphate buffered saline (PBS, SigmaAldrich, Oakville, ON) and the bone marrow was flushed with complete cell culture medium Dulbeccos Modified Eagle Medium (SigmaAldrich) supplemented with 20% heatinactivated fetal bovine serum (SigmaAldrich) and 1% penicillinstreptomycin fungizone (PSF, Invitrogen, Burlington, ON) using a 26gauge needle attachedtoa10mLsyringe.Theplasticadherentcellswereculturedat37 Cinatmospherecontaining20%O2,5%CO2inhumidifiedincubators. For adipogenic differentiation, passage 2 BMCs were cultured for 21daysinadipogenicinductioncellculturemedium(DulbeccosModified Eagle Medium, lowglucose, supplemented with 10% fetal bovine serum, 1%PSF,0.5mMIBMX,60Mindomethacin,0.5Mhydrocortisoneand1

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g/mL insulin). After 21 days, the cells were stained with Oil Red O (SigmaAldrich) to reveal intracellular lipid droplets indicating the differentiationofBMCsintoadipocytes. For osteogenic differentiation, passage 2 BMCs were cultured in osteogenic induction cell culture medium (Minimal Essential Medium, alpha modification, lowglucose, supplemented with 10% fetal bovine serum, 1% PSF, 10 mM Bglycerophosphate, 0.2 mM ascorbic acid, 0.01 Mdexamethasone).After21daysinculture,thecellswerestainedwith fresh Alizarin Red solution (SigmaAldrich) in order to reveal the extracellularcalciumdepositscharacteristicofosteoblasts. Forchondrogenicdifferentiation,passage2BMCswereculturedas pellets (200,000 cells/ pellet) in15 mL centrifugetubes for 21days. We used a commercially available chondrogenic differentiation kit (R&D Systems, Minneapolis, MN). The chondrogenic induction culture medium waspreparedaccordingtomanufacturer'sinstructions.After21days,the pellets were embedded in paraffin, cut into sections and stained with SafraninO(SigmaAldrich)torevealtheproteoglycandeposits indicative ofdifferentiationintochondroblasts. ForseparationofCD45+andCD45BMCssubpopulationsweused streptavidincoated magnetic beads (Dynabeads, Invitrogen) and biotinylatedCD45antibody(BDBiosciences,Missisauga,ON).

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2.3.2.LungFibroblasts(LFs)IsolationandCulture FibroblastswereisolatedfromadultWTBalb/Cmiceaccordingto a previously described method [51]. The lungs were washed twice with PBSandfinelymincedinDMEMsupplementedwith15%FBSand1%PSF. Tissuecultureplasticadherentcellsweregrownfortwopassages. 2.3.3.FluorescenceActivatedCellSorting(FACS) For the characterization of BMCs [37, 52, 54], the following antibodies were used: stem cell antigen1 (Sca1), CD11b, CD29, CD31, CD34, CD44, CD45, CD73, CD105 (Biolegend, San Diego, CA). The cells were analyzed using a BD FACScan flow cytometer (BD Biosciences, Mississauga,ON). For FACS analysis of lymphocytes from draining lymph nodes, mediastinal lymph node chains were harvested, minced and passed througha100mcellstrainer;forthequantificationofregulatoryTcells and macrophages in the lung, lymphocytes were isolated using a previouslypublishedmethod[50].Briefly,lungsofexperimentalanimals were digested using collagenase III (Worthington Biochemical Corporation, Lakewood, NJ) and the lymphocytes stained using IL10 antibody(Biolegend)andacommerciallyavailablekit(MouseTregFlow

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Kit, Biolegend) or CD11b (BD Biosciences) and analysed using a BD FACSCantoflowcytometer(BDBiosciences). 2.3.4.BMCsandLungFibroblastsCdMPreparation Passage 2 BMCs or lung fibroblasts (~80% confluent) were rinsed twicewithPBSandculturedinmediumwithoutFBSsupplementationfor 24hours.Thecellculturesupernatantswerecollected,concentrated(25 times)anddesaltedbycentrifugalfiltration(AmiconMillipore,Billerica, MA). Supernatants were generated from multiple batches of BMCs. Typically,3adultmicewereusedtoisolateprimarycellsfromfemursand tibiaeseededinone75cm2tissuecultureflask.Atpassage2,9identical flasks were generated from the original flask, yielding 600 L concentrated CdM / flask (used for animal treatment and cytokine quantification) and also cells for lineage differentiation and surface marker characterization (WT BMCs). BMCs (WT or APN KO) did not appear different between batches in terms of cell numbers, rate of growth/proliferation, viability (Trypan Blue counts). In order to further minimizevariability: BMCs were always isolated and cultured by the same operator, using the same protocol, reagents, mouse strain, age, gender, weight, nutritionstatusandmousesource;

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CdMwasalwayspreparedfromthesamenumberofcells;cellswould beinthelogphaseofgrowth,~80%confluenttoavoideffectsofsub oroverconfluencyoncellsorproteinyield;

CdM was never prepared twice from cells isolated at the same time; afterserumstarvationandsupernatantcollectioncellswerescreened for consistent viability between batches using Trypan Blue vital dye anddiscardedimmediately;

UponpreparationofCdM,CdMpreparedonthesameday(fromcells initiallyisolatedandpassagedatthesametime)waspooled,frozenat 80degreesCelsiusandthawedonlyonce,uponuse;

Again, for reproducibility purposes, the same frozen CdM (which wouldhaveotherwisebeenusedfortreatment)wasusedforcytokine measurements. Cytokine quantification was always preceded by total proteinquantification,toensuresufficientamountsandconsistency. For neutralization of APN in WT BMCs CdM, we used an APN

neutralizing antibody (R&D Systems). Cells were serumstarved for 24 hours in the presence of 100 ng/mL APN antibody [46] and CdM was preparedasdescribedbefore.

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2.3.5.AnimalModel Adult male BALB/c mice (810 weeks) were purchased from CharlesRiverLaboratories(Wilmington,MA).Theanimalswerehousedin specific pathogenfree conditions in the University of Alberta Health Sciences Laboratory Animal Services facilities throughout the entire durationoftheexperimentalprotocols.Allprocedureswereapprovedby the University of Alberta Animal Policy and Welfare Committee. Animals were allowed to adapt to housing conditions for a week before commencingexperimentalprotocol. Mice were sensitized to ovalbumin (OVA) by intraperitoneal injection of 10 g OVA (lypopolysaccharidefree chicken egg albumin grade V, SigmaAldrich) adsorbed on 2 mg aluminum hydroxide (Sigma Aldrich) in 0.2 mL saline [9]. Control animals received saline solution alone. ForOVAchallenges,micewerelightlyanesthetizedwithamixture containing ketamine (75 mg/kg) and acepromazine (2.5 mg/kg) and a volume of 25 L of a solution containing 5% OVA was administered intranasally. Intheacuteasthmamodel(shortprotocol)[9],micereceivedtwo intraperitoneal sensitization injections on days 1 and 6, followed by two intranasalchallengesondays12and14(Fig.21A).Inthechronicasthma

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model(longprotocol,adaptedfrom[57]),miceweresensitizedondays1, 8,15and29andchallengedondays22,24,27,31,34and36(Fig. 21B). A volume of 25 L concentrated, desalted BMCs CdM was administered intranasallyaftertheOVAchallenge. FortheAPNexperiments,recombinantAPN(rAPN,R&DSystems,5 g/gbodyweight)wasreconstitutedin25Lofsalineandadministered uponeachOVAchallenge,similartoBMCsCdMadministration. 2.3.6.BronchoalveolarLavageFluid(BALF)Analysis Mice not subjected to lung function testing were anesthetized by intraperitoneal injection of sodium pentobarbital. The trachea was cannulatedandinstilledwith5successivealiquotsof1mLsterile,icecold phosphatebufferedsaline(PBS,SigmaAldrich)solution.Therecoveryof the instilled fluid was typically greater than 80%. The total cells in the BALF were counted and cytospin preparations were obtained (Thermo Shandon, Pittsburgh, PA). Differential staining (Hema 3 Manual Staining System,ThermoFisherScientific,Nepean,ON)wasperformedinorderto identify the cell types present in the BALF based on their morphological features[9].

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2.3.7.LungFunctionTesting(LFT) At 48 hours after the last challenge, mice were anesthetized by intraperitoneal injection of a mixture containing ketamine and acepromazine. After anesthesia was achieved, pancuronium bromide (1 mg/kg, SigmaAldrich) was administered intraperitoneally in order to abolish spontaneous breathing by achieving muscular relaxation. An 18 gauge cannula was inserted into the trachea and secured in place. The lung function was assessed using a small animal ventilator (FlexiVent, Scireq, Montreal, QC). All data recordings were obtained while maintaining a positive endexpiratory pressure (PEEP) of 4 cm H2O. Aerosolized saline (to establish baseline responses), methacholine (2, 8 and32mg/mL)[8]andsalbutamol(0.5mg/mL)solutionsweredelivered intratracheally.Eachdosewasnebulizedfor10secondsusinganAeroneb nebulizer. The volume history was standardized to TLC after the administrationofeachdoseandtheselectedparametersweremeasured 12 times over 3 minutes. Only data with a coefficient of determination (COD) greater than 0.9 were used and the maximal responses correspondingtoeachdosewereselectedforanalysis. We employed the singlecompartment model (whole lung mechanics) of lung behavior [55]. The measurement of dynamic lung resistance(R)anddynamiclungelastance(E)wasobtainedusingapreset

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volumeperturbationduringwhichtheregularventilationwasinterrupted and a 2.5 Hz (150 breaths/ min) sine wave was applied. The data was automatically fitted to the equation of motion describing the single compartmentlinearmodeloflungbehavior: P(t)=RV(t)+EV(t)+P0

whereP(t)isthepressureattheairwayopening,V(t)istheairflow,V(t) is the volume of gas in the compartment and P0 is the resting applied pressure(i.e.PEEP). 2.3.8.CytokineQuantification Lungs from animals that had not been subjected to LFT or BALF collection were snapfrozen in liquid nitrogen and homogenized by sonication on ice in modified RIPA buffer (Millipore) supplemented with aprotinin,leupeptin,sodiumorthovanadateandphenylmethylsulphonyl fluoride(PMSF,allfromSigmaAldrich). Cytokine concentrations were determined using commercially available ELISA kits for detection of IL4, IL10, IL13 (eBioscience, San Diego, CA) according to manufacturers instructions. APN was quantified in the wildtype and APN KO BMCs CdM and LFs CdM using a commerciallyavailableELISAkit(Millipore).

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2.3.9.LungHistologicalAnalysis Thetracheawascannulatedandthelungswereinflatedwith10% zincformalin while maintaining a constant pressure of 20 cm H2O. The lungs were excised and placed in formalin for 24 hours, embedded in paraffin and cut into 5m sections according to a previously described method [54] in order to maximize the number of intact airways per section. Hematoxylin & eosin (H & E) stained lung sections were analyzed using a computercontrolled light microscope (Leica Microsystems Canada, Richmond Hill, ON). The airway smooth muscle layer thickness and peribronchial inflammatory infiltrate area were measured using the Openlab imaging software (Improvision, Coventry, UK).Allnonobliquelycut(maximumdiameter/minimumdiameter<2), intact mediumsized airways (100300 m diameter) were selected for airway analysis, according to a previously published set of criteria [17]. Theairwaysmoothmuscle(ASM)layerthicknesswasmeasuredusingthe formula: ASMlayerthickness=(outerairwaydiameterinnerairway diameter)/2

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where the outer airway diameter was measured to include the smooth muscle layer and the inner airway diameter was measured as lumen diameter+epitheliallayer. Peribronchial inflammatory area was delineated and quantified using the OpenLab selection and measurement tool. All quantifications wereperformedbyaninvestigatorblindedtotheexperimentalgroups. 2.3.10.StatisticalAnalysis StatisticalanalysiswasperformedusingtheStatView5.0software (Abacus Concepts, UK). The group means were compared using the analysisofvariance(ANOVA)onewayorrepeatedmeasurements(FLT), followedbyFisher'sprobableleastsignificantdifference(PLSD)posthoc test.Allvaluesareexpressedasmean(+/standarderror)andavalueof p<0.05wasconsideredsignificant.

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2.4.Results 2.4.1.BMCsDifferentiationAlongMesenchymalLineagesAndSurface MarkerProfile BMCsdifferentiatedintoadipocytes(Fig. 22A),osteocytes(Fig. 2

2B)andchondrocytes(Fig. 22C)anddisplayedasurfacemarkerprofile indicativeofaheterogeneouscellpopulation(Fig.23). 2.4.2. BMCs CdM Prevents Airway Inflammation in Experimental Asthma BMCs CdM significantly reduced the total number of cells in the BALF in OVACdM animals compared to untreated OVA mice in both the acute(n=8,p<0.0001, Fig. 24A) andthechronic(n=6,p=0.0001, Fig. 2 4B) asthma model. Polymorphonuclear cells (PMN), predominantly eosinophils(~50%oftotalBALFcells)weresignificantlyincreasedinthe BALF of OVA mice compared to control animals, whereas BMCs CdM significantly attenuated lung PMN influx (acute model: p<0.0001, Fig. 2 4C; chronic model: p=0.0006, Fig. 24D). BMCs CdM had no effect on control saline animals in the acute model (Fig. 24A). Control CdM from mouse lung fibroblasts (Fib CdM) had no effect when compared to

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untreated OVA animals in the acute model (Fig. 24B). Fib CdM significantlyloweredthetotalnumberofcellsandPMNintheBALFinthe chronicmodel(Fig. 24D)suggestingsomeantiinflammatoryproperties ofFibCdM. 2.4.3.BMCsCdMPreventsAHRinExperimentalAsthma Intheacutemodel,themethacholinedosedependentincreasesin dynamicwholelungresistance(Fig. 25A)andelastance(Fig.25B)were exacerbated in untreated OVA mice (n=11) compared to control saline (n=9). BMCs CdM treatment attenuated the dosedependent increase in both parameters dynamic wholelung resistance (Fig. 25A) and elastance (Fig. 25B) by 50%. In the chronic model, there were no differences between groups in the dynamic lung resistance (Fig. 25C). BMCCdMattenuatedtheincreaseindynamiclungelastanceinthechronic model by over 50% (Fig. 25D) Conversely, control Fib CdM had no beneficial effect compared to untreated OVA animals (Fig. 25AD) suggesting that the benefit was specific to the CdM of BMCs. BMCs CdM hadnodeleteriouseffectincontrolsalineanimals(Fig.25AD).

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2.4.4. BMCs CdM Improves Bronchial Responsiveness to Salbutamol and Prevents Chronic ASM Thickening and Peribronchial Inflammation The bronchodilator response to salbutamol was significantly bluntedinthechronicOVAmodelasdocumentedbyapersistentincrease in dynamic lung resistance (Fig. 26A) and elastance (Fig. 26B). The response to salbutamol remained blunted in OVAFib CdMtreated animals as quantified by the persistent increase in dynamic lung resistance (Fig. 26A) and elastance (Fig. 26B). Conversely, BMCs CdM restoredthebronchodilatorresponsetosalbutamolsimilartothecontrol groups(Fig.26AandB). MediumsizedbronchihadasignificantthickeningoftheASMlayer andanincreaseinperibronchialinflammatoryinfiltrateinOVAsensitized animalscomparedtocontrolsasquantifiedbyASMthickening(Fig.27A) and increased peribronchial inflammatory infiltrate (Fig. 27B) and shown in representative H&E sections for each group (Fig. 27CI). Increased ASM thickening (Fig. 27A) and peribronchial inflammatory infiltrate (Fig. 27B) were significantly reduced by 20% and 40% respectivelyinBMCsCdMtreatedOVAmicecomparedtountreatedOVA mice.FibCdMhadnobeneficialeffectontheseparameters.

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2.4.5. BMCs CdM Attenuates T helper2 (Th2) Lymphocyte Cytokine Response The Th2 cytokines IL4 and IL13 were significantly lower in the lungs of OVACdM mice compared to untreated OVA mice and OVAFib CdM mice in both the acute (Table 21) and the chronic (Table 22) model. The levels of the antiinflammatory cytokine interleukin10 (IL 10), were significantly increased in the lungs of OVA BMCs CdM mice comparedtountreatedOVAmice.IL10waspresentinBMCsCdM(29.4 3.26ng/mL)butnotdetectableinFibCdMbyELISA. 2.4.6.APNMediatesProtectiveEffectsofBMCsCdMonAHR We also found APN, an antiinflammatory adipokine, to be expressed in BMCs CdM but not in Fib CdM (Fig. 28A) suggesting that some of the beneficial effects of BMCs CdM could be mediated by APN. APN was produced predominantly by BMMSCs (5fold compared to CD45+BMCs)(Fig.28A). TotestforthecontributionofAPNtothebeneficialeffectsofCdM, we tested the effects of APN absence in BMCs CdM using a neutralizing antibodyandBMCsfromAPNknockout(KO)mice.APNinhibitionwitha neutralizing antibody efficiently decreased APN in BMC CdM (NeutCdM,

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Fig. 28A).APNinhibitionsignificantlyattenuatedthebeneficialeffectof BMC CdM on dynamic lung elastance in the acute asthma model (Fig. 2 8B). Likewise,APNwasundetectableinBMCsofAPNKOmice(Fig. 28A).In addition,APNKOBMCsCdMwasunabletopreventAHR(Fig. 28C)and bronchodilatorresponsiveness(Fig. 28D).Conversely,recombinantAPN (rAPN) treatment alone significantly improved AHR (Fig. 28C) and bronchodilatorresponsivenesstosalbutamol(Fig.28D). 2.4.7. APN Mediates Protective Effects of BMCs CdM on Airway Remodeling Representative H&E stained lung sections showed that compared tocontrolsalinemice(Fig. 29A),thechronicOVAmodelhadsignificant airwayremodeling(Fig.29B).AirwayremodelingwaspreventedinOVA CdM (Fig. 29C), but not by APN KO BMCs CdM (Fig. 29D). Treatment with rAPN in turn prevented airway remodeling (Fig. 29E). These data wereconfirmedbyquantitativeairwaymorphometryshowingattenuated ASMthickness(Fig. 29F)andperibronchialinflammation(Fig. 29G)in OVACdM and rAPNtreated mice but not in APN KO BMCs CdMtreated mice, further suggesting that APN contributes to the beneficial effects of BMCsCdM.

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2.4.8. BMCs CdM Induces Subsets of IL10Producing T Regulatory Cells(Tregs)andMacrophages FACS analysis indicated that, while CdM did not impact the proportion of CD4+CD25+Foxp3+ cells in the lung (Fig. 210A) or the draininglymphnode(Fig. 210B)lymphocytes,itledtotheupregulation ofasubpopulationoflunglymphocytescoexpressingthesurfacemarkers CD25 (Fig. 211A) and CD4 (Fig. 211B) and intracellular IL10, but lacking expression of the transcription factor Foxp3 (Fig. 211CF). This population was significantly upregulated in OVACdM mice compared to OVAandcontrolmice(Fig. 211G).FACSalsorevealedasubpopulationof IL10expressing CD11b+ macrophages (Fig. 211HK). This population wassignificantlyupregulatedinBMCsCdMandrAPNtreatedmice(Fig. 211L).

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2.5.

Discussion We show that airway delivery of CdM obtained from BMCs

prevents the development of AHR, airway inflammation and airway remodeling while restoring airway responsiveness to salbutamol in a murine model of acute and chronic asthma. BMCs CdM also attenuated Th2associatedcytokinerelease,inducedasubsetofantiinflammatoryIL 10secretingTregsandpromotedIL10productionbymacrophages.Our data support the potential for APN as a target for asthma therapy by showingthatAPNcontributedsignificantlytopreventAHR,inflammation and histological features of airway remodeling. We propose APN as a candidate for asthma therapy. BMCsderived soluble factors may be a viable alternative to wholecell delivery and could therefore serve as a novelapproachforthetreatmentofasthma. Recent evidence suggests that stromal stem cells prevent organ damage through a paracrine effect rather than cell replacement [45]. In addition, these cells display antiinflammatory and immunomodulatory properties[19,20,25,27,28,38].Thesepropertiesrenderstromalstem cells particularly appealing and amenable to the treatment of inflammatorydiseases. In vitro,thesecellshavebeenshowntoinhibitthe proliferation of immune effector cells such as B lymphocytes and T

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lymphocytes [19]. In humans, a phase II clinical trial suggested that stromal stem cells attenuate steroidresistant graftversushost disease after haemopoietic stem cell transplantation [27]. Inhaled and systemic steroidshavebeensuccessfullyemployedinasthmasincetherecognition of the contribution of airway inflammation to asthma pathogenesis, but refractoryasthmaisstillresponsiblefor250,000deathsworldwideevery year [31]. Refractory asthma is a pathological entity characterized by reducedorlackofresponsivenesstoconventionalbronchodilatortherapy [1, 23]. Here we show that BMCsderived CdM exerts potent anti inflammatory effects in acute and chronic murine asthma, preventing airway inflammation (Fig. 24) and AHR in both models (Fig. 25). In chronic asthma, BMCs CdM restored the response to salbutamol, a commonlyprescribedbronchodilator(Fig. 26),andpreventedASMlayer thickeningandperibronchialinflammation(Fig.27).Meanwhile,FibCdM failed to significantly impact AHR, bronchodilator responsiveness and airwayremodeling.ThissuggeststhatthebenefitswerespecifictoBMCs CdM. Asthma is an inflammatory disease caused by a complex set of interactions between multiple effector cell types. The presence of the antigentriggersTh2lymphocyteactivation,leadingtoanacutecascadeof events, which include recruitment of PMN to the lung parenchyma

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(typically eosinophils) [18] and secretion of Th2 cytokines such as IL4 and IL13 [13, 32, 62]. In our study, PMN recruitment was inhibited by BMCs CdM treatment, as indicated by BALF analysis in both acute and chronic asthma models (Fig. 24). Fib CdM also decreased total BALF cellularityandPMNnumbers.Thiseffectwaslimitedtothechronicmodel andreducedcomparedtoBMCsCdM.AsimilarpartialreductioninBALF cellularity and eosinophilia following skin fibroblast administration has beenreported[39].Theseobservationshighlighttheimportanceofusing appropriatecellcontrolsinordertofurtherourunderstandingaboutthe mechanismofactionofMSCsandCdM. In humans, chronic airway remodeling is characterized by airway wall thickening, mainly due to smooth muscle hypertrophy and hyperplasia, accompanied by the narrowing of the airway lumen with gobletcellhyperplasia,peribronchialandperivascularfibrosis[17].These structuralchangesinchronicasthmaticairwaysmaybeaconsequenceof prolonged inflammation [53]. These changes may contribute to fatal asthmarefractorytobronchodilatortherapy.Weevaluatedtheimpactof BMCs CdM administration on airway remodeling by quantifying the degree of ASM layer thickening and peribronchial inflammation in the chronic asthma model, where remodeling was expected to occur. Both parameterswerereducedinOVACdManimalscomparedtotheOVAand

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OVAFib CdM groups (Fig. 27C and D), suggesting that BMCs CdM prevents the development of airway remodeling. This histological improvement was accompanied by restoration of the bronchial responsiveness to salbutamol, a conventional bronchodilator (Fig. 26). Administration of a single dose of salbutamol on maximally constricted airwaysledtoasignificantdecreaseindynamicresistanceandelastance in the OVACdM similar to controls, while this response was blunted in untreated OVA mice and OVAFib CdM mice, a major feature in the occurrenceofrefractoryasthma. WesoughttodeterminethemechanismsthroughwhichCdMacts topreventthedevelopmentofasthmafeatures.Wefoundthatthelevelsof IL4andIL13,twoTh2cytokinesgenerallyelevatedinasthma[62],were attenuatedinthelungsofOVACdManimalscomparedtountreatedOVA mice (Tables 21 and 22). Our findings are consistent with recent reports showing that BMCs administration decreases lung inflammation andBALFlevelsofTh2cytokinesinanacute,ragweedinducedmodelof allergic asthma [39] and OVAinduced mouse models of AHR [5, 11, 21]. Moreover, in our study the levels of the antiinflammatory IL10 were significantly increased in the OVACdM lungs suggesting that BMCs CdM attenuates allergic Th2 inflammation through an IL10dependent mechanism.IL10hasbeenshowntocontributetothereductionofairway

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inflammation[41].BMCsCdMitselfactedasanexogenoussourceofIL10, however,wedidnotfindasignificantdifferencebetweenthelevelsofIL 10 in the lungs of BMCs CdMtreated control animals and untreated controls, suggesting that endogenous production of IL10 in the allergic inflammatory environment may be an important component of elevated IL10levelsfoundonlyinOVACdMlungs.Wethereforesoughttoidentify cellulareffectorsthatwouldmediatetheactionsofBMCssecretedfactors and also potential local IL10 sources. Regulatory T (Treg) cells have recentlybeenshowntodiminishinflammationinasthma[35]andBMCs areabletoinduceIL10secretionbyTregcells[10]andmacrophages[24, 40]. Naturally occurring Treg cells are most commonly defined as cells thatexpressthesurfacemarkersCD4,CD25andtheintracellularmarker Foxp3 upon activation [34]. We did not find significant changes in the proportionofnaturallyoccurringCD4+CD25+Foxp3+regulatoryTregcells inthedraininglymphnodesorlunglymphocytes(Fig. 210),suggesting that local BMCs CdM administration does not act on a systemic cellular immunity level in the acute model. This is consistent with reports indicating that shortterm exposure to ovalbumin may lead to decreased lungCD4+CD25+Foxp3+cells[6]orspleenCD4+Foxp3+ cells[59].Arecent studysuggestsupregulationofCD4+CD25+Foxp3+inthelungsofBALB/c mice following allogeneic BMCs treatment in an OVAinduced model of AHR [21]. However, in this report the allogeneic BMCs were also

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administered at the time of sensitization to OVA, not only upon antigen challenges. We did not attempt to prevent antigen sensitization, as we sought to mimic the clinical conditions in which the patient would have already been hypersensitive to specific triggers. Instead, we found that BMCs CdM prevented the OVAinduced downregulation of Treg cells expressingCD4,CD25andIL10andlackingexpressionofFoxp3 (Fig. 2 11AG),asubsetofinducibleTregcellsthathavebeendescribedtobeIL 10induced and secreting Treg cells (Tr1) [15, 16, 56]. This correlates with our finding that IL10 is present in BMCs CdM and consistent with otherreportsindicatingthepresenceofIL10inthehumanstromalstem cell secretome [58]. Simultaneously, using CD11b as a macrophage marker,wefoundasignificantupregulationofCD11b+coexpressingIL10 in OVACdM lungs (Fig. 211HL). It has been proposed that BMCs may contribute to reduction of allergic airway inflammation by promoting a switch from Th2 to Th1 phenotype in an OVAinduced murine model of AHR [11]. Recently, a population of CD11b+Gr1+F4/80+

immunoregulatory myeloidderived suppressor cells (MDSCs) have been showntocontributetobluntedTh2responsesfollowingpreallergenLPS exposure in a house dustmite murine model of AHR. MDSCs were also able to prevent airway eosinophilia and IL13 production when transplantedinvivopostOVAexposure.ThehypothesisthatIL10hasa

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directroleinreducingTh2inflammationissupportedbythefindingthat Th2cytokinelevelswererestoredwithneutralizationofIL10[3]. BMCs have also been shown to act via TGF production to alleviate airway inflammation in a ragweedinduced model of murine asthma[39]. In our quest to identify candidate factors mediating the beneficial effects of BMCs, we found APN, an antiinflammatory adipokine to be present in BMCs CdM. APN is a white adipose tissuederived anti inflammatory cytokine that is found in lower levels in obese patients. Obesity is associated with increased incidence and worse outcomes of asthma, suggesting that APN levels may serve as a biomarker in asthma [49].IthasbeenshownthatAPNKOmicedevelopmoreseverefeaturesof chronic allergic asthma, including airway inflammation and remodeling, comparedtowildtypeanimals[36].Inaddition,rAPNdecreasedAHRand neutrophilandeosinophilcounts,aswellasBALFIL13andIL5levelsin acute murine OVAinduced asthma [48]. Consistent with these observations, we found that administration of CdM from WT BMCs in which APN was neutralized (acute asthma model) or CdM from BMCs of APN KO mice (chronic model) was less effective than WT CdM in attenuating AHR (Fig. 28B and D) and remodeling (Fig. 29) in chronic OVAinduced asthma. Conversely, administration of rAPN alone restored airwayresponsivenesstonormallevels(Fig. 28CandD)andprevented

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ASMthickening(Fig. 29F)andperibronchialinflammation(Fig. 29G)in thechronicmodel. Given the relative scarcity of reports regarding the phenotype of BMCsfromtheBALB/cmousestrain[7,44]andtheheterogeneityofthe BMCs used in this study (Fig. 23), we sought to determine the cellular source of APN. We separated the BMMSCs by negative CD45based selection.BMMSCsdisplayedaCD45Sca1+CD29+phenotype,whereasthe positivelyselectedpopulation(CD45+BMCs)wereCD45+Sca1loCD29+(Fig. 212). The BMMSCs were found to be the major contributor to APN productioncomparedtoCD45+BMCs(Fig.28A). We sought to clarify the role of APN in our study and we tested whether APN alone could contribute to the upregulation of IL10 expressingcellularsubtypes.rAPNadministrationdidnotaffectTr1levels whencomparedtountreatedOVAanimals(Fig. 211AG).APNhasbeen showntoinduceIL10secretionbymacrophages[26,42],anothercellular source potentially responsible for the increased IL10 found in the OVA CdMlungscomparedtotheuntreatedOVAanimals.Inthechronicasthma model, rAPN induced the upregulation of IL10expressing macrophages (Fig. 211HL), similar to BMCs CdM administration. It is therefore possiblethatadministrationofBMCsCdMwouldinducetheTr1through IL10foundintheCdM,withasimilar,simultaneousupregulationofIL10 production by lung macrophages through APN. This would lead to a

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sustained production of endogenous IL10 that may contribute to attenuationofinflammatoryevents[41]. WeaimedtodeterminetheeffectsofCdMaloneinordertocome ascloseaspossibletotheclinicalsetting:arelativelyquickdeliveryofa cellfreepreparationthatcanbeadministeredviatheairways.Theformer paradigm for the benefit of BMCs administration focused on cell replacement;however,thisconceptencompassedtheconcernsrelatedto cell tumorigenic potential and allogeneic transplantation [2, 30]. We believed that the delivery of a cellfree preparation would somewhat alleviate concerns related to tumorigenesis. A recent report [29] has compared the effects of BMMSCs and BMMSCs CdM in endotoxin inducedlunginjury(aninflammatorymodel)anddidnotfindsignificant differencesbetweentheeffectsoftheCdMandtheadministrationofcells themselves. In summary, this is the first study to investigate the therapeutic potentialofMSCsCdMandtoproposeAPNasaMSCsderivedprotective factorinexperimentalasthma. Overall, our data not only highlights the therapeutic benefit of BMCsinasthma,butalsosuggestBMCsderivedsolublefactorsasanovel, practicalandclinicallyrelevantapproachforthetreatmentofasthmaand

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other inflammatory disorders, thereby alleviating the potential risks of whole cell therapy. Further identification of BMCsderived factors may holdpromisefornovelapproachesinthetreatmentofasthma.

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Table21.Cytokineprofileintheacuteasthmamodel(meanSEM) Interleukin4 Group (pg/mgprotein) 39.395.26 Interleukin13 (pg/mgprotein) 152.378.48 Interleukin10 (pg/mgprotein) 652.7588.64

Saline(n=11) SalineCdM (n=9) OVA(n=17) OVACdM (n=18) OVAFibCdM (n=7)

27.871.96

147.196.32

654.1388.02

66.374.81*,**

207.0412.99**

753.6355.65 1156.50 95.78**

51.624.16**

126.445.08

63.213.27**

190.4418.18*,**

992.9160.40*

IL4:**p<0.01OVAandOVAFibCdMvs.saline,salineCdM;OVACdMvs. salineCdM;*p<0.05OVAvs.OVACdM. IL13: **p<0.01 OVA vs. saline, salineCdM, OVACdM; OVAFib CdM vs. OVACdM;*p<0.05OVAFibCdMvs.saline,salineCdM. IL10:**p<0.01OVACdMvs.saline,salineCdM,OVA;*p<0.05OVAFib CdMvs.saline,salineCdM.

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Table 22. Cytokine profile in the chronic asthma model (mean SEM) Interleukin4 (pg/mgprotein) Interleukin13 (pg/mgprotein) Interleukin10 (pg/mgprotein)

Group

Saline(n=5)

19.183.12

99.424.90

662.0922.87

OVA(n=7)

131.9312.67**

165.6011.39**

840.5460.94

OVACdM (n=7) OVAFibCdM (n=7)

87.789.71*,**

118.565.56**

1365.95 100.24**

121.5710.97**

140.079.16*,**

862.3535.13

IL4:**p<0.01OVA,OVACdM,OVAFibCdMvs.saline;OVACdMvs.OVA; *p<0.05OVACdMvs.OVAFibCdM. IL13: **p<0.01 OVA and OVAFib CdM vs. saline; OVACdM vs. OVA; * p<0.01OVAFibCdMvs.OVA. IL10:**p<0.01OVACdMvs.saline,OVA,OVAFibCdM.

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2.7.Figures

Acute asthma model

Sensitizations: 20 g OVA + 2 mg Al(OH)3 i.p. 1 6 Challenges: 50 g OVA i.n. (in 25L saline) +/-25L CdM

Days

11

13 15

Endpoint analysis

Chronic asthma model

Sensitizations: 20 g OVA + 2 mg Al(OH)3 i.p. 1 8 15 Challenges: 50 g OVA i.n. (in 25L saline) +/-25L CdM

Days

22 24 27 29 31 33 36 38 Sensitization Endpoint analysis

Figure 21. Experimental protocols for the acute (A) and chronic (B) OVAinducedallergicasthmamodels.

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C C

D D

E E

F F

scale bar 65 m

Figure 22. Mesenchymal lineage differentiation of BALB/c BMCs. BMCs differentiate into adipogenic (A, arrows indicate Oil Red Ostained lipid droplets), osteogenic (B, arrows indicate Alizarin Redstained calcium deposits) and chondrogenic (C, arrows indicate Safranin O stained proteoglycans) lineages following three weeks in culture with specificinductionfactors.(DF)UndifferentiatedBMCsculturedinDMEM. Images representative from a series of n=3 experiments for each differentiationassay.

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Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control Isotype control

FSC-H

Sca-1

CD73

CD45

CD44

CD34

CD31

CD11b

CD105

FL-H

CD105 CD11b CD31 CD34 CD44 CD45 CD73 Sca-1

0

20

40

60

80

100

Percentage of positive cells

Figure23.FlowcytometriccharacterizationofBALB/cBMCssurface markerprofile.

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A
1600
Total cells in BALF (x 103)/mL

Acute asthma model C

Cells in BALF (x 103)/mL

1400 1200 1000 800 600 400 200 0

Saline (n=10) Saline-CdM (n=9) OVA (n=8) OVA-CdM (n=8) OVA-FibCdM (n=7)

700

*** ***

* ***

600 500 400 300 200 100 0

** ***

* *** *** ** *** *** ***

***

** ***

***

Eosinophils (x103)/mL

Neutrophils (x103)/mL

Monocytes / macrophages (*103)/mL

Chronic asthma model

B
900
Total cells in BALF (x 103)/mL

OVA-CdM (n=5) 700 600 500 400 300 200 100 0 OVA-FibCdM (n=5)

Cells in BALF (x 103)/mL

800

Saline (n=3) OVA (n=5)

** *** ** **

D
400 350 300 250 200 150 100 50 0 Eosinophils (x103)/mL Neutrophils (x103)/mL Monocytes / macrophages (*103)/mL

** *** * *** *** * * ***

* ***

Figure 24. BALF analysis. BMCs CdM significantly decreased the total cellnumber(Aacutemodel,Bchronic)andlungPMNinflux(Cacute, D chronic)inBALF. (A) Total cells in BALF (acute):***p<0.001OVA, OVAFibCdMandOVACdMvs.salineandsalineCdM,OVAvs.OVACdM; *p<0.05 OVAFibCdM vs. OVACdM. (B) Total cells in BALF (chronic): ***p<0.001OVAvs.saline,OVACdM;**p<0.01OVAvs.OVAFibCdMand OVAFibCdMvs.saline;*p<0.05OVACdMvs.saline. (C) Differential cell countsinBALF(acute):***p<0.001OVA,OVAFibCdMandOVACdMvs. salineandsalineCdM;**p<0.01OVAvs.OVACdM;*p<0.05OVAFibCdM vs. OVACdM (eosinophils only). (D) Differential cell counts in BALF

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(chronic). ***p<0.001 OVA vs. saline, OVACdM; (eosinophils and neutrophils) ***p<0.001 OVAFibCdM vs. saline; *p<0.05 OVA vs. OVA FibCdM and OVACdM vs. saline; (monocytes/macrophages) **p<0.01 OVACdM vs. OVAFibCdM and *p<0.05 OVAFibCdM vs. saline. The valuesareexpressedasmeansstandarderrorofthemean(SEM).Data representativefromaseriesofn=5separateexperiments.

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Figure 25. Invasive LFT. BMCs CdM significantly reduced the methacholineinduced increase in dynamic lung resistance (acute) and elastance (acute and chronic models). (A) Dynamic lung resistance (acute): *p<0.05OVAvs.OVACdM,OVAFibvs.salineandOVACdM,** p<0.01 OVA vs. saline. (B) Dynamic lung elastance (acute): *p<0.05 OVAFibvs.OVACdM,**p<0.01OVAvs.OVACdM,OVAFibvs.salineand salineCdM,***OVAvs.salineandsalineCdM.Dataforeachmethacholine

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doseexpressedasmeansSEMofthepeakpercentincreasecomparedto baselineresponse to aerosolized saline (n=4 separate experiments). n indicates the number of animals in combined experiments. (C) Dynamic lung resistance (chronic). (D) Dynamic lung elastance (chronic). * p<0.05OVAandOVAFibCdMvs.OVACdM,salineCdM;OVAFibCdMvs. saline; **p<0.01 OVA vs. saline. Data for each methacholine dose expressed as means SEM of the peak percent increase compared to baselineresponse to aerosolized saline (n=5 separate experiments). n indicatesthenumberofanimalsincombinedexperiments.

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Figure 26. Bronchodilator response to salbutamol. Bronchodilation inresponsetosalbutamolwasbluntedinthechronicasthmamodel.BMCs CdM restored the bronchodilator response to salbutamol. (A) Dynamic lung resistance: *p<0.05 OVA and OVAFib CdM vs. saline and saline CdM. (B) Dynamic lung elastance:*p<0.05OVAvs.saline,OVAFibCdM vs.salineandsalineCdM;**p<0.01OVAandOVAFibCdMvs.OVACdM. The values are expressed as means SEM. (n=5 separate experiments). nindicatesthenumberofanimalsincombinedexperiments.

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Figure 27. Airway remodeling in chronic asthma. (A) Bronchial smooth muscle layer thickness: *p<0.05 saline vs. salineCdM, OVA CdM vs. OVAFib CdM; **p<0.01 OVA and OVAFib CdM vs. saline and salineCdM, OVACdM vs. OVA and salineCdM. (B) Peribronchial inflammatory infiltrate area: **p<0.01 OVACdM vs. saline, saline CdM, OVA, OVAFib CdM. ***p<0.001 OVA and OVAFib CdM vs. saline, salineCdM. (CG) Hematoxylin and eosin (H & E) staining showing the

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characteristic features of airway remodeling of chronic asthma including increasedASMlayerthickeningandperibronchialinflammatoryinfiltrate inchronicOVAexposedmice.BMCsCdMsignificantlyreducedboth.Scale bar: 65 m. Data representative from a series of n=5 separate experiments.DataexpressedasmeansSEM.nindicatesthenumberof airwaysanalyzedincombinedexperiments.

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Dynamic lung elastance

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Figure 28. Effects of APN on AHR. (A) APN concentration in CdM preparations. *p<0.05WTCdMvs.CD45CdM;CD45vs.APNKOCdM, CD45+CdM,FibCdM,NeutCdM;***p<0.001WTCdMvs.NeutCdM;**** p<0.0001 WT CdM vs. APN KO CdM, CD45 CdM, Fib CdM. Results from two separate experiments.Data expressed as means SEM. n indicates thenumberofindependentCdMbatches. (B) Invasive LFT in the acute asthma model. NeutCdM failed to prevent acute OVAinduced AHR. * p<0.05OVAvs.saline,OVACdM.(C)InvasiveLFTinthechronicasthma model.BMCsCdMandrAPNsignificantlyreducedtheincreaseindynamic lung elastance. **p<0.01 OVA vs. OVACdM, saline, ***p<0.001 OVA vs. OVArAPN.DataforeachmethacholinedoseexpressedasmeansSEMof 118

the peak percent increase compared to baselineresponse to aerosolized saline(n=5separateexperiments). nindicatesthenumberofanimalsin combined experiments. (D) Bronchodilator response to salbutamol. Bronchodilation in response to salbutamol was blunted in the chronic asthmamodel.BMCsCdMandrAPNrestoredthebronchodilatorresponse to salbutamol *p<0.05 OVA vs. OVACdM, OVArAPN and saline. The values are expressed as means SEM. (n=5 separate experiments). n indicatesthenumberofanimalsincombinedexperiments.

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Saline

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Figure 29. Effects of APN on chronic airway remodeling parameters. (AE) Airway remodeling parameters in chronic asthma.

Representative H & Estained lung sections showing the characteristic feature of airway remodeling in the chronic asthma model including increasedASMlayerthickeningandperibronchialinflammatoryinfiltrate inchronicOVAexposedmice.BMCsCdMandrAPNsignificantlyreduced both. (F) Bronchial smooth muscle layer thickness: *p<0.05 OVA vs. OVACdM, OVArAPN, **p<0.01 OVA vs. saline, OVAKO CdM vs. OVA 120

CdM, OVArAPN, ***p<0.001 OVAKO CdM vs. saline. (G) Peribronchial inflammatory infiltrate area: *p<0.05 OVA and OVAKO CdM vs. OVA CdM,OVACdMvs.saline,**p<0.01OVAandOVAKOCdMvs.OVArAPN, ****p<0.0001 OVA and OVAKO CdM vs. saline. Scale bar: 65 m. Data representativefromaseriesofn=5separateexperiments.Thevaluesare expressedasmeansSEM.nindicatesthenumberofairwaysanalyzed incombinedexperiments.

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Figure210.FACSoflunganddraininglymphnodeslymphocytes.(A) OVA or BMCs CdM administration did not significantly impact the proportion of CD4+CD25+Foxp3+ cells in draining lymph nodes or (B) lungs. Data expressed as means SEM. n indicates the number of animalsincombinedexperiments.

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CD25

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Percentage of CD11b+IL-10+ cells

Figure 211. FACS of lung lymphocytes. (A) Gating for activated CD4+ lymphocytes (CD4+CD25+) followed by (B) gating for Foxp3 within this cell population. (CF) Representative scatterplots for CD4+CD25+Foxp3 IL10+ cells from each experimental group. (G) BMCs CdM increased the proportion of lung CD4+CD25+Foxp3IL10 cells.***p<0.001 saline and OVACdM vs. OVA and OVArAPN. (HK) Representative scatterplots for CD11b+IL10+ cells from each experimental group. (L) BMCs CdM increased the proportion of lung CD11b+IL10+ cells. *p<0.05 OVA vs. OVACdM and saline, ***p<0.001 OVArAPN vs. saline, ****p<0.0001 OVACdM vs. saline. Data representative from n=2 experiments. The valuesareexpressedasmeansSEM.

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SurfaceMarkerExpressionBALB/cBMCs

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Figure 212. Flowcytometric evaluation of CD45, Sca1, CD29 expressionbyWTBMCs,CD45+BMCsandBMMSCs.

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CHAPTER3STEMCELLCONDITIONEDMEDIUMPREVENTSACUTE LUNGINJURYINMICE:INVIVOEVIDENCEFORSTEMCELL PARACRINEACTION Aversionofthischapterisbeingreviewedforpublication. IonescuLI,ByrneRN,vanHaaftenTJ,VadivelA,AlphonseRS,ReyParra GJ,WeissmannG,EatonF,ThbaudB.StemCellConditionedMedium PreventsAcuteLungInjuryinMice:InVivoEvidenceforStemCell ParacrineAction.AmJPhysiolLungCellMolBiol

LIIperformedthemajorityoftheexperiments.RNBperformedpreliminary experiments.TvH,AV,RSA,GJRP,GWandFEprovidedtechnicalassistance.The manuscriptwaswrittenbyLIIandeditedbyBT.

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3.1.Abstract Mortalityandmorbidityofacutelunginjuryandacuterespiratory distresssyndrome(ALI/ARDS)remainhighinpartbecauseofthelackof pharmacologicaltherapiestopreventinjuryorpromoterepair.Stemcells have been proposed to regenerate damaged organs. Mesenchymal stem cells(MSCs)preventlunginjuryinvariousexperimentalmodels,despite a low proportion of donorderived cell engraftment. Increasing evidence suggestthatMSCsexerttheirbeneficialeffectsviaaparacrinemechanism. We hypothesized that soluble factors secreted by MSCs prevent lung injury in part by modulating macrophage function. We tested the protectiveeffectofMSCderivedconditionedmedium(CdM)comparedto wholeMSCs,lungfibroblastsandfibroblast(Fib)CdM in vivoand in vitro. Intratracheal MSCs and MSC CdM significantly attenuated

lipopolysaccharide(LPS)inducedlungneutrophilinflux,lungedemaand lung injury as assessed by an established lung injury score. MSC CdM increasedarginase1activityinLPSexposedalveolarmacrophages(AMs) andenhancedexpressionofYm1whiledecreasingiNOSinAMsfromLPS MSCandLPSMSCCdMmiceascomparedtocontrolmice,whichsuggests the occurrence of MSC CdMdriven alternative macrophage activation to an M2 healer phenotype. Comparative multiplex analysis betweenMSC CdM and lung fibroblastCdM demonstrated that MSC CdM contained a

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range of proteins that may confer therapeutic benefit in lung injury. In summary,MSCsexerttheirbeneficialeffectsthroughaparacrineactivity. MSC CdM prevents LPSinduced lung injury by attenuating lung inflammation and promoting a wound healing/antiinflammatory M2 macrophagephenotype.

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3.2.Introduction Despiteimprovementsinmanagement,acutelunginjury(ALI)and acute respiratory distress syndrome (ARDS) remain major causes of morbidity and mortality in critically ill patients of all ages [55]. The incidence of ALI/ARDS in the US is 138:100,000 persons per year and is anticipated to double in the next 25 years [55]. Treatment of ALI/ARDS remainsprimarilysupportive[34]. Bonemarrow derived mesenchymal stem cells (MSCs)

differentiateintocartilage,fatandbone,butalsointomuscle,liver,kidney, heartandbraincells[50].Thesepropertieshavebeenharnessedfororgan regeneration[58].Inthelung,bonemarrowderivedcells,includingMSCs, engraft and adopt a lung epithelial cell phenotype [2, 17, 27, 28, 47, 54, 62]. MSCs can also suppress local immune responses and have the capacitytoavoidimmunerejectioninallotransplantation[40, 24,29,30, 51].Theseimmunomodulatorypropertiesmightbeoftherapeuticbenefit inALI/ARDSandotherlungdiseasescharacterizedbyinflammation.Both intratracheal and systemic MSC administrations improve

lipopolysaccharide(LPS)inducedALIinmice[17,37].Thesefindingsare congruent with the therapeutic benefits of MSCs described in other lung injurymodels[2,21,31,46,54,62].Inallthesestudies,theengraftment rates of MSCs were low [2628, 54, 62]. Together with the rapid

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therapeuticbenefitsseenwithin48to72hoursofMSCdeliveryintheALI models [17, 37], these findings suggest that beyond cell replacement, MSCsmaybereleasingfactorsresponsibleforthebeneficialeffectsofcell therapy.Inaddition,sincetheuseofMSCsaswholecelltherapymayhold some risks to the patient [1, 32], we investigated the effects of MSC derivedconditionedmedium(CdM)inLPSinducedALIinmice.

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3.3.MaterialsandMethods AllprocedureswereapprovedbytheAnimalWelfareCommitteeof theUniversityofAlberta.Additionaldetailonallthemethodsisprovided intheonlinedatasupplement. 3.3.1. MSCs and Lung Fibroblasts Isolation, Culture and Characterization Bone marrow was harvested from adult (810 weeks) C57BL/6 mice (Charles River, ON, Canada), and MSCs were cultured and characterized as previously described [21]. The femur and tibia were excised and the marrow was flushed with Dulbeccos Modified Eagle Medium(DMEM;Invitrogen,Burlington,ON)containing10%fetalbovine serumand1%PSF.Theextractedmarrowwasdissociatedandplatedina tissue culture flask. After 24 hours, the media was aspirated, adherent cellswererinsedthreetimeswithPBSandreplenishedwithfreshmedia. Cells were grown to ~80% confluency, trypsinized, and reseeded at a density of 105 cells/cm2. Differentiation of MSCs was performed over 21 days on passage 23 cells [21]. MSCs were evaluated for expression of a panelofsurfacemarkersaccordingtoestablishedcriteria[12,15,37,49, 60].AntibodiesagainstthefollowingmarkerswereobtainedfromBecton

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Dickinson (BD, Mississauga, ON): stem cell antigen1 (Sca1, fluorescein isothiocyanate [FITC]), CD31 (phycoerythrin [PE]), CD11b (FITC), CD45 (FITC), CD44 (FITC), CD73 (PE), CD14 (FITC), CD34 (FITC), ckit (FITC), Flk1 (PE), CD106 (vascular cell adhesion molecule1 [VCAM1]), CD29 (PE) and from BioLegend (San Diego, CA): CD105 (PE). MSCs between passages711weredetachedfromculturesurfaces,counted,anddivided intoaliquotsof~0.51x106cells/samplein12x75mmpolystyreneround bottom tubes (BD Falcon). Cells were washed twice with flow buffer (0.05%sodiumazide,0.1%bovineserumalbumininPBS),incubatedwith therespectiveantibodiesat4Cwithgentleshakingfor30min,washed twice, resuspended in flow buffer and analyzed by flow cytometry (FACSCalibur, BD). Cellquest (BD) and FlowJo (version 5.7.2) software wereusedforanalyses. Lung fibroblasts were isolated from adult (810 weeks) C57BL/6 mice [59]. Fibroblast identity was confirmed by immunofluorescence stainingfortheintermediatefilamentproteinvimentin. 3.3.2.ConditionedMedium(CdM)Preparation Passage28MSCsandfibroblastsweregrownto>80%confluency. Medium (DMEM) was aspirated and cells were rinsed three times with PBS.CellswereculturedwithserumfreeDMEM(+PSF)for24hours.CdM

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was collected and filtered through a 0.2 m filter to remove cellular debris. Adherent cells were trypsinized, stained with trypan blue, and counted. The medium from 5 x 106 cells yielded 15 mL of primary CdM that was further desalted and concentrated ~25fold, yielding 600 L CdM, using ultrafiltration units with a 3 kDa molecular weight cutoff (Amicon UltraPL 3, Millipore, Billerica, MA). Similar to work by others [20], serumfree DMEM + PSF (desalted and concentrated 25fold) was thevehiclecontrol. 3.3.3.MurineLPSInducedALI EighttotenweekoldmaleC57BL/6micewereanaesthetizedwith 5%isofluraneandinjectedintratracheally(i.t.)with4mg/kgLPS(E. coli. 055:B5,SigmaAldrich,Oakville,ON).FourhourspostLPS,micewerere anesthetizedandreceiveda30Li.t.instillationofMSCs,LFs,MSCCdM, FibCdMorDMEM.WeensuredequivalencebetweencellbasedandCdM based treatment by administering the same number of cells (250,000 cells/30LDMEM)thatproduced30LconcentratedCdM. Mice (n>=5 per group per endpoint) were sacrificed via an intraperitonealinjectionofpentobarbitalat48hourspostLPSforeither bronchoalveolarlavagefluid(BALF)orlunghistologicalanalysis.

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3.3.4.BALFAnalysisandAlveolarMacrophages(AM)Isolation Lungswerelavagedwith2.5mLicecoldphosphatebufferedsaline (PBS)injectedat0.5mLincrementsviaa20gaugecatheterinsertedinthe trachea. BALF was centrifuged for 10 minutes at 400 g and BALF cells were enumerated using the Scepter automated cell counter (Millipore, Billerica, MA). Differential cell counts were performed on cytospin preparations (Thermo Shandon, Pittsburgh, PA) stained with Hema 3 Manual Staining System (Fisher Scientific, Nepean, ON) by counting 300 cellspercellsmearandmultiplyingbytotalcellnumberpermL. For AMs isolation, an established protocol was followed [65]. Briefly, BALF was centrifuged at 300 g for 10 minutes and the cellular pellet was washed with PBS, resuspended in red blood cell lysis buffer [8.3gNH4Cl,1gKHCO3,1.8mLof5%EDTAin1Lofdistilledwater)for5 minutes at room temperature and centrifuged again at 300 g for 10 minutes. The pellet was resuspended in RPMI1640 medium and plated atadensityof600,000cells/mLina24welltissuecultureplate.After2 hours,mediumwasremovedandadherentcellswerewashedthreetimes with PBS. AMs were stained with Hema 3 Manual Staining System for morphological assessment. AMs were evaluated for the expression of characteristicsurfacemarkers[18]usingantibodiesagainstCD11b(FITC)

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and CD11c (allophycocyanin [APC]) (BD Biosciences) by flow cytometry accordingtothesameprotocoldescribedforMSCcharacterization. 3.3.5.AssessmentofLungPermeability LungedemaduetoLPSinducedincreaseinlungpermeabilitywas measured using the wet / dry weight ratio of lung lobes as previously described[17].Briefly,lungswereweigheduponexcision(wet weights), homogenizedin1mLofwaterandplacedinadryingovenat55Cfor24 hours,dryweightsrecordedandwet/dryratiocalculated. 3.3.6.LungHistologicalAnalysis Lungs were inflated and fixed with 4% formaldehyde solution through a tracheal catheter at a constant pressure of 20 cm H 2O [21]. Lungswereprocessed,paraffinembeddedand4mthickserialsections were stained with hematoxylin and eosin (H&E). Images were captured (Openlab,Improvision,version5.0.2.)withLeicaCTRMICmicroscopeand 40highpoweredfieldsperlungwereexaminedbyablindedinvestigator toquantifythehistopathologyscore.Parametersassessedwere:alveolar septal congestion, alveolar hemorrhage, intraalveolar fibrin and intra alveolarinfiltrates.Ascorefrom0to3wasgivenforeachcriterionanda

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total score was established, all according to a previously published protocol [36, 37]. Briefly, lung injury score = [(alveolar hemorrhage points/no. of fields) + 2 x (alveolar infiltrate points/no. of fields) + 3 x (fibrin points/no. of fields) + (alveolar septal congestion/no. of fields)]/totalnumberofalveolicounted. 3.3.7.AMLPSExposureandAMPhenotypeCharacterization Freshly isolated AMs were cultured with RPMI1640 media containing10%FBSina12wellplate.Afterovernightadherence,RPMI 1640mediawasreplacedwitheitherDMEMalone,DMEM+LPS(1g/ml), or MSC CdM+LPS (1 g/ml). After 48 hours in culture, media was aspiratedandcellswererinsed3XwithPBS.Themonolayerofcellswas scraped,cellswerespundownandpelletswerefrozen.Arginaseactivity inAMwasmeasuredbydeterminingtheamountofureageneratedbythe enzyme [9, 57]. Briefly, the frozen AM pellets were thawed and homogenizedin100lpersampleoflysisbuffer(0.1%TritonX100with protease inhibitors: phenylmethylsulphonyl fluoride [PMSF] (1 mM), leupeptin (0.5 g/ml), aprotinin (5 g/ml), EDTA (2 mM)). The samples weresonicatedtwicefor35secondsandstoredat80C.Sampleswere thawedand50Lofthehomogenatewasaddedto50lTrisHCl(25mM, pH 7.5) containing MnCl2 (5 mM) and incubated at 56 C for 10 min to

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activatetheenzyme.50Lofthisheatactivatedsupernatantmixturewas incubated with 50 L Larginine (0.5 mol/L) at 37C for 60 min. The reaction was stopped by adding 400 L of an acid mixture (1 H2SO4: 3 H3PO4:7H2O).25Lofisonitrosopropiophenone(9%dissolvedin100 %ethanol)wasaddedtotheabovemixtureandincubatedat100Cfor45 minforcolordevelopment.Themixturewascooledatroomtemperature inthedarkfor10min.Astandardcurveforurea(030g)wasprepared. Ureaconcentrationinthehomogenatewasmeasuredusingacolorimeter at550/540nmwith200Lofthealiquot.Arginaseactivitywasexpressed asunits/mgprotein/hour. Forfurthermacrophagephenotypecharacterizationbymulticolor flow cytometry, AMs were isolated from experimental animals and allowedtoadheretoplasticasdescribedabove.Thefollowing antibodies wereused:CD11b(PacificBlue),CD11c(PE,bothfromBiolegend),rabbit antimouse inducible nitric oxide synthase (iNOS, Abcam) and donkey antirabbit secondary (FITC, Biomeda Corporation, Foster City, CA), goat antimouse Ym1 (R&D Systems) and donkey antigoat secondary (Cy5, Biomeda). The staining was performed as described for MSC characterization,withtheadditionofa30minuteincubationofthecellsat 4 C in Cytofix / Cytoperm Buffer (BD), followed by resuspension in Permeabilization Buffer (BD) before incubation with primary antibodies. AnalysiswasperformedusingFACSCantowithFACSDivasoftware(BD).

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3.3.8.AntibodyarrayofMSCCdM MSCCdMandFibCdMwereprepared,concentratedanddesalted asdescribedabove.Sampleswereanalyzedforapanelof96factorsusing an antibody array (RayBio Mouse Antibody Array G Series 1000, RayBiotech Inc., Norcross, GA). Relative intensity of each factor in MSC CdMandFibCdMisreportedcomparedtoapositiveandnegativecontrol, aswellasfoldchangeofspecificfactorsinMSCCdMversusFibCdM. 3.3.9.StatisticalAnalysis Data are expressed as mean standard error of the mean (SEM). Group comparisons were analyzed by oneway ANOVA with a Fishers PLSD post hoc test. An unpaired ttest was used when appropriate. Data was assessed with statistical software Statview (version 5.0.1, SAS Institute,Cary,NC).Valueswereconsideredsignificantwithp<0.05.

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3.4.Results 3.4.1. MSC Display Functional Characteristics and Surface Marker PhenotypeofMurineMSCs Bone marrowderived MSCs differentiated into adipogenic, osteogenic and chondrogenic mesenchymal lineages (Fig. 31). Cell surface antigen phenotype was assessed by flow cytometry. MSCs expressed high levels of Sca1 (92.93% of cells) and CD29 (89.99%), moderatelevelsofCD105(33.92%),CD106(14.36%),CD11b(12.10%), andCD45(11.62%)andwereconsiderednegativeforCD14(0.30%),ckit (0.53%),CD34(1.13%),CD73(1.49%),Flk1(3.01%)andCD31(4.08%of cells)(Fig.32). 3.4.2. MSC CdM Decreased Lung Inflammation and Lung Vascular PermeabilityinLPSInducedLungInjury LPSsignificantlyincreasedthetotalcellandneutrophilcountinthe BAL after 12 hours compared to uninjured controls (Fig. 33A). This inflammatoryinfluxwasattenuatedbyMSCsandMSCCdMtreatment,but notbytreatmentwithDMEM,thevehiclecontrol,FiborFibCdM(Fig. 3 3A). MSCs and MSC CdM, but not DMEM, Fib or Fib CdM prevented the

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LPSinduced increase in lung vascular permeability, as assessed by lung wet/dryratio(Fig.33B). 3.4.3.MSCCdMFailedtoPreventLPSInducedBodyWeightLoss All mice given LPS were lethargic and had reduced activity and decreased body weight over 48 hours compared to control mice ( Fig. 3 4A). 3.4.4.MSCCdMImprovedLPSInducedLungInjury Histological assessment of lung injury using a semiquantitative histopathology score revealed that mice treated with LPS had increased septal thickening, alveolar hemorrhage, alveolar infiltrates, and fibrin strandscomparedtocontrols,whereastreatmentwithMSCsorMSCCdM significantlyattenuatedthesefeatures(Fig.34BandC). 3.4.5. MSC CdM Determined Alternative Activation of AMs Following LPSExposureInVitroandInVivo Isolated AMs were macroscopically (insert Fig. 35A) and phenotypically(CD11c+CD11b)(Fig. 35A)consistentwithAMs. In vitro,

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AMs were exposed to LPS in order to test the role of MSC CdM on the induction and maintenance of the M2 phenotype in AMs. LPS increased arginase activity in AMs as compared with nonLPSexposed cells, and MSC CdM further elevated these levels compared to LPSexposed cells culturedwithDMEM(Fig.35B). InordertofurtherinvestigatetheeffectsofMSCCdM in vivo,AMs were isolated from LPSexposed animals that had received cell or CdM treatment.MSCCdMinducedaniNOSYm1+phenotypeinAMsfromboth control (11.6% vs. 0.8%) and LPStreated animals (69% vs. 6.8%). This effect was enhanced in LPSMSC recipients (79.9%). Fib and Fib CdM markedly induced an iNOS+Ym1 phenotype (52% and 41.9%, respectively) compared to control AMs (1.3%), similar to the proportion foundinLPSDMEMAMs(41.8%)(Fig.36). 3.4.6. MSC CdM Contains Soluble Factors That May Convey TherapeuticBenefit The levels of 96 different factors were compared between MSC CdM and Fib CdM (Fig. 37). Cluster analysis of both CdMs revealed the following distribution: chemokines (31%), binding proteins (27%), cytokines (21%), growth factors (14%), cytokine antagonists (4%) and molecules involved in extracellular matrix repair (3%) (Fig. 37).

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Significant foldvariations of certain factors were found (Fig. 38). MSC CdM contained higher concentrations of stem cell factor (SCF), IL 12p40/p70,stromalderivedfactor1alpha(SDF1)andosteoprotegerin and lower concentrations of IL6, macrophage inflammatory protein3 alpha(MIP3)andthymusandactivationregulatedchemokine(TARC).

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3.5.Discussion We provide evidence that the therapeutic benefits of MSCs are attributabletoaparacrinemechanism. Invivo,administrationofMSCCdM aloneattenuatedlungneutrophilinfluxandimprovedlunghistology.This effect was comparable with administration of an equivalent number of MSC and absent in mice treated with control cells (lung fibroblasts) or controlcellCdM.MSCCdMinducedanM2phenotypeinAMsexposedto LPS in vitroand in vivoinLPSexposedanimals.WealsosuggestthatMSC CdM contains soluble factors capable of attenuating lung injury. Identification of these soluble factors secreted by MSCs may yield new therapeuticoptionsforALI/ARDS. Recent studies have shown that MSCs modulate immune cell function[23,40,51]andhavecellprotectiveeffectsthroughthereleaseof cytokines and growth factors [14, 20, 31]. MSC CdM decreased hypoxia induced cell death and improved tube formation of human aortic endothelialcells[20].CdMderivedfromMSCsengineeredtooverexpress the prosurvival gene Akt protected cardiomyocytes from hypoxia induced cell death and limited myocardial infarct size in vivo [13]. MSC CdMimprovedhealinginanexcisionalwoundsplintingmodelinmice[7], oxygeninducedAT2andpulmonarymicrovascularendothelialcellinjury in vitro[62],neonataloxygeninducedlunginjury in vivo[2]andreversed

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hepatocyte death and increased survival in GalN induced fulminant hepatic failure [41]. Moreover, human MSC CdM attenuated endotoxin induced lung injury in an ex vivo perfused human lung model [31] and mouseMSCCdMpreventedthedevelopmentofmurineasthma[21]. In the current study, the therapeutic benefit seen with CdM mirroredtheprotectiveeffectsdescribedwithwholecelltherapy.Similar topreviousfindingswithwholecelltherapyafterLPSinjury[17,37],we observedthatasingleintratrachealinjectionofMSCCdMorMSCs4hours after LPS administration decreased lung neutrophil influx, lung permeabilityandimprovedthelunghistopathologyscoreascomparedto control media, lung fibroblasts or their CdM. These findings open new therapeutic options by identifying potential healing molecules contained inMSCderivedCdMandunderstandingtheirmechanismofaction. OurdatasuggestthatMSCCdMpromoteaM2healermacrophage phenotype. In the development of ALI/ARDS, neutrophils and macrophages are activated in order to eliminate pathogens, but also contribute to tissue injury through the release of antimicrobial compounds[63].Macrophagesarephenotypicallyheterogeneousbecause they respond to stimuli in their microenvironment and they also differ genetically [16]. Classically activated macrophages (M1) kill invading microorganisms and tumors and promote type I immunity by secreting high levels of proinflammatory cytokines and low levels of anti

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inflammatory cytokines [33]. In contrast, alternatively activated macrophages (M2) secrete lower levels of proinflammatory cytokines and higher levels of antiinflammatory cytokines. M2 macrophages promote type II immunity and are thought to dampen the immune response and to promote wound healing, angiogenesis, and debris scavenging [10]. M1 and M2 macrophages can be characterized by receptor expression, effector function, cytokine and chemokine production [33] or by a set of marker genes: M1 macrophages generate nitric oxide (NO) by upregulation of inducible nitric oxide synthase (iNOS), whereas M2 macrophages express Ym1 (also known as T lymphocytederived eosinophil chemotactic factor [ECFL] and chitinase 3like 3 [CHI3L3]), FIZZ1 (found in inflammatory zone1, also known as resistinlike molecule alpha [RELMalpha]), and arginase1 (Arg1) [16, 43]. The upregulation of arginase1 expression and activity is crucial in themetabolicswitchfromM1toM2phenotypeinmice[52].InourLPS induced inflammation model, we screened for the effect of MSC CdM on AMphenotype in vitro.AMsisolatedfromhealthymiceandsubsequently exposed to LPS followed by MSC CdM had higher Arg1 activity characteristicofM2macrophages.Inordertomorethoroughlytestforthe occurrenceofalternativemacrophageactivation in vivo,weisolatedAMs fromexperimentalanimalsandfoundthatLPS,aswellasFibandFibCdM, increasedthenumbersofmacrophagesexpressingiNOSintheabsenceof

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Ym1 (a pattern corresponding with the M1 phenotype), while MSCs and MSCCdMupregulatedYm1+macrophageslackingiNOSexpression(M2). These data support the immunomodulatory capacity of MSC CdM shiftingtheimmuneenvironmentfromprotoantiinflammatory viathe inductionofahealerM2AMphenotypefromakillerM1macrophage. Our results are in line with several recent reports indicating that MSCs exert antiinflammatory properties via macrophage reprogramming [25, 42, 44]. However, the murine MSC populations differ amongst these reports, mainly due to the current lack of consensus in establishing the universal murine MSC phenotype [6, 49]. Comparison is also limited by differences in experimental protocols and variations in CdM preparation methods. TheparacrinemechanismofactionofMSCsopensnewtherapeutic perspectives. Indeed, several paracrine mediators that can mediate restorativeeffectsofMSCshavebeenidentified,includinginterleukin10, IL1 receptor antagonist (IL1ra), and keratinocyte growth factor (KGF). IL1rawasidentifiedasanMSCderivedparacrinefactorthatreducedthe severityofbleomycininducedlunginjury[46].AllogeneichumanMSCsor theirCdMattenuatedendotoxininducedlunginjuryinan exvivoperfused human lung model partly through KGF, a wellknown growth factor to reduce lung injury [31]. In our study, we found that MSC CdM contained higher levels of KGF and HGF than Fib CdM. IL10 was shown to be

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responsible for the therapeutic benefits of exogenous MSCs in murine sepsis [44]. Our group has recently reported that BMSCderived adiponectin contributes to the antiasthmatic effect of BMSC CdM in a murine ovalbumininduced asthma model [21]. Human cord blood derived MSCs, but not fibroblasts, produce high levels of angiotensin converting enzyme 2, an exopeptidase recently shown to be lung protective [53]. Further identification of soluble factors may lead to the developmentofcriticallymissingpharmacologicaltherapiesforALI/ARDS andotherinflammatorydiseases[5]. Inanattempttobroadenthesearchforcandidatesolublefactors, weperformedamultiplexanalysisscreeningfor96factorspresentinMSC CdM and showed that their secretory profile differed from that of lung fibroblasts.Inasimilarmanner,Schinktheetal.screenedhumanMSCsto attain the first largescale description of factors they secrete and categorized these into functional groups: antiapoptotic,

immunosuppressive, proproliferative and angiogenic modulating [58]. Amongst factors that may account for the alternative macrophage activation, we found that MSC CdM contained several known M2 activators.Interleukins(IL)4,10and13wereidentifiedinourmultiplex screenofMSCCdM(Fig.37).ThesecytokinescanactivatetheJAKSTAT6 signalingpathwayandpromoteSTAT6bindingtothepromoterregionof targetgenes,whichultimatelydrivestheexpressionofM2specificgenes

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[23].Althoughthelevelsoftheseinterleukinswerecomparablebetween MSC CdM and Fib CdM, they may contribute to alternative macrophage activationinanorchestratedfashion,togetherwithothercomponentsof MSCCdM.FibCdMalsocontainedhigheramountsofthepotentclassical (M1)activatorIL6,whileMSCCdMhadhigherlevelsoffactorsthathave recently been shown to contribute to the promotion of an M2 environment. One of these factors is insulinlike growth factor1 (IGF1), whichhasbeenfoundtoaidincreatinganM2favorableenvironment[7]. Another factor, adiponectin, that may exert similar M2activating effects [43] was found in our MSCs CdM from both WT C57/BL6 and Balb/C mousestrains,butwasundetectableinFibCdMbyELISA[21]. Of the 96 different cytokines screened, MSC CdM contained markedly higher levels of potential protective factors compared to Fib CdM,suggestingthatadditionalmechanismscouldexplainthetherapeutic benefit of MSC CdM in this model. Stem cell factor (SCF) is increased almost 3fold in MSC CdM compared to Fib CdM. SCF improves survival, proliferation, and differentiation of hematopoietic stem and progenitor cellsandantiapoptoticeffectsreportedinkidneytubularepithelialcells [3, 12]. Another molecule, FcRIIB, is increased 14fold in MSC CdM comparedtoFibCdM.FcRIIBmediatestheantiinflammatorybenefitsof intravenous gammaglobulin (IVIG) in a murine model of immune thrombocytopenia[56].WealsofoundthatIL12p40,anaturalantagonist

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ofIL12,whichisacytokineresponsiblefortheproductionofIFN,was increased 7fold in MSC CdM compared to Fib CdM. IL12p40 has protective effects in bacterial pneumonitis in mice [18] and selectively inhibitsairwayhyperresponsivenessandperibronchialfibrosisinmurine asthma [46]. A twofold increase in soluble TNF receptor II (sTNFRII), an inhibitor of TNF and a possible regulator of inflammatory activity, wasobservedinMSCCdMcomparedtoFibCdM.sTNFRIImaycontribute todampeningoftheactivityofTNFinLPSinjury[38]. Inconclusion,ourstudyprovidesdirectinvivoevidencethatMSCs exerttheirtherapeuticbenefitthroughaparacrineactivity.Identification of pneumoprotective soluble factors in MSC CdM holds promise for the discoveryofnewpharmacologicaltherapiesforlungdiseases.WhileMSCs may have a unique ability to monitor the microenvironment of injured tissues and respond appropriately, therapies with the proteins or cytokines produced by activated MSCs may be more practical than cell therapies [1, 32]. In vitro priming of MSCs to optimize the production of protective factors may combine the advantages of both cell and small moleculetherapy.

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3.7.Figures

Figure 31. Mesenchymal lineage differentiation of C57BL/6 BM MSCs.MSCsdifferentiatedalongadipogenic,osteogenicandchondrogenic lineages.Toprow:differentiatedMSCs;bottomrow:controlMSCs.Leftto right panels: Oil Red O staining (adipocytes), Alizarin Red (osteocytes), SafraninO(chondrocytes).Sizebar:30m.

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Figure 32. Flowcytometric characterization of C57BL/6 BMMSCs surface marker profile. Representative flowcytometry histograms (A) and quantification of MSCs surface marker expression (B). Values are expressedasmeanSEM.

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Figure 33. BALF and lung permeability analyses. (A) BALF total cell andneutrophilnumbers.LPSDMEMmice(n=10)hadsignificantlymore inflammatory cells than uninjured controls (n=5) and controlCdM (n=6) mice.TreatmentwithMSCs(n=5)orMSCCdM(n=10),butnotFib (n=5) or Fib CdM (n=5), significantly attenuated lung cells influx. BAL fluid of micetreatedwithDMEMhad2timesmorePMNsthanthosetreatedwith 163

MSC CdM. There were no differences in MN number. *p<0.05 control, controlCdM vs. LPSMSC CdM; **p<0.01 control, controlCdM vs. LPS DMEM,LPSFib,LPSFibCdM,LPSMSC;LPSMSCvs.LPSDMEM,LPSFib, LPSFibCdM. (B) Lung permeability evaluation. LPSDMEM(n=5)lungs had increased wet / dry weight ratios compared to control (n=5) and controlCdM (n=5) lungs. LPSMSCs (n=4) and LPSMSC CdM (n=5), but notLPSFib(n=4)orLPSFibCdM(n=5),hadsignificantlydecreasedwet/ dry weight ratios compared to LPSDMEM. *p<0.05 LPSDMEM vs. LPS MSC; LPSFib and LPSFib CdM vs. LPSMSC CdM; **p<0.01 control, controlCdMvs.LPSDMEM,LPSFib,LPSFibCdM,LPSMSCCdM;LPSFib andLPSFibCdMvs.LPSMSC.

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Figure 34. LPSinduced body weight loss and lung histological assessment. (A) Body weight loss following LPSinduced lung injury. No effect of treatment on LPSinduced body weight loss. Control (n=13), controlCdM (n=6), LPSDMEM (n=36), LPSFib (n=9), LPSFib CdM (n=10),LPSMSC(n=8),LPSMSCCdM(n=35).*p<0.01controlgroupsvs. eachLPSgroup.(B)Lunginjuryscore.LPSMSC(n=5)andLPSMSCCdM (n=8) lungs had improved lung injury score compared to LPSDMEM (n=8),LPSFib(n=5),LPSFibCdM(n=8).**p<0.01controlgroupsvs.each LPS group, LPSMSC and LPSMSC CdM vs. LPSDMEM, LPSFib and LPS

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Fib CdM. (C) Representative images of lungs from experimental animals. Sizebar:130m.

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Figure 35. AM polarization following in vitro LPS exposure. (A)AMs were98.2%CD11c+andCD11b. Insert:representativeimageofHema3 stained AMs. (B) AMs exposed to LPS for 24 h had greater arginase activity compared to control AMs. MSC CdM enhanced arginase activity comparedtoDMEM(n=4/group).*p<0.05controlvs.LPSDMEMandLPS MSCCdM;LPSDMEMvs.LPSMSCCdM.*p<0.05.

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Figure36.AMpolarizationfollowinginvivoLPSadministration. (A)Representativescatterplotsof4colorstainedAMsfromexperimental lungs.(B) AMsfromLPSDMEM,LPSFib,LPSFibCdMlungsdisplayedan iNOS+Ym1 phenotype. AMs from LPSMSC, LPSMSC CdM showed an iNOSYm1+phenotype(n=5/group).

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Figure 37. MSC secretome analysis.Antibodyarraybasedcomparison betweenMSCCdMandFibCdM.

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Figure 38. Factors up or downregulated in MSC compared to LF secretome. Significant differences in MSC vs. Fib CdM levels of factors highlighted.

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CHAPTER4GENERALDISCUSSIONANDCONCLUSIONS ThischapterwaswrittenbyLIIandeditedbyBT.Fragmentsofthis chapterhavebeenpublishedaspartof: ColtanLI,ThbaudB.Chapter30:Lung.inRegenerativeMedicine, Steinhoff,Gustav(Ed.),1stEdition.,2011.ISBN9789048190744

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4.1.Overview Since the initial observations that stem cells engraft into the lung [15,16],stemcellbasedtherapies(usingmostlywholeBMderivedcells orMSCs)havebeenstudiedextensivelyinvariousanimalmodelsoflung diseases.Anoverwhelmingproportionofthesestudiesshowedtheability ofthesecellstopreventlunginjury,despitealowrateofcellengraftment. Thecurrenthypothesisisthatstemcellsactviaaparacrinemechanismto protect resident lung cells from injury, rather than through engraftment andcellreplacement.Recentinsightisnowconstructinganemergingview of celltocell communication mediated by transfer of exosomes/ microvesicles or whole organelles. Microvesicles are small membrane fragments enriched in bioactive molecules [29]. Since the observations that ESCs can donate exosomepackaged mRNA and proteins to reprogramhematopoieticprogenitors[28],increasingevidenceindicates thatMSCsdonatemicrovesicles[27]andmitochondria[1,6] in vitroand in vivo to protect against ALI [11] and acute and chronic kidney injury [10]. The microvesicles could contain a variety of molecules, including microRNA [7] and factors involved in cellular processes such as proliferation, migration, adhesion and morphogenesis [14]. The identification of factors produced and released into the local microenvironment or directly transferred to neighboring cells by MSCs

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may yield new therapeutic avenues for lung diseases that currently lack efficient treatment strategies, thereby alleviating the potential risks associatedwithwholecelldelivery. The studies presented highlight the ability of murine adult BM derivedstromalcellstoprevent: (i) allergic airway inflammation, AHR and remodeling in an OVA inducedmousemodelofasthma; (ii) lunginflammationinanLPSinducedmousemodelofALI throughparacrinemediatedactionsontwoimmunecelleffectortypes:a flexiblesubsetofregulatoryTcellsandlungmacrophages. The targeted approach used in the first study indicates that the

therapeutic effects of BALB/c BMCs CdM in Th2 allergic airway inflammationaremediatedbyIL10andAPN.IL10hasbeenpreviously shown to alleviate airway inflammation [23] and to contribute to anti inflammatory properties of BMderived stromal cells [22]; the present studyproposesthatexogenousIL10actsonIL10inducedandsecreting Tr1 to determine additional, endogenous production of IL10; APN also contributes to the generation of endogenous IL10 through APN reprogrammedlocalmacrophages.Overall,twoantiinflammatoryfactors contribute through different effectors to amplify and sustain the local productionofoneofthesefactors.

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In the context of LPSinduced inflammation, a wider approach

(CdM screening) of C57Bl/6 BMMSCs CdM was adopted in order to investigate the CdMmediated reprogramming of local macrophages. Severalfactorswithpotentiallyrelevantimmunomodulatoryactivitywere detected in MSCs CdM at significantly different levels compared to Fib CdM. Independent from the 96factor screening, APN, which has also recently been shown to determine the alternate (M2) activation of macrophages[24],wasalsofoundtobepresentinC57Bl/6MSCsCdMat levelscomparabletothoseinBALB/cBMCsCdM.APNwasnotdetectedin Fib CdM. Fib CdM was used in both studies to determine whether the observed effects of BMCs and MSCs CdM were indeed stem cellspecific; minorimprovementswereobservedfollowingFibCdMadministrationin asthmaticmice,inlinewithprevioussimilarfindings[22]. The effects of BMMSCs CdM in LPSinduced ALI also largely

reproduce the effects of wholecell therapy. Although previous reports indicate comparable effects of CdM and wholecell therapy in other lung injury models [4, 19], these findings may be considered at odds with a previous report in which BMMSCs CdM administration was insufficient formacrophagereprogramming[22].Themethodusedhereforpreparing CdM, however, differs from this report, in which cells were cultured in complete medium for 3 days and the resulting supernatant was further diluted 1:1 with fresh medium for experiments. The presented studies

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employed serumstarvation of MSCs for 24 hours and the CdM was prepared by concentrating the supernatants 25fold for all the experiments. This suggests that the concentrations of various factors responsibleinmacrophagereprogramminginourCdMcouldbedifferent from(ifnothigherthan)theconcentrationsinthestudymentioned. 4.2.StudyLimitations ThesamemethodwasusedtoisolatetheBMderivedstromalcells from experimental animals. Although the method used ensured minimal cellpopulationvariabilityamongstbatcheswithineachmousestrain,the innatedifferencesamongstvariousmousestrainsiswelldocumented[5, 11, 25, 26, 30]. This partially accounts for the current lack of consensus with respect to defining mouse MSCs in a fashion that would mirror the setofestablishedcriteriaforhumanMSCs[8];also,thisaccountsforone of the inherent limitations of studies involving the use of mouse MSCs. BothBALB/cBMCsandC57Bl/6BMMSCsmettheplasticadherenceand mesenchymal(trilineage)differentiationcriteria,butdifferedintermsof surface marker expression profile in a manner that has been previously reported [26, 30]. For instance, the low amount of Sca1 expression by BALB/c BMCs compared to C57Bl/6 BMMSCs has been described in direct comparison studies [26]. Notably, whereas BALB/c mice are

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traditionallyconsideredTh2biased[9,11,13,32]andthereforewidely employedforgenerationofmurineasthmamodels,theC57Bl/6strainis used on a larger scale and the BMMSCs from this strain conform to a greaterextenttoestablishedMSCscriteriaforhumanMSCs.Thesecriteria [8] have often been used as a substitute tool to compare the molecular phenotypeofdifferentBMderivedstromalcellpopulations. TheTh2biasoftheBALB/cmousestrainandthemorefrequent useofC57Bl/6miceforthedevelopmentofdiseasemodelsalsopartially justifiedalargerscaleapproachtopotentialbeneficialfactorsinC57Bl/6 MSCsCdM.Oneofthestudyspecificlimitationsderivesfromtherelative scarcity of commercially available multifactor screening arrays, which narrows the relevance of this array to a relatively limited number of knownantiinflammatoryfactors. Anothergenerallimitationstemsfromthefactthatmurinemodels ofasthmareproduceonlyasubsetoffeaturesfoundinhumanasthma[18, 21]. Theuseofgeneticallyengineeredanimalsinwhichagenehasbeen

constitutively modified is another general limitation. The absence of a single factor may often impact the levels of expression of numerous related factors. Our study involving the use of APN KO BMCs sought to partially account for possible additional consequences of innate lack of

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APN by investigating the effects of recombinant APN administered alone inOVAexposedanimals. 4.3. Conclusions and Future Perspectives on Lung Regenerative Therapies Recommendations for future directions for lung stem cell biology havebeenpreviouslysummarized[31].Thefutureaimsandperspectives ofthisresearchfallwithinthegeneralprojectionsforthefield.Stemcell biology has been developing exponentially over a relatively short period oftime.Thecellreplacementparadigmwasoriginallyatthecoreofstem cell research; this may still hold promise for genetic diseases like cystic fibrosis,whereadegreeofCFTRfunctionalityofnomorethan 510%of normal would alleviate CF symptoms; it is reasonable to estimate that a similar engraftment rate may be safely achievable and may ensure this degree of improvement. Stem cells engineered to express the corrected gene could be differentiated in vitro or in vivo and confer sufficient gene function; however, wholecell administration calls for careful consideration of risks associated with cell transplantation. The dawning insightintotheintimatemechanismsofcellularcommunicationbasedon exosomaltransferindicatesthatpreconditioningMSCswithlungderived microvesicles before transplantation may enhance acquisition of AT2

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phenotype by transplanted MSCs [2]; however, further research is mandatory, given that routine maintenance of MSCs in culture can also exposethesecellstoforeign(rodent,bovine)microvesicles[17].Inrecent years,thechangeoffocustowardtheparacrineactivityofstemcellshas partially attenuated concerns linked to wholecell therapy; whereas it is not impossible that the CdM by itself may still carry insufficiently characterized protumorigenic, proapoptotic and proinflammatory factors that may become predominant in certain conditions, determination of specific beneficial factors would eventually lead to preparationscontainingonlythesefactors. Theneedtocharacterizethesecretoryprofileofstemcellsextends totheneedtolinkthisprofiletothemolecularphenotypeof thesecells. Definitive characterization of both endogenous and exogenous stem cell population will certainly be a natural addition to the field. While much more needs to be learned about the mechanisms of normal lung development, injury and repair, stem cell biology and the long term efficacyandsafetyofstemcellbasedtherapies,thepromisinganimaland sparseclinicalobservationssuggestthatitmightbetimetoinitiateclinical trialsusingstemcellbasedapproachesfordevastatinglungdiseasesthat currentlylackeffectivetherapies.Thereisampleprecedentinmedicineof establishedtreatmentsforwhichthemechanismofactionisstillnotfully

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understood. Carefully conducted trials for patients in desperate need for improvementmayteachusmorethanadditionalpreclinicalstudies. TheprojecteddevelopmentsofiPSCresearchwillfacilitatefurther investigation of cellbased approaches for therapeutic purposes but also forunderstandingofdiseaseprocessesanddrugtesting.Thechallengeof lungregeneration,e.g.repairofestablishedlungdamage,relevantforlung fibrosisandemphysema,forexample,remainsandmayrequireadditional strategiesincombinationwithstemcellbasedapproachestorebuildthe lung.Itishopedthatinsightintolungstemcellbiologywillfacilitateand expandbioengineeringapproachesforlungregeneration.

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4.4.References 1. AcquistapaceA,BruT,LesaultPF,FigeacF,CoudertAE,leCozO, ChristovC,BaudinX,AuberF,YiouR,DuboisRandJL,Rodriguez AM(2011)Humanmesenchymalstemcellsreprogramadult cardiomyocytestowardaprogenitorlikestatethroughpartialcell fusionandmitochondriatransfer.StemCells29(5):81224.doi: 10.1002/stem.632 2. AliottaJM,SanchezGuijoFM,DoonerGJ,JohnsonKW,DoonerMS, GreerKA,GreerD,PimentelJ,KolankiewiczLM,PuenteN, FaradyanS,FerlandP,BearerEL,PasseroMA,AdediM,ColvinGA, QuesenberryPJ(2007)Alterationofmarrowcellgeneexpression, proteinproduction,andengraftmentintolungbylungderived microvesicles:anovelmechanismforphenotypemodulation.Stem Cells25(9):224556 3. AmariglioN,HirshbergA,ScheithauerBW,CohenY,LoewenthalR, Trakhtenbrot L, Paz N, KorenMichowitz M, Waldman D, Leider Trejo L, Toren A, Constantini S, Rechavi G (2009) Donorderived braintumorfollowingneuralstemcelltransplantationinanataxia telangiectasiapatient.PLoSMed6(2):e1000029 4. Aslam M, Baveja R, Liang OD, FernandezGonzalez A, Lee C, Mitsialis SA, Kourembanas S (2009) Bone marrow stromal cells 180

attenuate lung injury in a murine model of neonatal chronic lung disease..AmJRespirCritCareMed180(11):112230 5. ChamberlainG,FoxJ,AshtonB,MiddletonJ(2007)Concisereview: mesenchymalstemcells:theirphenotype,differentiationcapacity, immunological features, and potential for homing. Stem Cells 25(11):273949 6. ChoYM,KimJH,KimM,ParkSJ,KohSH,AhnHS,KangGH,LeeJB, Park KS, Lee HK (2012) Mesenchymal stem cells transfer mitochondriatothecellswithvirtuallyno mitochondrialfunction butnotwithpathogenicmtDNAmutations.PLoSOne7(3):e32778 7. CollinoF,DeregibusMC,BrunoS,SterponeL,AghemoG,ViltonoL, TettaC,CamussiG(2010)Microvesiclesderivedfromadulthuman bonemarrowandtissuespecificmesenchymalstemcellsshuttle selectedpatternofmiRNAs.PLoSOne5(7):e11803 8. Dominici M, Le Blanc K, Mueller I, SlaperCortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, and Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy8:315317 9. Duguet A, Biyah K, Minshall E, Gomes R, Wang CG, Taoudi Benchekroun M, Bates JH, Eidelman DH (2000) Bronchial

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responsiveness among inbred mouse strains. Role of airway smoothmuscle shortening velocity. Am J Respir Crit Care Med 161(3Pt1):83948 10. GattiS,BrunoS,DeregibusMC,SordiA,CantaluppiV,TettaC, CamussiG(2011)Microvesiclesderivedfromhumanadult mesenchymalstemcellsprotectagainstischaemiareperfusion inducedacuteandchronickidneyinjury.NephrolDialTransplant 26(5):147483 11. GuedersMM,PaulissenG,CrahayC,QuesadaCalvoF,HachaJ,Van HoveC,TournoyK,LouisR,FoidartJM,NolA,CataldoDD(2009) Mouse models of asthma: a comparison between C57BL/6 and BALB/cstrainsregardingbronchialresponsiveness,inflammation, andcytokineproduction.InflammRes58(12):84554 12. IslamMN,DasSR,EminMT,WeiM,SunL,WestphalenK,Rowlands DJ,QuadriSK,BhattacharyaS,BhattacharyaJ(2012)Mitochondrial transfer from bonemarrowderived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med 18(5):75965. doi:10.1038/nm.2736 13. JainAV,ZhangY,FieldsWB,McNamaraDA,ChoeMY,ChenGH,Erb DownwardJ,OsterholzerJJ,ToewsGB,HuffnagleGB,OlszewskiMA (2009) Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus

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neoformansinfection.InfectImmun77(12):538999 14. KimHS,ChoiDY,YunSJ,ChoiSM,KangJW,JungJW,HwangD,Kim KP,KimDW(2012)Proteomicanalysisofmicrovesiclesderived fromhumanmesenchymalstemcells.JProteomeRes11(2):83949 15. Kotton DN, Ma BY, Cardoso WV, Sanderson EA, Summer RS, Williams MC, Fine A (2001) Bone marrowderived cells as progenitors of lung alveolar epithelium. Development 128, 5181 5188 16. KrauseDS,TheiseND,CollectorMI,HenegariuO,HwangS,Gardner R, Neutzel S, Sharkis SJ (2001) Multiorgan, multilineage engraftment by a single bone marrowderived stem cell. Cell 105, 369377 17. KubikovaI,KonecnaH,SedoO,ZdrahalZ,RehulkaP,HribkovaH, RehulkovaH,HamplA,ChmelikJ,DvorakP(2009)Proteomic profilingofhumanembryonicstemcellderivedmicrovesicles revealsariskoftransferofproteinsofbovineandmouseorigin. Cytotherapy11(3):33040,1pfollowing340 18. Kumar RK, Foster PS (2002) Modeling allergic asthma in mice: pitfallsandopportunities.AmJRespirCellMolBiol27(3):26772 19. LeeJW,FangX,GuptaN,SerikovV,MatthayMA(2009)Allogeneic humanmesenchymalstemcellsfortreatmentofE.coliendotoxin inducedacutelunginjuryintheexvivoperfusedhumanlung. Proc

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NatlAcadSciUSA106,1635716362 20. LepperdingerG,BrunauerR,JamnigA,LaschoberG,andKassemM (2008)Controversialissue:isitsafetoemploymesenchymalstem cellsincellbasedtherapies?ExpGerontol43:10181023 21. MeursH,GosensR,ZaagsmaJ(2008)Airwayhyperresponsiveness inasthma:lessonsfrominvitromodelsystemsandanimalmodels. EurRespirJ32(2):487502 22. NmethK,LeelahavanichkulA,YuenPST,MayerB,ParmeleeA,Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E (2009) Bone marrow stromal cells attenuatesepsisviaprostaglandinE(2)dependentreprogramming of host macrophages to increase their interleukin10 production. NatMed15(1):4249 23. Ogawa Y, Duru EA, Ameredes BT (2008) Role of IL10 in the resolutionofairwayinflammation.CurrMolMed8(5):437445 24. OhashiK,ParkerJL,OuchiN,HiguchiA,VitaJA,GokceN,Pedersen AA,KalthoffC,TullinS,SamsA,SummerR,WalshK(2010) Adiponectinpromotesmacrophagepolarizationtowardananti inflammatoryphenotype.JBiolChem285(9):61536160 25. PeisterA,MelladJA,LarsonBL,HallBM,GibsonLF,ProckopDJ (2004)Adultstemcellsfrombonemarrow(MSCs)isolatedfrom 184

differentstrainsofinbredmicevaryinsurfaceepitopes,ratesof proliferation,anddifferentiationpotential.Blood103(5):1662 1668 26. PhinneyDG,KopenG,IsaacsonRL,ProckopDJ(1999)Plastic adherentstromalcellsfromthebonemarrowofcommonlyused strainsofinbredmice:variationsinyield,growth,and differentiation.JCellBiochem72(4):57085 27. PlotnikovEY,KhryapenkovaTG,GalkinaSI,SukhikhGT,ZorovDB (2010)Cytoplasmandorganelletransferbetweenmesenchymal multipotentstromalcellsandrenaltubularcellsincoculture.Exp CellRes316(15):244755 28. RatajczakJ,MiekusK,KuciaM,ZhangJ,RecaR,DvorakP,Ratajczak MZ(2006)Embryonicstemcellderivedmicrovesiclesreprogram hematopoieticprogenitors:evidenceforhorizontaltransferof mRNAandproteindelivery.Leukemia20(5):84756 29. RatajczakJ,WysoczynskiM,HayekF,JanowskaWieczorekA, RatajczakMZ(2006)Membranederivedmicrovesicles:important andunderappreciatedmediatorsofcelltocellcommunication. Leukemia20(9):148795

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30. SungJH,YangHM,ParkJB,ChoiGS,JohJW,KwonCH,ChunJM,Lee SK,KimSJ(2008)Isolationandcharacterizationofmouse mesenchymalstemcells.TransplantProc40(8):264954 31. Weiss DJ, Bertoncello I, Borok Z, Kim C, PanoskaltsisMortari A, Reynolds S, Rojas M, Stripp B, Warburton D, Prockop DJ (2011) Stemcellsandcelltherapiesinlungbiologyandlungdiseases.Proc AmThoracSoc8(3):22372 32. Whitehead GS, Walker JK, Berman KG, Foster WM, Schwartz DA (2003) Allergeninduced airway disease is mouse strain dependent.AmJPhysiolLungCellMolPhysiol285(1):L3242

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APPENDIX 1 INDUCED PLURIPOTENT STEM CELLS (IPSC) DIFFERENTIATE INTO ALVEOLAR EPITHELIAL CELLS IN VITRO AND PREVENTHYPEROXIAINDUCEDLUNGINJURYINVIVO Lavinia Iuliana Ionescu1, Arul Vadivel2, James Ellis3, Bernard Thbaud1,2
1DepartmentofPhysiology,UniversityofAlberta,Edmonton,Canada

2DepartmentofPediatrics,UniversityofAlberta,Edmonton,Canada

3Hospital

for Sick Children, Ontario iPS Facility, University of Toronto,

Toronto,Canada Correspondingauthor: BernardThbaud,MD,PhD

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ABSTRACT Bronchopulmonarydysplasia(BPD)isthechroniclungdiseasethat follows mechanical ventilation and oxygen therapy in about 30% of premature infants with birth weight < 1000g. The delivery of supplemental oxygen saves the lives of these infants, but arrests lung development in the late canalicular stage, resulting in decreased alveolarizationandimpairedangiogenesischaracteristicofBPD.Thelung is a complex structure composed of more than 40 types of cells with differentembryologicorigin.Hyperoxicinjuryofalveolarepithelialtype2 (AT2) cells, the putative distal lung progenitors and the sole source of pulmonary surfactant, can lead to arrested lung development and permanent respiratory dysfunction. Recent evidence indicates that administration of embryonic stem cell (ESC)derived AT2 may benefit lung repair; ethical and immunological concerns that have so far limited the clinical relevance of ESC may be alleviated with the use of induced pluripotent stem cells (iPSC), ESClike cells engineered from adult somaticcells.IncreasingevidencesuggeststhatiPSCmaydifferentiate in vivo and restore the functionality of damaged organs such as heart and liver. WehypothesizedthatiPSCdifferentiateintoAT2cells in vitroand alleviatehyperoxiainducedlungdamageinvivo.

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Mouse ESC and fibroblastderived iPSC cultured as embryoid bodieswithactivinAandsmallairwaygrowthmediumacquiredlungcell specificmarkers:prosurfactantproteinC(AT2)andaquaporin5(AT1),as indicatedbyimmunofluorescenceandflowcytometry. Inan in vivomodelofBPD,newbornmiceexposedto95%O2from postnatal day (P) 4 to P14 received cells (ESC, iPSC or lung fibroblasts LF) intratracheally at P4 (prevention) or P14 (rescue), followed by lung function testing and histological analysis at P28. ESC and iPSC could prevent, but not rescue, hyperoxiainduced decreases in lung resistance and elastance. ESC and, to a lesser extent, iPSC, prevented, but did not rescue,arrestedalveolargrowth,whileLFhadnoeffect. In vitroevaluationofpotentialparacrineactionsrevealedthatESC and iPSC conditioned medium (CdM) significantly accelerated wound closurerateinconfluentAT2comparedtoLFCdM.iPSCandESCCdMalso protectedagainstAT2hyperoxicinjurywhileLFCdMhadnoeffect(MTT assay). Insummary,mouseiPSCdifferentiateintoalveolarepithelialcells, accelerate wound healing and prevent hyperoxic injury in vitro, while improvinglungfunctioninamousemodelofBPD.Thepossibilityofusing readilyavailableiPSCtogenerateAT2holdsgreatpromisefor thefuture ofcellbasedtherapyforlunginjury.

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INTRODUCTION Pretermdeliveryisamajorandgrowinghealthcareproblem[10], affecting 10% of all births and accounting for more than 85% of all perinatalcomplicationsanddeath [35].Recentadvancesinperinatalcare favored the survival of premature infants born at increasingly earlier stages of gestation [11], which is synonymous to earlier stages in lung development.Theimmaturelungsofextremelyprematureneonates(born at < 28 weeks of gestation) require ventilator support and sustained deliveryofhighconcentrationsofoxygen(O2);however,theseinfantsare at high risk for longterm injury to lung, brain and eyes [39] due to this support in itself. Each year, ~10,000 babies suffer from

bronchopulmonarydysplasia(BPD)[5],achroniclungdiseasethatfollows ventilator and O2 therapy for acute respiratory failure after premature birth[26].BPDischaracterizedbyanarrestinlungdevelopment,which encompasses simplification of alveolar structure and blunting of lung capillary growth. BPD has long term respiratory [1] and neuro developmental [6, 18] consequences that reach beyond childhood and resultinincreasedhealthcarecosts[23].Structuralabnormalitiesclosely mimicking human BPD (fewer and larger airspaces and decreased capillarydensity)havebeendemonstratedinanewbornratmodelofBPD

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causedbyexposuretohyperoxiaduringtheirfirst14daysofpostnatallife [30,33,43]. Amongtheresidentlungcellpopulations,typeIIalveolarepithelial cells (AT2) are the sole source of pulmonary surfactant and differ both phenotypically and functionally from their type I counterparts. AT2 cells proliferate and generate AT1 cells following injury and have therefore long been considered the putative AT1 progenitors [31]. AT1 have also beenshowntodifferentiateintoAT2 in vitro[9],whichsuggeststhatAT1 andAT2maybealternateprogenitorcellsdependingonthetypeoflung injury. Hyperoxic injury of AT2 can lead to permanent, lifethreatening respiratorydysfunction.Recentevidenceindicatesthatadministrationof embryonicstemcell(ESC)derivedAT2maybenefitlungrepair;however, ethical and immunological concerns limit the clinical relevance of ESC. These concerns may be alleviated with the discovery of induced pluripotent stem cells (iPSC) [36, 41], ESClike cells engineered from adult somatic cells. iPSCderived AT2 may have therapeutic benefits in diseases such as BPD and there is increasing evidence that iPSC may differentiate in vivo andrestorethefunctionalityofdamagedorganssuch as heart [27], brain [42] and liver [7, 20]; moreover, there are reports indicating that pluripotent stem cells cultured in specific directing conditions can give rise to definitive endoderm lineagescommitted cells

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[4, 8, 12, 17, 32], including cells with liver [16] and lung and thyroid [21,24]specifications.

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MATERIALSANDMETHODS AllprocedureswereapprovedbytheAnimalWelfareCommitteeof theUniversityofAlberta. ESCandiPSCCultureMaintenance iPSCweregeneratedfromCD1mouseembryonicfibroblastsusing the EOS lentiviral vector selection system [14, 15]. ESC and iPSC were initially cultured on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs, The Jackson Laboratory) and gradually adapted to feederfree conditions in 6well plates coated with a solution of 0.1% gelatin in deionized water (Millipore, Billerica, MA). Cells were grown in highglucose Dulbeccos Modified Eagle Medium (DMEM) supplemented with 15% ESqualified fetal bovine serum (FBS), Lglutamine, non essential aminoacids [NEAA], sodium pyruvate, betamercaptoethanol (all Invitrogen, Burlington, ON) and leukemia inhibitory factor (LIF, Millipore). Culture medium was replaced daily. For iPSC selection, puromycin(1g/mL,Invitrogen)wasaddedtotheculturemedium.ESC andiPSCweregrownongelatincoatedplatesforatleast4weekspriorto assessmentofinvitroexperimentalendpointsorinvivoadministration.

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ConditionedMedium(CdM)Preparation For in vitrostudiesCdMfromESC,iPSC,MSCandLFwasprepared

by culturing the cells in serumfree medium for 24 hours. The resulting CdMwasstoredat80Cuntiluse. WoundClosure(Scratch)Assay Alveolar epithelial cells (RLE6TN cell line, ATCC) were seeded at 25,000cells/wellina24wellplateandallowedtoreachconfluency.The cell monolayer was scratched in a straight line using a P200 pipette tip [19]. Cells were rinsed with PBS, complete culture medium (F12, Invitrogen) as control or serumfree DMEM or cellderived CdM were added.Thesurfaceareaofthewoundwasrecordedatthetimeofscratch (T0)andmonitoredat4,8and12hoursafterscratch(OpenLabsoftware). CellViability(MTTAssay) Alveolarepithelialcells(RLE6TN)wereseededasspecifiedabove. Upon reaching confluency, cells were exposed to 95% O2 for 48 hours (XVivo,Biospherix).Followinghyperoxicculture,cellswerewashedtwice with PBS and a solution of 3(4,5dimethylthiazol2yl)2,5

diphenyltetrazolium bromide (MTT, Invitrogen) (0.5 mg/mL in serum free DMEM) was added. Cells were incubated for 2 hours at 37C. MTT wasremovedand200Lofdimethylsulfoxide(DMSO,Sigma)was used

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todissolvetheresultingformazancrystals.Samplesweretransferredtoa 96well plate and a colorimetric plate reader was used to measure absorbanceat550nm,adirectindicatorofcellnumber[22]. InVivoMurineBPDModelandCellAdministration Newborn CD1 mice were exposed to 95% O2 from postnatal day (P) 4 to P14. On P4 prevention protocol) or P14 (rescue protocol), the animalsreceivedasingleintratrachealinjectionofcells(iPSC,ESC,MSCor lung fibroblasts, LF 1.2 x 105 cells / mouse in 25 L DMEM). Control animalsreceivedanequalvolumeofDMEM. LungFunctionTesting(LFT) LFTwasperformedatP28usingacomputercontrolledsmall animalventilator(flexiVent,Scireq,Montreal,QC)aspreviouslydescribed. LungHistologicalAnalysis Lung fixation and histological analysis were performed as

previouslydescribed[37]. InVitroDifferentiationProtocol For differentiation, mouse ESC and fibroblastderived iPSC were transferred to lowadherence culture plates (Corning) on day 0 of

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differentiation protocol and grown as embryoid bodies (EBs) for the entire duration of the protocol. All EBs were initially cultured in EB medium (highglucose DMEM with 15% regular FBS, Lglutamine, NEAA, sodium pyruvate and betamercaptoethanol); half of the EBs received supplementation with 100 ng/mL activin A for 4 days. EBs were then maintained in EB medium (control) or transferred to EB medium supplementedwithdecellularizedlungmatrixpowder(DLMP,2mg/mL) orsmallairwaygrowthmedium(SAGM,Lonza)foranadditional14days. SAGM was supplemented with 30 ng/mL recombinant human keratinocytegrowthfactor[38](KGF,R&DSystems)betweendays1418. On day 18, EBs were dissociated using 0.05% trypsin (Invitrogen) and plated overnight for immunofluorescence staining or homogenized for proteinandRNAextraction. LungDecellularizationandDLMPPreparation Lung decellularization followed a previously published protocol [28]. Briefly, adult (810 weeks) CD1 mice were anesthetized using sodiumpentobarbital(65mg/kgi.p.)andsternotomywasperformed.The trachea was cannulated, the cannula secured using surgical thread (Prolene) and the lungs and heart were mobilized from the thorax. Thoracic aorta and pulmonary artery were cannulated and successively perfused at a constant pressure of 20 mm Hg with: heparinized PBS (15

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min), 0.1%sodium dodecyl sulfate (SDS, SigmaAldrich,Oakville,ON)in deionizedwater(120min),deionizedwateralone(15min),1%TritonX 100 (Sigma) in deionized water (10 min), PBS supplemented with 100 U/ml penicillin G, 100 U/ml streptomycin, amphotericin B (Invitrogen) (72 h). Complete decellularization was confirmed using hematoxylin & eosin (H&E) staining and immunoblotting. Decellularized lungs were lyophilized and the resulting DLMP reconstituted in EB medium at a concentrationof2mg/mL. ImmunofluorescenceStaining EBsweredissociatedasasinglecellsuspensionbytreatmentwith trypsin (0.25%, Invitrogen) followed by passage through a 1000 L pipettetipanda23gaugesyringeneedle.Theresultingcellswereplated ontopolyLlysine(Sigma)coatedglasscoverslips(Fisher)andallowedto adhereovernight.Cellswerestainedwithfluorescencelabeledantibodies (Abcam)againstmurineprosurfactantproteinC(proSPC)andaquaporin 5 (AQP5). Slides were examined using an inverted microscope (Leica) runningOpenlabsoftware(Improvision,Coventry,UK). Immunodetection(WesternBlot) EBs were homogenized using a Dounce homogenizer and the protein content extracted by centrifugation at 14,000 g for 15 minutes.

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Immunoblotting was performed using proSPC, AQP5 and betaactin (Abcam)antibodieswithcorrespondingsecondaryantibodies(Abcam). FluorescenceActivatedCellSorting EBs were dissociated into a singlecell suspension and the cells stained with proSPC or AQP5 antibody and corresponding secondary antibodies. Data acquisition and analysis were carried out using the FACSCanto II system running FACSDiva software (BectonDickinson).

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RESULTS iPSCCdMAcceleratesInVitroWoundClosure ESC and iPSC CdM, as well as MSC CdM, significantly accelerated wound (scratch) closure rate in confluent alveolar epithelial cells comparedtoLFCdM(Fig.1A). iPSCCdMProtectsAlveolarEpithelialCellsAgainstHyperoxicInjury ESCandiPSCCdMsignificantlyimprovedcellviability(asassessed bytheMTTassay)following48hoursofsustainedhyperoxiacomparedto LFCdM(Fig.1B) iPSCPreventButFailToRescueO2InducedFunctionalLungDamage LFT revealed that administration of iPSC at P4 restored O 2 impaired lung function, as assessed by measuring wholelung resistance and elastance at P28 (Fig. 2). iPSC prevented the significant decrease in lung resistance and elastance observed in O2exposed animals treated with DMEM alone (O2DMEM). Administration of iPSC at P14 failed to rescuelungfunction(Fig.3).

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iPSCPreventButFailToRescueO2InducedStructuralLungDamage Histological analysis indicated that iPSC administration at P4 preservesalveolarstructureinO2exposedanimals(Fig. 4).Theapparent improvementinmicetreatedwithiPSCatP14comparedtoO2DMEMor O2LFwasnotfoundsignificantbylungmorphometricanalysis(Fig.5) TumorDevelopmentFollowingiPSCAdministration At 18 months postcell administration at P14 (n=5), 3 iPSC recipients developed abnormal growths with apparent infiltration in kidney,spleenandcolon(Fig.6). LungDecellularizationforDLMPMediumPreparation Complete lung decellularization was confirmed by histological analysis and immunoblotting. H&E stainingdid not reveal remaining cell nuclei or nuclear fragments. Immunoblotting confirmed absence of pro SPC in decellularized constructs along with preservation of elastin (Fig. 8). ESC and iPSC Express ProSPC and AQP5 Following Culture in Lung CellDirectingConditions ImmunofluorescencestainingrevealedthatESC(Fig. 9A)andiPSC (Fig. 9B)canacquireproSPCandAQP5expressionfollowingcultureas

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EBs in lung lineagespecification conditions: AA (100 ng/mL) + SAGM. FlowcytometricanalysisindicatedthatASKyieldedhigherproportionsof proSPC or AQP5expressing iPSC (15% and 25%, respectively) than DLMPbasedmedium(10%and22%,respectively)(Fig.10).

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DISCUSSION In the present study, we sought to assess the differentiation and therapeutic potential of iPSC within the framework of BPD: impaired alveolar structure [30, 33, 43] with a corresponding functional impairment [13]. There is a large amount of evidence for the benefits of MSC administration of in numerous organ injury models, including hyperoxiclunginjury[3,38];however,thesebenefitscannotbeaccounted for by in situ cell differentiation and engraftment. There are only sparse reports indicating the potential of ESC to acquire lung cellspecific markers in vitro and to engraft into the lung to a very limited extent followinglunginjury;averyrecentstudyconfirmsthepotentialofiPSCto give rise to lung and thyroidmarkerexpressing progenitor cells [21]. Another recent report indicates that intravenous administration of iPSC prevents endotoxininduced lung injury [44]; however, the therapeutic impact of iPSC administration in hyperoxic lung injury has never been investigated. We tested the paracrine effects of iPSC in vitro by screening the iPSCCdMagainstESC,MSCandLFCdMinawoundclosureassayandinan in vitro hyperoxia exposure model. iPSC CdM (and generally stem cell derivedCdM, but not LF CdM) accelerated wound closure and preserved cell viability throughout 48hour O2 exposure, suggesting that iPSC may

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have beneficial properties relevant to our model that are mediated by paracrinefactors. WeassessedthetherapeuticpotentialofiPSCinamousemodelof BPDbyadministeringasinglei.t.injectionofiPSCbefore(prevention)or after (rescue) 10day neonatal O2 exposure. We controlled for iPSC specific effects by comparing iPSC effects to LF, ESC and MSC administration, as MSC have been previously shown to pose therapeutic benefits inrodent models O2induced lung injury [3, 38]. iPSC prevented the development of BPDreminiscent functional and structural impairment, but were unable to rescue lung function or structure after establishmentofinjury. WealsoreportapossiblelongtermeffectofiPSCadministrationat P14 in normoxic subjects (healthy). With the tumorigenic potential of pluripotent stem cells in mind [3], we monitored ESC and iPSCtreated mice for two years after i.t. injection of cells. Three subjects that had received iPSC and one ESC recipient developed tumors that were macroscopicallylocalizedonthelimbs.Weshowacharacteristicsynovial sarcomalike tumor (the earliest, developed by an iPSC recipient 18 months after cell administration). The tumor was accompanied by infiltration of the spleen (with splenomegaly), kidney (with bilateral nephromegaly) and colonic wall (Fig.6). It is important to note that teratomaformationisusuallyemployedtocertifycellpluripotencyinvivo;

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althoughtherearemajordifferencesinrouteofadministrationandtiming oftumorformationbetweenourstudyandtheusualcelladministration teratoma formation test, possible reactivation of the protooncogene c mycusedingeneratingtheiPSCusedinthisstudycouldformthebasisfor malignant tumor (teratocarcinoma) formation in iPSC recipients [3]. Whereasmethodsforreprogrammingsomaticcellswithouttheuseofmyc have been proposed [25, 45], these tumorigenesisrelated concerns may bealleviatedwiththeadministrationofpureiPSCderivedAT2. We sought to differentiate iPSC into alveolar epithelial cells by employingafewgeneraldifferentiationprinciples:1)removalofLIFfrom the culture medium; 2) aggregation of iPSC as EBs and 3) culture in the presence of Activin A (AA), a factor that promotes endodermal differentiation [8, 12, 17], for 4 days. Following AA exposure, EBs were transferred to SAGM or DLMPbased medium for two weeks. Previous reportsindicatetheabilityofSAGM,aspartofamultistepdifferentiation protocol, to determine lung markerexpression in ESC. Differentiation of iPSCwasassessedusinglungcellspecificmarkers:proSPC(intracellular AT2 marker) and AQP5 (AT1 surface marker). Immunofluorescent stainingbased screening of iPSCderived EBs on day 18 indicated that lungspecificationconditions(AA+SAGM)determinedproSPCandAQP5 expression.

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In the quest to maximize the yield of iPSCderived, proSPC expressing cells, we treated the EBs with 30 ng/mL KGF during SAGM exposure;recentevidencesuggeststhatKGFpromotestheproliferationof ESCderivedalveolarepithelial cellsinvitro[38].However,thetimingof KGF supplementation (days 48, 913 or 1418 of the differentiation protocol) did not seem to impact the proportion of proSPC or AQP5 expressingcells,asassessedbyFACS(datanotshown). The successful decellularization of the lung, with preservation of alveolar structure, has been described recently [28, 29]. We confirmed lung decellularization with absence of cell residues, including proSPC, andpreparedDLMPbasedculturemediumbyadditionoflyophilizedlung scaffolds(2mg/mL)totheregularEBsculturemedium.Thispreparation was able to induce proSPC and AQP5 expression in iPSC, but the yield waslowerwhencomparedtoiPSCculturedinASKconditions. In summary, we found that mouse iPSC differentiate into alveolar epithelialcells,acceleratewoundhealingandpreventhyperoxicinjury in vitro,whileimprovinglungfunctionwhenadministeredpreventivelyinan oxygeninducedmousemodelofBPD.Sincetheinitialreportsthatthree germlayercompetentiPSCcanbederivedfromterminallydifferentiated cells, iPSC have often been referred to as ESClike cells. However, the biology of reprogramming is far from being understood. From the very pointofreprogramming,differentapproacheshavealreadybeen usedin

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terms of gene cocktails or small molecules and transduction methods [25, 34, 45]. Efficiency and completeness of reprogramming are still subject to effervescent debate, as well as the risks of cell therapy in general and iPSC in particular. While there is still much to be learned about the biology of iPSC, the possibility of using readily available pluripotentstemcellstogenerateAT2holdsgreatpromiseforthefuture ofcellbasedtherapyforlunginjury. Acknowledgements: Ramneek Kumar, Jenny Shi, Katherine Fu, Andrew Qi,AlbertaDiabetesInstituteHistologyCore.

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FIGURES

Figure 1. iPSC CdM accelerates AT2 wound closure (upper panel) and preservesAT2viabilityduringhyperoxicexposure(lowerpanel).

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Figure 2. iPSCpreventedlungfunctionimpairmentinhyperoxiainduced lunginjury.

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Figure 3. iPSC failed to rescue lung function in hyperoxiainduced lung injury.

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Figure 4. iPSCpreservedlungstructureinhyperoxiainducedlunginjury (preventionprotocol).

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Figure 5. iPSCdidnotrescuelungstructurefollowinghyperoxiainduced lunginjury(rescueprotocol).

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Figure6.PotentiallongtermeffectsofiPSCadministration.

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Figure7.Differentiationprotocol.

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Figure 8. Decellularized lungs did not contain nuclear fragments or pro SPCresidues.

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Figure 9. ImmunofluorescencestainingofplasticadherentsinglecellEBs preparations.

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Figure 10. FACS and immunoblotting indicated that ESC and iPSC acquiredalveolarepithelialcellmarkers.

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