Doordonot.Thereisnotry.(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.Theabilityofpluriandmultipotentstemcells todifferentiate

alongvariouscellular lineageshasplaced themat thecoreof research that

seeks toprotect endogenous stemcell populationsor todeliver 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

thatmultipotent stem cell populations, such asmesenchymal stromal cells

(MSCs), exert paracrine activity that canmodulate local inflammatory and

immune responses in experimental lung diseasemodels including asthma,

acutelunginjury(ALI)andpulmonaryfibrosis.Thestudiespresentedhereby

demonstrate that factors secretedbyadultbonemarrowderivedMSCscan

preventthedevelopmentofinflammatorylungdiseasesinmousemodelsof

asthmaandALIandprovidemechanistic insight intotheantiinflammatory

propertiesofMSCs.

Theperspectiveofusingpluripotentstemcellsastherapeuticagents

hasbeenrevivedbythelandmarkdiscoveryofinducedpluripotency,where

embryonic stem cell (ESC)like cells can be generatedby reprogramming

terminallydifferentiatedsomaticcells.Whilethefieldofinducedpluripotent

stem cell (iPSC) biology is still in its effervescent infancy, this findingmay

relievemanyESCsrelatedethicalconcernsandmayopenthewayto large

scale production and evaluation of pluripotent stem cells for organ

regenerationand repair.Theadditional studypresentedprovidesproofof

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 andDr.Harissios Vliagoftis,my Supervisory Committee

members, who have thoughtfully contributed to designingmy research path; our

collaborators, particularly Dr. Zamaneh Kassiri; Dr. Lisa Cameron (University of

Alberta); Dr. KennethWalsh 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

LaboratoryAnimal Services),DrewNahirney (Dr.Duszyks laboratory),Dr.Wilma

SuarezPinzonfortheirworkandadvice.

Iwouldalso like to thank theDepartmentofPhysiology(Dr.KeirPearson,

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

CHAPTER3STEMCELLCONDITIONEDMEDIUMPREVENTSACUTE

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.MSCCdMDecreased Lung Inflammation and LungVascular

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 INTOALVEOLAREPITHELIALCELLS INVITROAND

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

Figure212.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

AHR airwayhyperresponsiveness

AM alveolarmacrophage

ANOVA analysisofvariance

APC allophycocyanin

APN adiponectin

ARDS acuterespiratorydistresssyndrome

Arg1 arginase1

AT2 alveolarepithelialtype2cells

ASM airwaysmoothmuscle

AT1 alveolarepithelialtypeIcell

AT2 alveolarepithelialtypeIIcell

BALF bronchoalveolarlavagefluid

BASC bronchoalveolarstemcell

Bmi1 BlymphomaMoMLVinsertionregion1homolog

BMC plasticadherentbonemarrowderivedstromalcell

BMSC bonemarrowderivedstromalcell

BOOP bronchiolitisobliteransorganizingpneumonia

BPD bronchopulmonarydysplasia

ckit protooncogenec(tyrosineproteinkinase)Kit

CCL chemokine(CCmotif)ligand

CCSP Claracellsecretoryprotein(also:CC10,Scg1b1)

CdM conditionedmedium

CD clusterofdifferentiation

CFTR cysticfibrosistransmembraneregulator

CGRP calcitoningenerelatedpeptide

CHI3L3 chitinase3like3(also:ECFL,Ym1)

COPD chronicobstructivepulmonarydisease

CXC chemokine(CXCmotif)

CXCL chemokine(CXCmotif)ligand

DALY disabilityadjustedlifeyears

DMEM DulbeccosmodifiedEaglemedium

DNA deoxyribonucleicacid

ECFL Tlymphocytederived eosinophil chemotactic factor (also:

CHI3L3,Ym1)

ECM extracellularmatrix

ECMO extracorporealmembraneoxygenation

EDTA ethylenediaminetetraaceticacid

ELISA enzymelinkedimmunosorbentassay

eNOS endothelialnitricoxidesynthase

EPC endothelialprogenitorcell

EpCAM epithelialcelladhesionmolecule

ESC embryonicstemcell

FACS fluorescenceactivatedcellsorting

FBS fetalbovineserum

FcRIIB FcreceptorforimmunoglobulinG

Fib fibroblast

FiO2 oxygenfraction

FITC fluorescein

FIZZ1 foundininflammatoryzone1(also:RELM)

Foxp3 forkheadboxp3

H&E hematoxylinandeosin

HELP HumanExVivoLungProject

HSC hematopoieticstemcell

IBMX isobutylmethylxanthine(1methyl3(2methylpropyl)7H

purine2,6dione)

IFN interferongamma

IGF1 insulinlikegrowthfactor1

IGFBP6 insulinlikegrowthfactorbindingprotein6

IL interleukin

IL1ra IL1receptorantagonist

iNOS induciblenitricoxidesynthase

iPSC inducedpluripotentstemcell

i.n. intranasal

i.p. intraperitoneal

i.t. intratracheal

i.v. intravenous

IVIG intravenousimmunoglobulin

JAK Janusactivatedkinase

KGF keratinocytegrowthfactor

KO knockout

LF lungfibroblast

LFT lungfunctiontesting

LIX lipopolysaccharideinducibleCXCchemokine

LPS lipopolysaccharide

M1 classically(canonically)activatedmacrophages

M2 alternativelyactivatedmacrophages

MAPK mitogenactivatedproteinkinase

MCP1 monocyte chemotactic protein1 (also: CCL2, small

induciblecytokineA2)

MCSF macrophage/monocytecolonystimulatingfactor

MDC macrophagederivedchemoattractant/chemokine

MDSCmyeloidderivedsuppressorcells

MIP2macrophageinflammatoryprotein2

MIP1macrophageinflammatoryprotein1alpha

MSC mesenchymalstemcells

NEB neuroendocrinebody

OVA ovalbumin

PaO2 partialarterialpressureofoxygen

PBS phosphatebufferedsaline

PE phycoerythrin

PEEP positiveendexpiratorypressure

PF4 plateletfactor4(also:CXCL4)

PI3K phosphoinositide3kinase

PLSD probableleastsignificantdifference

PMN polymorphonuclearcell

PMSF phenylmethylsulphonylfluoride

PNEC pulmonaryneuroendocrinecell

PTEN phosphataseandtensinhomolog

PSF penicillinstreptomycinfungizone(amphotericinB)

RANTES regulated on activation, normal T cell expressed and

secreted

RIPA radioimmunoprecipitationassay

RPMI1640 RoswellParkMemorialInstitutemedium1640

qRTPCR quantitative reverse transcriptase polymerase chain

reaction

RELM resistinlikemoleculealpha(also:FIZZ1)Sca1 stemcellantigen1

SCF stemcellfactor

SDF1 stromalderivedfactor1alpha

SEM standarderrorofthemean

SP sidepopulationcells

SPC surfactantproteinC

STAT6 signaltransducerandactivatoroftranscription6

sTNFRII solubleTNFreceptorII

TARC thymusandactivationregulatedchemokine

TGF transforminggrowthfactorbeta

TLC totallungcapacity

TGF transforminggrowthfactorbeta

Th1 Thelper1

Th2 Thelper2

Tr1 interleukin10inducedandsecretingregulatoryTcell

Treg regulatoryTcell

TNF tumornecrosisfactoralpha

VCAM1 vascularcelladhesionmolecule1

VEGFR2 vascularendothelialgrowthfactorreceptor2

WT wildtype

Ym1 see:CHI3L3,ECFL

1

CHAPTER1GENERALINTRODUCTION

ThischapterwaswrittenbyLIIandeditedbyBT.Fragmentsofthis

chapterhavebeenpublishedaspartof:

ColtanL,ThbaudB.Chapter30:Lung.inRegenerativeMedicine,Steinhoff,

Gustav(Ed.),1stEdition.,2011.ISBN9789048190744

2

1.1. Overview

Therespiratorysystemsupports thevital functionofbreathing. It

canbeviewedastheinterfacebetweentheoxygenrichenvironmentand

thecarbondioxideproducing livingorganism.Thefailureof the lungsto

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 alveolarwalls come in close contactwith 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

3

lung disease of prematurity (or bronchopulmonary dysplasia, BPD),

chronicobstructivepulmonarydisease(COPD)andcysticfibrosisarestill

lackingefficient treatments.According to theWHOWorldHealthReport

2000,lungdiseasescontributetoatotalof17.4%ofdeathsand13.3%of

disabilityadjusted life years (DALY) worldwide [180]. These facts

highlight the absolute necessity to study the potential applicability of

recentdevelopments in the fieldofregenerativemedicineas therapeutic

optionsforlungdiseases.

1.2. LungDevelopmentandRegeneration

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

4

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(embryonicstage).Duringthefollowing

pseudoglandular stage, rudimentary bronchi divide by dichotomous

branching; the resulting tubular structures are lined by columnar

epitheliumsurroundedbymesenchymal tissue.Thecanalicularstage is

characterizedby thebifurcationof the lastgenerationsofdistalbronchi.

In thisstagethere isalsocapillary invasionanddifferentiationof theair

5

spaceepitheliumintoalveolartype2cells(AT2,responsibleforsurfactant

production)andtype1cells(AT1,whichformthethinairbloodbarriers).

Next, during the saccular stage, the peripheral air spaces expand in

length andwidth, and, around 36weeks of gestation these spaces form

sacculesattheexpenseoftheinterveningmesenchyme.Alveolarization,

thefinalstageoflungdevelopment,beginsintheneartermlungpriorto

birth but primarily occurs postnatally, during the first 23 years of life,

andmaycontinuebeyondchildhood,albeitataslowerrate.

Thealveolusisthedefinitivegasexchangingunitofthelung.Alveoli

are, in part, formedby subdivision (septation) of the saccules. Septation

involvesbuddingof septal crests,which is followedbyelongationof 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).

Microvascularmaturation,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 alveolarwall decreases by about 20% and the distance

between alveolar gas and capillary blood diminishes by about 25%.

Morphometricstudiesshowthatfrombirthtoadulthoodthealveolarand

6

capillarysurfaceareasexpandabout20foldandthecapillaryvolume35

fold.

Whilethehistologicalchangesarewelldescribed[76,87,190],much

moreneedstobelearnedaboutthemechanismsthatregulatenormallung

developmentinordertoharnesstheseprocessesfortherapeuticpurposes

[168]. This is particularly relevant for the perinatal care of extremely

premature infantswho 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

7

transmembrane regulator (CFTR), an ion channel normally present in

epithelialcells.Thismonogenicdiseasehasbeenclassicallyregardedasa

purely electrophysiological disease due to the CFTR dysfunction;

however, recent findingssuggest thatCFTR isalsoexpressed in immune

effector cells, such as macrophages [22], which opens new therapeutic

perspectives that place more emphasis on the inflammatory aspects of

cysticfibrosis.

Aside fromandalsoalike cystic fibrosis,numerous lungdiseasesare

currentlylackingefficienttherapies:whileALI/ARDSorasthmahavean

overwhelminginflammatorycomponent,othertypesofinjurymayhavea

more obscure etiology (emphysema, COPD, pulmonary fibrosis). BPD

impairslungdevelopment,yet,inrecentyears,advancesinperinatalcare

havepermitted the survival of extremelyprematurebabieswhose lungs

areinearlierdevelopmentalstages;whereastheoldBPDhadastronger

fibrotic component, the new BPD is an arrest in alveolarization with

minimalinflammation[11].Theprolongedstalemateinfindingsolutions

forpatientssufferingfromtheseincurablediseaseshasbroughtstemcell

researchat thecoreofRMtoday. Thehallmarkabilitiesofstemcells 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.

8

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

itsmicroenvironmentbyacquiring traitsspecifictoothercell typeswas

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

visionof thebonemarrowas anorigin and reservoirofblood cellswas

elaboratedby theRussianscientistAlexanderMaximow.Hedevelopeda

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

9

thespleensofirradiatedmicethathadreceivedunirradiatedmarrowcells

[153]. Before the end of that decade, the first allogeneic bone marrow

transplantations[54]weretoprovidetheclinicalproof for theexistence

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 amodel 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 SamuelWeiss 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

10

fibroblast (CFUF) [47]; later, these cellswere going to be assigned the

termmesenchymalstemcells(MSCs)[4,115].

Stemcellsaredefinedascells thathaveclonogenicandselfrenewal

potential and are able todifferentiate alongmultiple 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

consideredtoberestricted to theiroriginalgerm 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.

11

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 nowwidely used tomodel the

effectsofanabundanceoffactors.Todate,ESCsarebettercharacterized

thanadultstemcells.Yet,thelineagerelationshipbetweenembryonicand

adultstem/progenitorcellshasnotbeenclearlydescribed.

Oneof thedefining featuresof stemcells is theirability to divide

either symmetrically, generating two identical daughter cells or

asymmetrically,givingrisetoanidenticaldaughterstemcellandamore

specialized, lineagecommitted progenitor cell [121, 149] that lacks the

selfrenewalabilitybutpossessesahigherproliferationratecomparedto

itsparentstemcell.

It becomesobvious that a tight regulatory control of thebalance

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 thespecializedprogenitorbehindas the identical daughtercell

movesdistallywiththebuddinglungtips[121].

Regenerativeapproachesmaythereforefollowseveraltherapeutic

directions:

12

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) Cellreplacementbyexogenousstemcells, as stem cells

may be able to regenerate damaged organs by

differentiatingandengraftinginvivo.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/reconstructionofdamagedorgansusing

stem cells as a source of terminally differentiated cells for

tissueengineering.

13

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/progenitorcellscontributetomaintenanceof lungcellpopulations

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

14

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 theirDNA [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], consistentwithprevious findingsdemonstrating 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

15

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].Kottonetal.

[77]andKrauseetal. [78]showedthatbonemarrowderivedstemcells

cangiverisetodaughtercellsintheairways.Thisabilityofthecellsto

engraft and differentiate has led to the hypothesis that they may

reconstituteinjuredtissuesbyreplacingthedamagedcells.Thereisnowa

large body of evidence in support of the hypothesis that bonemarrow

derived multipotent MSCs can differentiate into airway [166, 177] or

alveolar[159]epithelialcellsinvitro,engraftanddifferentiateinvivoand

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].TheabilityofadultMSCs todifferentiate into lungcellshas

16

rendered them particularly important candidates for lung regeneration

approaches; today, MSCs are the most widely used stem cells in

regenerativemedicine.Theestablishmentofaminimalsetofcriteria 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], type1 diabetes [43, 53, 162] andneurodegenerative 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 apicalmembrane of epithelial cells. The stem

cells would be engineered to overexpress functional CFTR and act as a

deliveryvehicletothedamagedtissues,includingthelung[22].Thesame

17

approachwouldbeapplicableforothermonogenicdiseasesthatseverely

affect the lung such as alpha1 antitrypsin deficiency (which leads to

irreversible emphysemalike lesions) or surfactant protein B deficiency

(whichresults in fatal respiratory failure innewborns).Moreover, ithas

been suggested that stem cells could act asdrugdelivery vehicles [107]

based on observed therapeutic effects inmyocardial 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

18

the primarybeneficial component. This has led to the current view that

stemcells act throughaparacrinemechanismby secreted factors [116].

Indeed,MSCssecreteantiapoptotic,angiogenic,andimmunomodulatory

factors. Since the initial report indicating paracrinemediated protective

effectsofMSCsoverexpressing theprosurvivalgeneAkt 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 ofMSCsmay also have therapeutic potential in other

inflammatorydiseases [68, 116, 136]. Several factors found in theMSCs

secretome, among which are interleukin10 [98], transforming growth

factorbeta (TGF) [92], stanniocalcin1 [19] and keratinocyte growth

factor (KGF) [82], have been proposed tomediateMSCs crosstalkwith

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.

19

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 forresearchandhasopenedthewayto largescaleproductionand

evaluationofpluripotentstemcellsforlungregenerationandrepair.

Whenmaintained 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 fromwhich theymaybe furtherdifferentiated into lung cells

using specific factors [128, 161]. Another method employed was the

exposure of ESCs to microenvironments mimicking lung conditions

(coculturewith lungmesenchyme 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

20

cultureswitharelativelylowyieldoflungcells.Invivoadministrationof

ESCs or progenitor cells derived fromESCs 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 relapseof certain tumorsowing to the fact that these cellsmightbe

resistant to many conventional cancer therapies [111, 113, 182]. The

identificationofputativeresidentstemcells in lungtumors[75] leadsto

the question whether the resident cells that survive pollutantinduced

injurymayinfactbesuchacancerstemcell.Theexistenceofcancerstem

cellsinthelungissupportedbyworkindicatingthatCD133+isamarker

ofselfrenewingcellsthatsustaintumorpropagationinmice[39].These

cellsareresistanttocisplatintreatment[17];however,theproportionof

21

cells expressing this marker lacks prognostic value [132]. Other work

suggeststhatactivationofthekrasgene,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 orwhether 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, aswell as the proposed contributions of bonemarrow

derived cells to cancer progression [48] has yet to be investigated.

Althoughmuch work is still needed to identify and characterize cancer

22

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 patientswith

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 notmatch the demand

andoneofthefactsthatcontributetothisshortageisthatonlyabout20%

of donor organs are considered acceptable for transplantation [118].

Improper oxygenation capacity (reflected by a PaO2 below 300mm Hg

23

afteroxygenationwithaFiO2of100%for5minandPEEPgreaterthan5

cmH2O) leads to rejectionofdonor lungs.HELP involves the conceptof

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

transplantoflungsharvestedfromadonorandreconditionedexvivowas

performed successfully in 2007 [141]. The impact of this promising

strategyremainstobeevaluated.

1.6.2.ArtificialLungNovaLung

Theartificiallungisarelativelynewmethod,similarinconceptto

dialysisanddesignedtosupportrespiratory functionwhile thepotential

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

24

not available. Other advantages of this system over ECMO are reduced

anticoagulation and avoidanceof longtermmechanical 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 silicomodel of the alveolar

capillaryinterfacehasbeendevelopedemployingbiomaterialsandhuman

alveolar epithelial cells at airliquid interface, along with human

pulmonarymicrovascular endothelial cells [60]. This type of biomimetic

microsystems could facilitate drug screening and toxicology studies by

allowinghighthroughputprocessing.Ona larger scale, so far bothESCs

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 lineagesorbioengineered lung components, including

recellularization of a human tracheal scaffold with MSCsderived

chondrocytes,followedbysurgicalimplantationasamainbronchus[86].

However, the lack of conclusive information with respect to the

25

tumorigenic potential of stem cells, especially ESCs, known for their

karyotypic instability, togetherwith 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

polyacrylamidehavebeenused inattempts toengineer 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

evenenhancedperil in the caseof recipientswhohavedevelopedgraft

26

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

iPSCsdevelopteratomasinvivoandtherearealsoreportsindicatingthat

transplantationofneural stemcells led to thedevelopmentof 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 termsofbothphenotypeand function. ForMSCs,minimal criteria for

defininghumanMSCs,establishedbytheInternationalSocietyforCellular

Therapy[37],havereducedsomeofthevariationswithregardstocellular

compositionofMSCpopulationsisolatedaccordingtodifferentprotocols.

Lung injurypreventionobtainedwithMSCs in various animalmodelsof

lungdisease, togetherwith theireaseof isolationandculture,aswellas

their immunomodulatory properties make these cells very promising

candidatesforclinicaltrials.

27

Thusfar,MSCshavebeentransplantedinhumansaspartofwhole

bonemarrow transplantation for various disorders (including leukemia

and genetic diseases of the immune system). Gendermismatched

transplantation(maledonorbonemarrowtofemalerecipient)hasproven

tobeausefultoolinassessingtheimpactofstemcelltransplantationon

otherorgans thanbonemarrow.Donormalecellswere identified in the

lungsofrecipientsasepithelialandendothelialcells[147]andalsointhe

liver[152],heart[35],brain[29,96]andkidney[114].Also,inthereverse

casewheremaleswere recipients of sexmismatched organ transplants,

theYchromosomeindicatingrecipientoriginwasidentifiedinavariable

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

tolerabilityandsafetyofprogenitorcells forthetreatmentofpulmonary

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

pilotstudieshavesupportedthefeasibilityof thisapproachinidiopathic

pulmonaryhypertension[167,193].

28

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.

29

Celltype Location Phenotype Lunginjurymodel

Remarks

Basalandparabasalcells

proximalairwayepitheliumsubmucosalglandularductsandintercartilaginouszone

cytokeratin5/14+

polydocanolorSO2induced[21]

clonogeniccapacity,multilineagedifferentiation[134];repopulateairwayepitheliumpostinjury[59]

TypeA(new,variant)Claracells

bronchioalveolarjunction;proximityofneuroepithelialbodies(NEBs)[49]

Claracellsecretoryprotein(CCSP)+;nosecretorygranules,nosmoothER

naphthaleneorozoneinduced

retainlabeledDNAprecursors[121];repopulateinjuredairwayepitheliumwithbothmature,quiescentClaracellsandciliatedepithelialcells

Bronchioalveolarstemcells(BASC)

Sca1+/CD34+/CD45/CD31[74,75]

maycoexpressSPC

TypeIIalveolarepithelialcells(AT2)

SPC proliferateandgenerateAT1cellsfollowinginjury[4,123].AT1differentiateintoAT2invitro[33]:alternateprogenitorcellsdependingontypeoflunginjury

Sidepopulation(SP)[146]

heterogeneouspopulationeffluxtheDNAdyeHoechst[50]

derivedfrombonemarrow,identifiedinlung[88];differentiatealongendodermandmesodermderivedlineages[89,143];endothelialpotential(newbornmice);decreasedinoxygeninducedarrestedalveolargrowth[65]

Pulmonaryneuroendocrinecells(PNECs)

foundwithtypeAClaracellsinNEBsassociatedregenerativefoci[126].

calcitoningenerelatedpeptide(CGRP)

naphthaleneinduced

oxygensensing[188];ifbothnaphthalenesensitiveandresistantClaracellsareablatedairwayepitheliumdoesnotregeneratePNECsarenotairwayepithelialprogenitors[59]

Lungepithelialprogenitors

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

giverisetobronchialandalveolarepithelium[94]

Multipotentlungprogenitors

ckit+

selfrenewing,clonogenic[72]

Table11.Summaryofputativeendogenouslungstemcellpopulations.

30

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