Murine bone marrow mesenchymal stromal cells respond efficiently to oxidative stress despite the low level of heme oxygenases 1 and 2

M

ANTIOXIDANTS&REDOXSIGNALING Volume29,Number2,2018

ªMaryAnnLiebert,Inc.DOI:10.10 89/ars.2017.7097

F ^ORUM O ^RIGINAL R ^ESEARCH C OMMUNICATION

MurineBoneMarrowMesenchymalStromalCellsRespondEffi cientlytoOxidativeStressDespitetheLowLevel

ofHemeOxygenases1and2

WitoldNorbertNowak,¹He vi

darTaha,^1,2N e l i

Kachamakova-Trojanowska,¹J

acekSte˛pniewski,¹ JoannaAgataMarkiewicz,¹A n n a

Kusienicka,¹K r z y s z

tofSzade,¹A g a t a

Szade,¹K a

rolinaBukowska- Strakova,^1,3Ka

rolinaHajduk,¹D a

mianKlo´ska,¹A l e k s a n

draKopacz,¹A n n a

Grochot- Prze˛czek,¹K a t h r i n

Barthenheier,¹Ca

milleCauvin,¹J

o´zefDulak,^1,4a n d

AlicjaJo´zkowicz¹

Abstract

Aims:Mesenchymalstromalcells(MSCs)areheterogeneouscellsfromadulttissuesthatareabletodiff er-

entiatei n v i t r o i n t o a d i p o c y t e s , o s t e o b l a s t s , o r c h o n d r o c ytes.S u c h c e l l s a r e w i d e l y s t u d i e d i n r e g e n e r a t i v e medicine.However,thesuccessofcellulartherapydepends onthecellsurvival.Hemeo xygenase-1(HO-

1,encodedb y t h e H m o x 1 g e n e ) , a n e n z y m e c o n v e r t ingh e m e t o b i l i v e r d i n , c a r b o n m o n o x i d e , a n d F e

,i scytoprotectiveandcanaffectstemcellperformance.Therefore,ourstudyaimedatassessingwhether Hmox1iscriticalforsurvivalandfunctionsofmurinebonemarrowMSCs.

Results:BothMSCHmox1

an

dHmox1

showe

dsimilarphenotype,differentiationcapacities,andproduction ofcytokinesorgrowthfactors.Hmox1

an

dHmox1

cell sshowedsimilarsurvivalinresponseto50lmol/Lhemineveninincreasedglucoseconcentration,conditionsthatwere unfavorableforH mox1

bon

emarrow-derivedproangiogeniccells(BDMC).Hmox1

+

M

SCsbutnotfibroblastsretainedlowROSlevelsevenafterprolongedincubationwith50lmol/Lhemin,althoughbothce lltypeshaveacomparableHmox1expressionandsimilarlyincreaseitslevelsinresponsetohemin.MSCsHmox1

treate

dwithheminefficientlyinducedex-

pressionofavastpanelofantioxidantgenes,especiallyenzymesoftheglutathionepathway.

InnovationandConclusion: Hmox1overexpressionisa popularstrategytoenhanceviabilityandperfor- manceofMSCsafterthetransplantation.However,murineMSCsHmox1

d

onotdifferfromwild- typeMSCsinphenotypeandfunctions.MSCHmox1

sho wbetterresistancetoheminthanfibroblastsandBDMCsandrapidlyreacttothestressbyupregulationofquintess entialgenesinantioxidantresponse.Antioxid.RedoxSignal.29,111–127.

Keywords:stemcells,antioxidantgeneresponse,heme,mesenchymalstemc ell

Introduction

esenchymals t r o m a l c e l l s (MSCs),a l s o k n o w n a s mesenchymalstemcells,ormultipotentstromalcells

areaheterogeneouspopulationofconnectivetissuecellsthatcontainso s t e o b l a s t a n d a d i p o c y t e p r o g e n i t o r s , f i b r o b l a s t s , a ndsmoothmusclecells(5).InvitrocriteriaforhumanMSCs

includeadherenceto the plasticinstandard culturecon di-

tions,differentiationinvitrotoadipocytes,osteoblasts,andcho ndrocytes(9).MSCsshouldexpressCD73,CD90,andCD105 markersbutnotCD45,CD34,CD14,CD11b,CD79a,CD19,an dHLA-

DR(9).MSCswerefurtheridentified,alsoinvivo,byCD271an dCD106(human)(33),CD146(44),nestin(34),Sca-

1(mouse)andPDGFRa

1DepartmentofMed ic al Biotechnology , Facu lt y ofBiochemistry, Biophysicsand Bio te ch no lo gy, JagiellonianUnive rsi ty,Krak o´w,Poland.₂

DepartmentofAnimalProduction,CollegeofAgriculture,UniversityofDuhok,Duhok,Iraq.

3DepartmentofClinicalImmunology,InstituteofPediatrics,JagiellonianUniversityMedicalCollege,Krako´w,Poland.

4Kardio-MedSilesia,Zabrze,Poland.

111

NOWAKETAL.

112

Innovation

Enhancementofstemandprogenitorcellsantioxidantcap acityistheaimofmanystudiesfocusingonthecel- lulartherapies.Manyofsuchstrategiesproposetheoverex pressiono f H m o x 1 a s a p r o t e c t i o n a g a i n s t c e l l stress.Forthefirsttime,thisarticle showsthatmesen -

chymalstromalcells(MSCs)lackingHmox1canmore efficientlythanothercellsdealwithoxidativestressin- ducedw i t h h e m i n , u s i n g t h e m e c h a n i s m i n v o l v i n g t h e upregulationofglutathionepathway.Highresista ncetostressanduniqueabilitytoactivateantioxidantrespon sesuggestthatMSCmaynotneedadditionalprotectionbyH mox1o v e r e x p r e s s i o n .

(36),leptinreceptorLepR(8,67),orhighexpressionofCXCL1 2(41).

MSCswereshowntobeimmuneevasiveorimmunomod- ulatory,dependingonthemicroenvironment(2).Themecha- nismofimmunosuppressioniscomplexandinvolvesmanyfacto rs,thatis,prostaglandinE2,nitricoxide,andTGFb(39).

Finally,theeffectofhemeoxygenase-1onMSCdiffer- entiationt o a d i p o c y t e s a n d o s t e o b l a s t s w a s a l s o s t u d i e d . Abrahamandco-

workersshowedintheseriesofpublicationsthatenh ancedexp re ssi o nofH O-1inMS Csresultsini m -

proveddifferentiationtoosteoblasts,whereasitsinhibiti onpromotesadipogenesis(3,56–

59).Ontheotherhand,Zarjouetal.reportednodifferencesindiffer entiationpotential

betweenMSCHmox1^{+/+a n d}Hmox1^-/-

( 6 6 ) .Alsoinotherstudies,overexpressionofhemeoxyge

nase-1inMSCsdid

notaffecttheirdifferentiation(18,68).

Dataontheinfluenceofhemeoxygenase-

1onMSCsareoftencontradictory.Conjointly,copperortinproto porphyrinswereusedinmanystudiestomodulateHO-

1activity,althoughtheywereshowntohavemanyhemeoxygena se-

independenteffectsinvariouscelltypes(17,23).MSCsareessent ialfortheproperfunctionofstemcellnichesinbonemarrow,andla ckofhemeoxygenase-

1wasshowntopotentlyaffecto therbonemarrow- derivedcells,thatis,pro-angiogeniccells(PACs)

(16).Therefore,wedecidedtocharacterizemurinebonemarrow- derivedMSCslackingthefunctionalHmox1gene,withthefocus ontheirresponsetooxidativestress.

Results AlthoughMSCsarecommonlybelievedtodealwithoxidativestr

essefficiently(55),thebiggestobstacletothetherapeuticuse ^{Hmox1+/+ orHmox1-/-} bonemarrowMSCsshow ofM SCsist heirp oorsurvivalandengraftmenta ftert hetranspl

antation(11).Therefore,manystudiesfocusontheen-

hancementoftheirantioxidantactivitywithoverexpressionofvar iousgenes,forexample,Hmox1(54,63).

Hemeoxygenase-1(HO-

1,encodedbytheHMOX1gene)isa n e n z y m e d e g r a d i n g h e m e t o c a r b o n m o n o x i d e ( C O ) , biliverdin,andFe^2+ions.Duet oitsenzymaticactivity,heme

oxygenase-

1influencescellsurvival,resistancetotheoxi-

dativestress,andangiogenesis(10).Wehaverecentlyshownthatproang iogeniccellsisolatedfromthebonemarrowof

Hmox1knock-

outmicepresentimpairedproliferation,mi-

gration,a n d f o r m a t i o n o f c a p i l l a r i e s ( 1 6 ) . W h a t i s m o r e , overexpressionofhemeoxygenase-

1canleadtotheblockofdifferentiation,thatis,inmyoblasts(27).

RatMSCstransfectedwiththeplasmidcodingforhumanhem eoxygenase-

1showeddecreasedapoptosisinhypoxiaandhigherresistanceto H2O2(54).Inourhands,pigbonemarrow-

derivedcellstransducedwithadenoviralvectorsencodinghemeo xygenase-1(AdHO1)werecharacterizedbybetteran-

giogenicactivityinvitroandimprovedleftventricularejectionfra ction30minafterinfarctioninp igs(63).Treatmentwithcobaltpr otoporphyrinIX(CoPP),hemeoxygenase-

1activator,enhancedproliferationofhumanmesenchymalstemc ellsandproductionofVEGF;whereastinprotoporphyrinIX(Sn PP),

hemeoxygenase-

1inhibitor,hadanoppositeinfluence(20).Further,CoPP- treatedMSCsacceleratedwoundhealinginaxenogeneicmodelo fdiabeticmice(20).

Modulationo f h emeoxygenase-

1a ctivityw ithS nPPi nhumanMSCsaffectedtheirabilitytoinhi bitTcellprolifera-

tioninvitro.Interestingly,theeffectofSnPPwasnotobservedinrat MSCs,andTcellproliferationwasrestoredonlywhenconcomita nttreatmentofnitricoxidesynthase2wasused(7).Moreover,hem eoxygenaseinhibitiondecreasedtheabilityofMSCstoinduceTr1 andTh3regulatorycellsandtoelevatelevelsofIL-

10andTGFb,respectively.MSCspreconditionedwithamixedly mphocytereactionshoweddecreasedHO-

1levelsaswellasimmunomodulatoryactivity(37).

113

MURINEBMMSCSA R E RESISTANTTOOXIDATIVESTRESS similarphenotypeanddifferentiation

First,wecomparedthephenotypesofmurinebonemarrowstromalcells Hmox1^+/+orHmox1^-/-

inculturebyusingflowcytometry.Regardlessofthegenotyp e,60%ofthecellsinculturewereCD45^-CD31^-

(data

notshown).Therefore,cellsusedforalltheexperimentswerepurified fromtheremainingCD45+f r a c t i o nwithMACSsorting.Obtai nedcellslackedexpressionofendothelialmarkersCD31andC D34,whereasCD117(c-kit)wasexpressedonlyonasmallsub- fractionofcells(Fig.1A).IsolatedMSCsexpressedposit ivemarkers

thatwereattributedtothemesenchymalstem/stromalcells,th atis,CD29,CD90,CD105,Ly-6A/E(Sca-1)

(Fig.1B),andCD140a(P DGF R a)(F i g . 1C , D) . H m o x1^-/-

bonem ar r o w -

derivedPACshadimpairedproliferation(16).However,theprolife rationo f M S C H m o x 1^+/+orH m o x 1^-/-

w ass i m i l a r , evenwhencellsweregrownunderstressconditi onsinhigh

glucoseconcentration(Fig.2A).

Further,b o n e m a r r o w s t r o m a l c e l l s i s o l a t e d f r o m l o n g bonesofmiceHmox1^{+/+o r}Hmox1^-/-

f o r m e dsimilarnum-

bersofcoloniesthatconsistedoffibroblastoidcells(Fig.2B)

orc e l l s a b l e t o b e d i f f e r e n t i a t e d t o o s t e o b l a s t s ( F i g . 2 C ) . MSCsw e r e s h o w n t o d i f f e r e n t i a t e i n t o a d i p o c y t e s , o s t e o -

blasts,andchondrocytes(6).BothHmox1^{+/+a n d}Hmox1^-/- MSCsdifferentiatedintoosteoblastsandadipocytes,which wereevidencedwithstainingforosteopontinorFabp4,re- spectively(SupplementaryFig.S1A,B;SupplementaryDataareavailabl eonlineatwww.liebertpub.com/ars).

BecauseHO-

1wassuggestedtoplayacrucialroleintheregulationofMSCad ipogenesis(59),wefocusedontheef-

fectsofHmox1knockoutonthegenesassociatedwithlipid metabolism.I n t e r e s t i n g l y , d i f f e r e n t i a t i o n o f m u r i n e b o n e marrowMSCstoadipocytesinducedsimila rchangesinthegeneexp re ssi on i n bot h Hm ox1^+/

+a n d

Hm ox1-/-c e l l s

(SupplementaryFig.S1C).

However,H m o x 1^+/

+MSCsw e r e t h e o n l y o n e s t h a t i n - creasedexp re ssi o n ofmiR-21-

5p,t h em i cro RNA th at, vi a TGFbsignaling,regulatesadipogenes is(26)(Supplementary

FIG.1 . M S C H m o x 1^{1/1o r}H m o x 1^{2/2h a v e}s i m i l a r p h e n o t y p e . E x p r e s s i o n o f M S C p o s i t i v e m a r k e r s C D 2 9 , C D 9 0 , CD105,andSca-1(A),MSCnegativemarkersCD31,CD34,andckitinHmox1^{+/+o r}Hmox1^-/-

M S C s(B).Phenotypeofnonsortedb o n e m a r r o w s t r o m a l c e l l s i n p a s s a g e 4 : f r a c t i o n o f CD45^-CD31^-

cellsinculture(C),fractionofSca-1⁺CD140a^+cellswithinCD45^-CD31^-population(D),orSca- 1⁺CD140a⁺CD105^+cellswithinCD45^-CD31^-populationinHmox1^+/+orHmox1^-/-cells.(C,D,N=9–

10).MSCs,mesenchymalstromalcells.

Fig.S1D).LevelsofothertestedmicroRNAsassociatedwithadipogenesis ,namelymiR-31-5p,miR-15 0-5p, miR-301 a- 5p,miR- 378a-3p,ormiR-378a-

5p,remainedunchangedincellsofbothgenotypes(Supplementar yFig.S1E–

I).Further,basalexpressionofalltestedmicroRNAs,includingmiR-21- 5p,whichincreasedduringadipocytedifferentiationonlyin Hmox1^+/+cells,wassimilarinMSCHmox1^+/+andHmox1^-/- (datanotshown).

EffectsofchangedexpressionofHMOX1inhumanMSCsontheadipo genesisorosteogenesiswerestrongerwhencellswereculturedinhighg lucoseconcentration(3).Therefore,

wet e s t e d m a r k e r s o f a d i p o g e n e s i s i n M S C H m o x 1^+/+or

Hmox1^-/-

differentiatedinloworhighglucoseconcentration.Interestingly,adipogen icdifferentiationofmurinebone

marrow-derivedHmox1^+/+o

rHmox1^-/-MSC sdidnot

changewhencellswereculturedunderhighglucosecon- ditions.BothHmox1^+/+andHmox1^-/-MSCsupregulated

fattyacid-bindingprotein4—amarkerofadipocytedif- ferentiation(Fig.2D).

MSCsw e r e s h o w n t o b e p r e c u r s o r s o f f i b r o b l a s t s a n d myofibroblastsand,therefore,contributetothetumorstrom a

(35)ordevelopmentoffibrosis(30).Hemeoxygenase- 1canaffectbothtumormicroenvironment(62)andkidneyfibrosis (47).Therefore,weinvestigatedwhetherthelackofHmox1genei nMSCsmayinfluencetheirabilitytoformmyofibro-

blasts.Hmox1^+/+andHmox1^-/-MSCsweredifferentiatedto myofibroblastswithTGFb1treatmentfor6days.Cellschang edt h e i r m o r p h o l o g y ( S u p p l e m e n t a r y F i g . S 1 J ) a n d upregulateda -smoothm u s c l e a c t i n (Acta2) ( F i g . 2 E ) . O f note,u p r e g u l a t i o n o f A c t a 2 w a s l o w e r i n m y o f i b r o b l a sts

derivedfromHmox1^-/-

c e l l s .

Transcriptl e v e l s o f f i b r o b l a s t - s p e c i f i c p r o t e i n 1 ( Fsp1)

(Fig.2 F ) t e n d e d t o d e c r e a s e i n H m o x 1^+/

+a n d

H m o x 1^-/-

MSCs;

h o w e v e r , t h i s t r e n d r e a c h e d s i g n i f i c a n c e o n l y i n

FIG.2 . M S C H m o x 1^{1/1o r}H m o x 1^{2/2s h o w}s i m i l a r a b i l i t y t o d i f f e r e n t i a t e t o a d i p o c y t e s , osteoblast sa n d s m o o t h musclecells.ProliferationofMSCHmox1^{+/+o r}Hmox1^-/-

i nloworhighglucosemediumassessedwithBrDUassay(A).Abilityto formfib ro b last oid (B)o rosteoblast (C )co lon ie sbybone marrow cellsiso lat ed from Hmo x1^+/+orHmox 1^-/-MSCsassessedwithfibroblastoidorosteoblastcolony- formingunitassay,respectively.Datashownasmean+SD,N=3.

ExpressionofFabp4inHmox1^{+/+o r}Hmox1^{-/-M S C s}differentiatedtoadipocytesinloworhighglucoseconditions(D) GeneexpressionwasassessedwithqRT-PCR.Dataareshownasmean+SD,N=4–7.ExpressionofActa2(E),Fsp1(F) measuredw i t h q R T - P C R i n c o n t r o l a n d d i f f e r e n t i a t e d M S C H m o x 1^+/

+o rH m o x 1^-/-.D a t a a r e s h o w n a s m e a n –SD,

*p<0.05,* *p<0.01,* * * *p<0.0001c o n t r o l v e r s u s T G F b 1 ;^{$ $}p<0.01H m o x 1+/+v e r s u sH m o x 1^-/-,N =3.q R T - P C R ,

quantitativereal-timePCR.

Hmox1^-/-

cells.T o s u m m a r i z e , w e s h o w h e r e t h a t M S C s , regardl essofthehemeoxygenase-

1,showsimilarphenotypeanddifferentiationability,eveninstresscon ditions.

LackofHmox1doesnotchangeMSCsimmunomodulat oryactivityorproductionofcytokines

Akiyamaetal.showedthatMSCsinjectedintravenouslycande creasenumbersofcirculatingCD3^+andincreaseTcellap- optosis(1).Mougiakakosetal.suggestedthatinhibitionofHO- 1activitycandecreaseMSCsabilitytoinduceTregulatorycells(3 7).Therefore,totesttheeffectofcompleteknock-

outofHmox1onimmunomodulatoryactivityofMSCs,wein-

Further,lackofthefunctionalHmox1genedidnotchangetheprofileof cytokinesandgrowthfactorsproducedbyMSC.Conditionedmediafrom MSCHmox1^+/+andHmox1^-/-

containedsimilaram ount sofG-C SF ,I L-

6, LIF,L IX, CXCL1,CXCL2,CCL5,andVEGF(Fig.3D–

K).Levelsofeotaxin,GM-CSF,IGNc,IL-1a,IL-1b,IL-2,IL- 3,IL-4,IL-5,IL-7,IL-9,IL-10,IL-12p40,IL-12p70,IL-13,IL-15,IL- 17,MIP-1a,MIP-1b,M-CSF,MIP-2,MIG,andTNFawere underth ethreshold ofdetection.Th erefore, weconclu dedthatlackoftheHmox1genedidnotchangethetestedMSCsec retomeanddidnotaffecttheabilityofMSCstoinduceTcelldeathbot hinvitroandinvivo.

jectedwild-typeC57Bl6·FVBmicewithMSCHmox1^+/+or

Hmox1^-/-andanalyzedapoptosisandactivationofTcells. ^Hmox1+/+ andHmox1^-/_- MSCsshowhighresistance NumbersofcirculatingCD3^+Tcellsdidnotchangeinmiceinje

ctedwithMSCs(Fig.3A).However,numbersofapoptoticAnnex inV^+CD3^+cellstendedtoincreaseaftertheMSCin-

jection,butthist rendd idnotr eachstatisticalsignificance(Fig.3 B).I njectionofMSCHmox1^+/+orHmox1^-/-

didnotaffectthenumbersaswellofactivatedCD3⁺CD4⁺CD25^hig

hTcells( Fig.3 C).Invitro,primarymurineCD3^+Tcellsco- culturedwithMSCHmox1^{+/+o r}Hmox1^-/-

showedasimilarcellcycle(SupplementaryFig.S2A–

C),percentageofKi67^+cells(SupplementaryF ig.S 2D)andannexi nV^+cells(Sup-

plementaryFig.S2E).

tooxidativestressinducedwithH2O2o r hemin

Bonemarrow-derivedproangiogeniccells(BDMC)lack- ingHmox1genewerecharacterizedbyhighersensitivitytooxi dativestress,thatis,inducedwithhemin(16).Therefore,wed e c i d e d t o t e s t w h e t h e r i t i s a l s o t r u e f o r M S C s . S u r - prisingly,H m o x 1^{+/+a n d}H m o x 1^-/-

M S C s

s h o w e d n o d i f -

ferencei n t h e v i a b i l i t y w h e n t r ea t e d f o r 6 hw i t h H2O2 or50lmol/Lh e m i n i n b o t h l o w an d h i g h g l u co s ec o n d i t i o n s

(Fig.4A,B).Moreover,treatmentwith10ng/llofTNFafor24hdi dnotyieldsignificantdifferences intheviabilityofMS CswithorwithoutHmox1(SupplementaryFig.S3A,B).

FIG.3. LackofHmox1inMSCdoesnotaffecttheirimmunomodulatoryproperti esor secretoryprofile. Number ofcirculatingCD3^+Tcells(A),CD3⁺AnnexinV^+apoptoticTcells(B),oractivatedCD3⁺CD4⁺CD25^highTcells(C)incontrolmiceorinject edi.v.withMSCHmox1^+/+orHmox1^-/-(N=3–4).ConcentrationofG-CSF(D),IL-6(E),LIF(F),LIX(G),CXCL1(H),MCP- 1(I),RANTES(J),andVEGF(K)inconditionedmediafrom Hmox1^{+/+o r}Hmox1^-/-MSCassessed

withmultiplexassayonLuminexplatform.Dataareshownasmean–SD,Mann–Whitney,N=4.

Subsequently,wecheckedthetoxicconcentrationsofheminf orMSCH mox1^+/

+andHmox1^-/-.Surprisingly,heminwasmoretoxicforHmox1^-/-

MSCsthanforHmox1^+/

+cellsnotuntilata concentrationof200lmol/L(Fig.4C).Howeve r,

conditionedmediafrombothM SCHmox1^+/+andHmox1^-/-

showedsimilartotalantioxidantcapacityassessedwithtotalanti oxidantcapacity(TAC)(Fig.4D),2,2¢-azino-bis(3-ethyl- benzothiazoline-6-sulphonicacid)(ABTS),orferric-

reducingantioxidantpower(FRAP)assays(SupplementaryFig .S3C,D).ThelatterresultsuggeststhatthereasonfortheMSCre- sistancetooxidativestressisratherintrinsic.

First,wesupposedthatlowsensitivityofHmox1^{+/+a n d} Hmox1^-/-

MSCstohemin,especiallyincomparisontoBDMC,c o u l d b e r e l a t e d t o t h e h i g h e x p r e ssiono f h e m e

oxygenase-

2.However,theexpressionofHmox1waslowerinMSCsHmox1

+/+than

inPACsHmox1^+/+(Fig.4E).Si-

milarly,theexpressionofHmox2waslowerinMSCsthan inP A C c e l l s a n d s i m i l a r i n H m o x 1^+/+andH m o x 1^-/-

cells

(Fig.4 F ) . T h e n , w e h y p o t h e s i z e d t h a t l o w s e

n s i t i v i t y o f Hmox1^-/-

M S C

cellscouldbecausedbythelowuptakeof hemefromtheculturemedium.Therefore,weanalyzedheme

uptakewithtwomethods—

directmeasurementofhemeincellsstimulatedfor2hwith50 lmol/Lhemin,andthenafter2hinfreshmedium(Fig.5A),andwit hthemeasurementoftinprotoporphyrinIXfluorescence.

Hmox1^+/+andHmox1^-/-

MSCsdisplayedcomparablelevelsofcellularhemeatalltimepoi ntstested(Fig.5B).Moreover,bothcellgenotypesshowedcom parableSnPPfluorescence

proportionalt o t h e c o n c e n t r a t i o n o f S n P P i n t h e c u l t u r e medium(SupplementaryFig.S4A).Therefore,higher resis-

tancetoheminincomparisontoPACcannotbeexplaine d

bythecompensationofHm ox1functionbyHmox2or bychangeduptakeofhemin.

SincebonemarrowPACsaremostlyofmonocyticorigin, andt h e r e f o r e d i s t a n t f r o m M S C s , w e d e c i d e d t o f u r t h e r focusonthedifferencesinstressresponsebetweenMSCs andfibroblasts.I n t e r e s t i n g l y , m o u s e t a i l -

t i p f i b r o b l a s t s h a d a phenotypes i m i l a r t o M S C s , t h a t i s , t h e y e x p r e s s e d S c a 1 , CD106andpartofthemsh owedpositiveforCD140a(Sup-

plementaryFig.S5).BothfibroblastsandMSCs,regardlessof Hmox1,werecharacterizedbythesimilarexpressionof hemetransportersHcp1(Slc46a1)andHrg1(Slc48a1)

FIG.4 . M SC,r egardlessoft helowe xpressionofb othh emeo xygenases1 o r2 , showh ighr esistancet oh emino r hydrogenp erox ide.L D H r e l e a s e i n M S C H m o x 1^+/+orH m o x 1^-/-

treatedw i t h H2O2(A)o r h e m i n ( B ) i n l o w o r h i g h glucosem e d i u m . C e l l d e a t h i n M S C H m o x 1^{+/+o r}H m o x 1^-/-

t r e a t e d

w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f h e m i n f o r 6 h,

assessedwith7-AADstainingandflowcytometry(C).Dataareshownasmean–SD.***p<0.001two-wayANOVAwithBonferronipost- test,N=3.TotalantioxidantcapacityofconditionedmediafromMSCHmox1^{+/+o r}Hmox1^-/-,measuredwithTACkit(N=4) (D).( E)Expressiono fHmox1,( F)Hmox2in m urinebonemarrowP ACa ndM SCsisolatedfrom

Hmox1^+/+orHmox1^-/-mice,****p<0.0001MSCversusPAC;two-wayANOVAwithBonferronipost-test,N=3.7-AAD, 7-aminoactinomycinD;ANOVA,analysisofvariance;LDH,lactatedehydrogenase;PAC,pro-angiogeniccell;TAC,total antioxidantcapacity.

(SupplementaryFig.S4B,C).MSCsHmox1^-/-

weretheonlyonestoincreaseinresponsetohemintreatmentandt heex-

pressionofhemeexporterFLVCR1(Fig.5C),whichma yprotectthemfromthehemeoverload.

Tof u r t h e r e l u c i d a t e t h e e f f e c t s o f h e m e o n M S C s , w e analyzedgenesinvolvedinhemesynthesis,ofwhichAlas1is regulatedby h em e . Tr ea tm e nt w i t h h em in (50 lmol/L)d e -

creasedAlas1expressioninalltreatedcells.Therefore,wec oncludedt h a t h em e i nt h e c u l t u r e m ed i u m e n t e r s t r e a t e d

cellsr e g a r d l e s s o f H m o x 1 e x p r e s s i o n a n d a f f e c t s k n o w n heme-regulatedp at hways.In H mox1^-/-

fibroblasts,

alreadycontrolcellswerecharacterizedbylowerAl as1levelsthan

Hmox1^+/

+c e l l s

( F i g . 5 D ) . E x p r e s s i o n o f U r o s i n h e m i n - treatedHmox1^-/-

fibroblasts

waslowerthanincorrespondingMSCcells(SupplementaryFi g.S4D).

Whati s m o r e , H m o x 1^-/-

fibrobl asts

t r e a t e d w i t h h e m i n

decreasedCpoxexpressionwhereasitremainedunchangedin othercelltypes(SupplementaryFig.S4E).Therewerenodifference

sintheexpressionofHmbs,Alad,Ppox,andFech(SupplementaryFig.

S4E,F).Hmox1wassimilarlyupregu- latedinbothHmox1^+/

+MSCs

andfibroblasts(Fig.5E).TheexpressionofHmox2wasnot affectedbyhemintreatment

anddidnotdifferinanyofthetestedgroupsofcells(Fig.5F).Tosumup,MSCs Hmox1^-/-showhigherresistancetoH2O2

andheminthanpreviouslytestedPACs(16).However,lowsens itivitytoheminisnotcausedbychangesinhemeuptakeorsynthesisa nditisnotrelatedtoHmox2.

Heminincreasescellular H2O2inHmox 1^-/-MSCs

andfibroblasts

ToassesstheeffectsofheminonoxidativestressintheMSCs Hmox1^+/

+andHmox1^-/-,weanalyzedthelevelsofcellularhydrogenpero xidebyusingH2DCFDAstaining.Tail-

tipfibroblastsisolatedfromthesameHmox1^+/+orHmox1^-/- micewereusedasnonprogenitorinternalcontrolcells.After6ho fincubationwithhemin(50lmol/L),levelsofH2O2wereincreas edinHmox1^-/-

MSCsandfibroblastsandhigherthaninrespectivewild- typecells(Fig.5G).Aftera24-hincubation

period,levelsofH2O2remainedlowinHmox1^+/

+cellsandwerehigherinHmox1^-/-fibroblaststhaninHmox1^-/-

MSCs(Fig.5H).ThissuggeststhatmurineMSCsmightbeequip ped

withananti-

oxidantprotectivemechanism,whichworksbetterthaninfibrob lasts.

Nevertheless,after48hofstimulationwithhemin,bo thHmox1^{-/-M S C s}andHmox1-/-f i b r o b l a s t s

weredead.Inter- estingly,atthislatetimepoint,levelsofcellularH2O2w e r e

higheri n h e m i n - t r e atedH m o x 1+/+fibroblasts

t h a n i n

FIG.5 . H e m i n i n c r e a s e s i n t r a c e l l u l a r h y d r o g e n p e r o x i d e i n H m o x 1^{1/1o r}H m o x 1^{2/2a n d}d e c r e a s e s A l a s 1 e x - pressionbutdoesnotaffectlevelsofhemeimporters.Schemeoftheexperimentfortheassessmentofintracellularhemeconte nt(A).HemecellularcontentmeasuredwithspectrophotometryinMSCHmox1^+/+orHmox1-/-stimulated

for2hwith hemin(T2)andthenafter2hinhemin-

freemedium(T4),N=3(B).ExpressionofFLVCR1(C),Alas1(D),Hmox1(E),andHmox2( F ) i n H m o x 1^{+/+o r}H m o x 1^-/-

M S C so r f i b r o b l a s t s s t i m u l a t e d f o r 6 hw i t h 5 0 lmol/Lh e m i n . L e v e l s o f H2O2measuredwithH2DCFDAafter6(G ),24(H),or48h(I)inHmox1^{+/+o r}Hmox1^-/-

M S C s

orfibroblastsstimulatedwithhemin(50lmol/L).Dataareshownasmean–

SD,*p<0.05,**p<0.01,***p<0.001,****p<0.0001hemin-treatedcells

versuscontrol;^##p<0.01,^####p<0.0001MSCversusfibroblasts,^$p<0.05,^$$p<0.01,^$$$p<0.001,^$$$$p<0.0001Hmox1^+/+

versusHmox1^-/-.Two-wayANOVAwithBonferronipost-test,N=3.

Hmox1^+/+MSCs(Fig.5I).Therefore,weconcludedthatbothHmox1^+/

+andHmox1^-/-

MSCcellsshowedhigherresistancetoheminthanrespectivetail- tipfibroblasts.

MSCslackingHmox1efficientlyinducean tioxidantgeneresponse

Al owerconcentrationo f H2O2i nM SCHmox1^-/-than Hmox1^-/-fibroblastsafters hortincubationw ithh eminsug-

gestedthatMSCsmightmoreeffectivelyrespondtothepro- oxidativeinsult.Therefore,weevaluatedtheexpressionofavas tp anelo f a ntioxidantg enesi nM SCsa ndH mox1^+/

+orHmox1^-/-fibroblastsin responseto50lmol/Lhemin.Treat- mentwithhemindidnotchangetheexpressionofthemajorregul atorofantioxidantgeneresponseNfe2l2(Fig.6A),which encodesfortheNrf2transcriptionfactor.LevelsofSqstm1,whi chisbothtargetandregulatorofNrf2(21),wereincreasedinMSC sandHmox1^-/-fibroblastsbuttheupregulationwas

higherinMSCs(Fig.6B).

FIG.6 . MSCHmox1^1/1showmoreefficientantioxidantresponseaftertreatmentwithheminthanfibroblastsHmox1^1/1.ExpressionofN fe2l2(A),Sqstm1(B),Cat(C),Nqo1(D),Sod2(E),Sod3(F),Prdx3(G),Txn1(H),Prdx6(I),Fth1(J),andFpn(K)inHmox1^+/+orHmox1^-/-

MSCsorfibroblastsstimulatedfor6hwithhemin(50lmol/L).Dataareshownasmean–

SD,*p<0.05,**p<0.01,***p<0.001,****p<0.0001hemin-treatedcellsversuscontrol;^#p<0.05,^##p<0.01,

####p<0.0001MSCversusfibroblasts,^$p<0.05,^$$p<0.01,^$$$p<0.001,^$$$$p<0.0001Hmox1^+/+versusHmox1^-/-,Two- wayANOVAwithBonferronipost-test,N=3.

118

119

MURINEBMMSCSA R E RESISTANTTOOXIDATIVESTRESS

ExpressionofCatwasthehighestinhemin-

treatedMSCHmox1^-/-,theonlyonestochangeCatlevels(Fig.6C).The n,Hmox1^-/-

f i b r o b l a s t streatedwithheminhadlowerexpres- sionofNqo1thanHmox1-/-M S C s ,theonlycellstoupre- gulateNqo1andwithitsexpressionhigherthanHmox1^+/+

MSCs(Fig.6D).Similarly,expressionofSod2waschangedonlyi n H m o x 1^{-/-M S C}( F i g . 6 E ) w h e r e a s h e m i n -

t r e a t e d Hmox1^-/-

fibroblasts

hadlowerSod3levelsthancorre- spondingMSCcells(Fig.6F).

Further,expressionofPrdx3andTxn1wasenhancedwithhe minonlyi nH mox1^-/-

MSCc ells( Fig.6 G,H);whereasPrdx4,Prdx5,andTxnrd3were notaffected(Supplementary

Fig.S6A–C).Interestingly,Prdx6,theonly1-

Cysmemberofperoxiredoxinfamily(13),waspotentlyupregul atedinhemin-treatedH mox1^-/-

MSCs.Prdx6expressionwast henh igherinMSCHmox1^-/-

thaninallothercells(Fig.6I).

MSCsHmox1^{-/-w e}retheonlyonesthatupregulatedfer- ritinheavychain1(Fth1)expressioninresponsetothehemin treatment(Fig.6J).LevelsofFpnwere,ontheotherhand,incr easedonlyinHmox1^+/

+M SCs(Fig.6K).HemindidnotaffectNox4expressioninanycells(

SupplementaryFig.S6D).

Interestingly,onlyHmox1^{-/-M S}CsdecreasedGpx1expres- sioninresponsetohemin(SupplementaryFig.S6E),whereasexpr essionofGpx3andGpx4wasunchanged(Supplementary

Fig.S6F,G),andexpressionofGpx8wasdecreasedinbothMSC celltypes(SupplementaryFig.S6H).Ontheotherhand,controlfibr oblastsexpressedlowerlevelsofGpx8thanre-

spectiveMSCs(SupplementaryFig.S6H).

Tosummarize,bothMSCsandfibroblastsHmox1^{-/-u p -} regulatedSqstm1andPrdx6inresponsetohemin.Moreover,thee x p r e s s i o n o f b o t h g e n e s w a s h i g h e r i n t r e a t e d M S C Hmox1^{-/-t h a n}fibroblastsHmox1^-/-.MSCHmox1^-/-

b u tnotfibroblastsHm o x1^-/-

elevated

l eve ls o f t r a n s cr ip t s f o r C at,Nqo1,Prdx3,Txn1,an dFth1,whichconfirmedtheirmore

efficientantioxidantresponse.

Importantly,MSCHmox1^-/-weretheonlycellstopo- tentlyupregulatequintessentialgenesinvolvedintheglu- tathionepathway,namelyGclc,Gclm,Gss,andGsr.MSC Hmox1^-/-alsoelevatedGstp1(Fig.7A–

E),whichformsaheterodimerwithPrdx6(13),alsoincreasedin hemin-

treatedMSCHmox1^-/-.LevelsofGstk1,anotherglutathi- oneS-

transferase,remainedunchangedinallcelltypes(Fig.7F).Leve lsoftheupregulatedglutathionepathwaygeneswerehigherinh emin-treatedHmox1^-/-MSCcellsthaninhemin-

treatedHmox1^+/+MSCsandHmox1^-/-fi-

broblasts.ThelatteronesincreasedexpressiononlyofGclman dGstp1(Fig.7B,F).

Finally,tofunctionallyvalidatetheuniqueinfluenceofhemi nonglutathionepathwaygenesinHmox1^-/-

MSCs,weanalyzedreducedglutathione(GSH)tooxidizedglu- tathione(GSSG)ratio,changesintotalGSHandGSSGinMSC s,andf ibroblastsofbothHmox1^+/+andHmox1^-/-

phenotypes.Cellsweretreatedwithhemin(50lmol/L)

for6h,likeingeneexpressionexperiments.However,wetheni ncubatedthecellsfortwomorehoursinhemin-

freecompletemediumtoallowforGSHrecovery.TheGSH/GSS Gratiothatallowsassessingcellularoxidativestress

wasdecreasedinbothHmox1^+/+andHmox1^-/-

MSCsaswellasinHmox1^-/-fibroblasts.Further,Hmox1^-/-fibro- blastswerecharacterizedbylowerGSH/GSSGratiothan Hmox1^-/-

MSCcells,whichadditionallyhada slightlylowerratiothanHmo x1^+/+MSCs(Fig.7G).

NOWAKETAL.

120

Noteworthy,Hmox1^-/-MSCsweretheonlyonesthatin- creasedtotalGSHinresponsetohemin,whereastheycausedad ecreaseintotalGSHinHmox1^+/

+MSCs(Fig.7H).LevelsofGSSGwereincreasedinHmox1^+/

+MSCs,Hmox1^-/-MSCs,

andHmox1^{-/-f i}broblasts.TheywerehigherinbothHmox1^-/- cellt ypesthanint heirr espectiveHmox1^+/

+counterparts.However,levelsofGSSGincreasedmoreinHm ox1^{-/-f i}bro-blaststhaninHmox1^-/-MSCs(Fig.7I).

MSCsisolatedfromHmox1^{-/-m i c e}showed,incompari- sont o f i b r o b l a s t s a n d P A C , h i g h e r r e s i s t a n c e t o h e m i n . However,h e m i n s t i l l i n c r e a s e d c e l l u l a r c o n c e n t r a t i o n o f

H2O2inbothMSCsandfibroblastsHmox1^-/-.Weshowhereforthe firsttim eth atmurin eMSCs Hmox1^-/-

c o u l d

betterthanm ousetail-

tipfib ro b last si n duc eant ioxi d an tge ne re- sponse,especiallygenesinvolvedintheglutathionepathway.

Importantly,changesintheglutathionepathway,observedonthemRNAl evel,werefurthervalidatedfunctionallybythemeasurement ofreducedandoxidizedcellularglutathione.

Onemayspeculatethatsuchafastresponsetostressfactorscan,in part,contributetothepresenceofonlyminoreffectsofH m o x 1 k n o c k o u t o n o t h e r f u n c t i o n s o f m u r i n e b o n e

marrow-derivedMSCs.

Discussi on

Murinebonemarrow-

derivedMSCsisolatedfromHmox1^+/+orHmox1^-/-

miceshowedsimilarphenotype,proliferation,anddifferenti ation.Inourhands,neitherpro-

liferationnordifferentiationtoprimarylineageswasaffectedbyi n c r e a s e d g l u c o s e c o n c e n t r a t i o n s i n t h e c u l t u r e m e d i a . However,weshowhereforthefirsttimethatincomparisontot a i l -

t i p f i b r o b l a s t s , m u r i n e M S C s a r e m o r e e f f i c i e n t i n antioxidantresponse.

AvailablereportsontheroleofHmox1inMSCdifferen- tiationareofteninconsistent,whichmayresultfromtheuseofcob altortinprotoporphyrinstomodulateHmox1activityordifferenc esbetweenmouseandhumanMSCs.Suchspecies-

dependentvariationswerereported,forexample,forimmunomodul atoryactivitiesofMSCs,whichareregulatedbynitricoxidesynthase inmurinecellsandindoleamine-2,3-

dioxygenaseinhumancells(51).Weperformedourexperi- mentsonmurinebonemarrow-derivedMSCsisolatedfrom wild-typeo r H m o x 1^-/-

m i c e .I m p o r t a n t l y , t h e u n a f f e c t e d phenotypeo fHmox1^{-/-M S C s}aswellasnochangesinthe

differentiationtoadipocytes,osteoblasts,andchondrocyte s

werepreviouslyshownbyZarjouetal.

(66),whoalsousedcellsisolatedfromHmox1-/-m i c e .

Ontheotherhand,Barbagalloetal.reportedthatexpres- sionofhemeoxygenase-

1changesduringthedifferentiationofhumanMSCstoosteoblast s(3)andtreatmentwithoste-

ogenicgrowthpeptideincreasesHMOX1expressioninhu- manb o n e m a r r o w M S C s ( 5 6 ) . M o r e o v e r , h u m a n M S C s stimulatedwithCoPPduringtheosteogeni cdifferentiationhadupregulatedosteonectin,osteogenicgrowthp eptide,andosteocalcin.C o P P d e c r e a s e d a d i p o g e n i c d i f f e r e n t i a t i o n o f MSCs( 3 , 5 6 ) , w h e r e a s d o w n r e g

u l a t i o n o f H M O X 1 w i t h siRNAresultedinenhancedadipo genesis.

Humanb o n e m a r r o w M S C s t r e a t e d w i t h e p o x y e i c o s a - trienoicaciddisplayeddecreasedlevelsofBach1,arepressor ofHMOX1expression(52),andincreasedHMOX1mRNA;whereasl evelsofPPARcandC/EBPa,involvedinadipo-

genesis,weredecreased(57).Vanellaetal.reportedlateron

FIG.7.M S C Hmox11/1s t r o n g l y

induceexpressionofglutathionepathwayenzymesandincreaseGSHlevelsafterstimulat ionwithhemin.ExpressionofGclc(A),Gclm(B),Gss(C),Gsr(D),Gstp1(E),andGstk1(F)inHmox1^{+/+o r}Hmox1^-/-

MSCsorfibroblastsstimulatedfor6hwithhemin(50lmol/L).RatioofGSHtoGSSG(G),changesintotalGSH(H),andGSSG(I)i nHmox1^+/+orHmox1^-/-MSCsandfibroblastsstimulatedfor6hwithhemin(50lmol/L)andthenkeptin

freshmediumfor2h.LevelsofGSHandGSSGweremeasuredwithGSH/GSSG-Glo™Assay.Dataareshownasmean–SD,

*p<0.05,* *p<0.01,* **p<0.001,* ***p<0.0001h emin-

treatedc ellsv ersusc ontrol;^#p<0.05,^{# #}p<0.01,^####p<0.0001MSCversusfibroblasts,^$p<0.05,^$$p<0.01,^$$$p<0.001,^$$$

$p<0.0001Hmox1^+/+versusHmox1^-/-,Two-wayANOVAwith

Bonferronipost-test,N=3.GSH,reducedglutathione;GSSG,oxidizedglutathione.

thatinhibitionofadipogenesisinducedwithCoPPand i n-

creasewithtinmesoporphyrincouldbelinkedtomodulationofthecano nicalWntsignaling(59).

Useo f h e m i n , h e m e o x y g e n a s e -

1 s u b s t r a t e , a n d p o t e n t inductorl edtotheop positeco n clusions.Hemin increasedadipogenesisi n m u r i n e 3 T 3 L 1 p r e a d i p o c y tesa n d h u m a n bonemarrowMSCs butalsoincreasedoxidativestressandinducedD N A d a m a g e i n m u r i n e p r e a d i p o c y t e s ( 4 3 ) . I m -

portantly,allobservedchangesingeneexpressioninduced withh e m i n c o u l d b e r e v e r s e d w i t h a n t i o x i d a

n t T e m p o l . Therefore,theauthorsconcludedthatoxidatives tressisthe

keyfa ct o r that r eg ula te s d ifferent iatio n p at t e r n s in h emin-

treatedc e l l s ( 4 3 ) . H u m a n b o n e m ar r owMS C s t r a n s d u ce d witha d e n o v i r a l v e c t o r s e n c o d i n g f o r H m o x 1 s h o w e d n o changesindifferentiationpatternbuti mprovedviabilityinhypoxia( 1 8 , 6 8 ) . H o w e v e r , a d e n o v i r a l v e c t o r s g i v e o n l y transientexpressionofthetr ansgene,whichwaslostafter2weeksofculture(18).

Primaryr a t o s t e o b l a s t s s h o w e d r e d u c e d e x p r e s s i o n a n d activityofalkalinephosphataseandreducedosteocalcinan dRunx2expressionlevelswhentreatedwithheminorwhe ntransducedw i t h a d e n o v i r a l v e c t o r s h a r b o r i n g t h e H m o x 1

gene(32).Similarresultswereobtainedwithcarbonmon- oxider e l e a s i n g m o l e c u l e C O R M -

2 o r b i l i r u b i n o r w h e n Hmox1e x p r e s si o n w a s i n d u c e d w i t h p r o s t a g l an d i n J 2 (PGJ2).E f f ec t s o f h em i n o rP GJ2 co u ld b e r ev e r se d w i t h hemeoxygenase- 1inhibitorzincprotoporphyrin(32).

Inanotherstudy,calcificationofhumansmoothmusclecellsw asinhibitedwhenc ellsweres timulatedwithh eme( 65).Import antly,theeffectofhemea ndhemeoxygenase-

1wasmediatedbyferritin(65).Inourstudy,MSCsisolatedfrom Hmox1^-/-micehadbasallevelsofH2O2similartothoseofwild- typecellsandpotentlyelevatedFth1inresponsetohe-

min.Further,Hmox1^-/-MSCscouldinduceanefficientanti- oxidantresponsetohemin,whichisastrongstressfactorforcellsl ackingheme-

degradingenzyme.Onemayspeculatethatrelativelyhighresista ncetooxidativestressobservedinMSC

Hmox1^-/-

can,atleastinpart,explaintheobservedlackofdifferencesinthep atternofdifferentiationtobasiclineages.

MSCHmox1^{+/+o r}Hmox1-/-t e n d e d

toinduceTcellap- optosiswheninjectedinvivo.However,thechangedidnotreac hstatisticalsignificance.BothpreviousreportsanalyzingtheroleofHmox1ini mmunosuppressiveactivityofMSCsusedSnPPtoinhibitHmo x1activity(7,37)andhumanandrat(7)orhumanMSCs(37)an ddidnotassessTcellapo-

ptosisinvivo.Moreover,aninvivoexperimentconductedonther o l e o f H m o x 1 i n M S C s o n t h e p r o t e c t i o n f r o m g r a f t rejecti onl a ck e d t h e c o n t r o l g r o u p o fa n i m a l s t r e at e d w i t h S nPPonly,withoutMSCs(7).TheroleplayedbyHmox1inhumancell sremainedambiguous,sinceMougiakakosetal.failedtoshowth edirectrelationbetweenHmox1levelsandimmunomodulatoryac tivityofMSCs(37).

Surprisingly,bothHmox1^+/+andHmox1^-/-

MSCsshowedhighr e s i s t a n c e t o H2O2o r ,e v e n m o r e u n e x p e c t e d l y , t o

hemin,i r r e s p e c t i v e o f t h e c o n c e n t r a t i o n o f g l u c o s e i n t h e

medium.C el l s d e v o i d o f t h e h em e o x y g e n a s e - 1 , e n z y m e -

degradingheme,wereuptonowshowntobehighlysensitivetohemin(16 ).Freehemeistoxictothecellsandincreasesoxidatives t r e s s t h a t m a y l e a d t o l i p i d p e r o x i d at i o n , D N A damage,and proteinaggregation[reviewedin(28)].

Inourstudy,weshowforthefirsttimethatMSCsHmox1^-/-

wereresistanttoheminconcentrations,whichpotentlyinducedc elldeathinbonemarrowPACcells(16).

Moreover,MSCsHmox1^-/-

expressedlowerbasallevelsofHmox2t hanP ACc ells,a ndHmox 2remainedunaffectedb y hemint reatment.S ubsequently,H mox 2-dependenth eme

degradationcannotbeconsideredarescuepathway.Finally,H mox1^+/

+MSCswerecharacterizedbylowerHmox1levelsthanPACcel lsandhadsimilarHmox1expressionincom-

parisontofibroblasts.

WehypothesizedthathighMSCresistancetohemincouldresultfromt helowimportoffreeheme.Interestingly,hemeuptakeinHmox1^+/

+a n d

Hmox1^{-/-M S C s}didnotdiffer.The

latterresultsweremirroredbytheexpressionofSlc46a1andSlc48a1

—

hemet r a n s p o r t e r s . E x p r e s s i o n o f S l c 4 8 a 1 w a s shown,however,toberegulatedbyBach1(61),whichr e-

pressesHmox1andrespondstoincreasedhemeconcentra- tion.I n o u r h a n d s , h e m i n i n c r e a s e d e x p r e s s i o n o f h e m e exporterFLVCR1inHmox1^-/-

M S C s

butnotinHmox1^+/+

MSCso r f i b r o b lasts,r e g a r d l e s s o f t h e i r g e n o t y p e . I n c r e a s e d FLVCR1waspreviouslyreportedinkidneysofHmox1^-/-

mice(4 8) . Th e r e f or e , wem i g h t speculate t h a t an in cr ea se i n

FLVCR1can,atleastinpart,accountfortheMSCHmox1^-/- resistancetohemin.

Asexpected,hemindecreasedexpressionof5¢-

aminolevulinatesynthase1inalltestedcelltypes.Alas1isahemes ynthesisrate-

limitingenzyme,whoselevelsaretightlyregulatedbecauseofthepresenceof hemeregulatorymotifinitspromoter(38).Otherenzymesinvolvedi nthehemesyn-

thesisweremostlynotchangedanddidnotdifferbetween Hmox1^+/+andHmox1^-/-cells.Regarding th eironmetab o- lism,hemin-treatedHmox1^+/+MSCsupregulatedferroportin.

FpnexpressiondidnotchangeinMSCHmox1^-/-

c e l l s ,

buttheysh owedat ren dtow ar d h igherferropo rti nlevel st han wild-

typec o n t r o l s . O n t h e o t h e r h a n d , H m o x 1^-/-

b u t

n o t Hmox1^+/

+M S C s

oranyoffibroblastcellsincreasedferritinthatcapture slabile ironand,therefore,protectscellsfromoxidati vestress[reviewedin(28)].

Although6 ho f t reatmentwith50lmol/Lh emindidn otincr easecelldeathinHmox1^-/-MSCs,itelevatedconcentra- tionso f cellularh ydrogenp eroxide.L evelso f H2O2were higherinH mox1^-/-fibroblastst hani n Hmox1^-/-MSCsa lso after24hoftreatmentwithheminand,importantly,inHmox1⁺

/+fibroblaststhaninHmox1^+/+MSCsafter48ofcul-

ture.However,neitherinfibroblastsnorinMSCswefoundany differencesinbasallevelsofH2O2.

Previously,higherH2O2levelswerereportedinHmox1^-/- thaninHmox1^+/

+i P Scells(31).Increasedproteincarbon-

ylationandlipidperoxidationwerealsoreportedinliversandkidneyso f H m o x 1^-/-

m i c e

( 4 2 ) . B a s a l i n t r a c e l l u l a r R O S levelsinhu manMSCsandfibroblastsweresimilarandlowerthanROSinINS- 1insulinoma(55).BothMSCsandfibro-

blastswerecharacterizedbysimilarlevelsofSOD1,SOD2,CAT ,andGPX1mRNA,andhigheractivitiesofcatalaseandglutathionepero xidase-1thaninINS-1cells(55).Ofnote,in

ourstudy,theconcentrationofhemin,whichwastoxicfo rMSCHmox1^-/-

cells,

alsocausedsomeincreaseincelldeathinHmox1^+/+MSCs.

MSCsHmox1^-/-

upregulatedinresponsetoheminasetofgenesinvolvedinantioxida ntdefense,namelySod2,Prdx3,Prdx6,Cat,Gclc,Gclm,Gss,Gsr, andGstp1,allofwhichcanberegulatedbytheNrf2transcriptionfactor.N otably,hemin

inducedexpressionofenzymesinvolvedinbothsynth esisandmetabolismofglutathione.Bilirubin,whichi srapidlyformedbybiliverdinreductasefrombiliverdin,aprod uctof

hemeoxygenaseactivity,isastrongantioxidant(50)thathasproperties thatar e complem entary t o glutathione (46). Al- thoughglutathionehasamuchhighercellularconcentration thanbilirubin,itprotectsmainlyhydrophilicproteins.Ontheotherhand ,lipophilicbilirubincanprotectlipids.However,inourhands, MSCHmox1^-/-cellsdevoidofhemeoxygenase-1

didnotchangetheexpressionofhemeoxygenase- 2,anothersourceofcellularbiliverdin.

Increasede x p r e s s i o n o f c -

glytamylcysteinel i g a s e andglutathionesynthetaseleadst oenhancedproductionofglu-

tathione,whereasupregulatedglutathionereductaserestoresGSHfr omGSSG.Inourexperimentalsetting,GSHtoGSSGratio,whichisanind icationofcellredoxstatus,decreasedin

alltestedcellstreatedwithhemin.However,thedecreasewasmuchstronge rinH m o x 1-/-f i b r o b l a s t sthaninHmox1^-/-MSCs.H m o x 1^-/-

fibroblastsw e r e c h a r a c t e r i z e d b y h i g h e r totalGSSGleve ls,whereasHmox1^-/-MSCsweretheonlyto

increasetotalGSH.

Ofnote,HUVECcellswithlongallelesoftheHMOX1promote r,andthuslowerlevelsofHMOX1,treatedwithH2O2hadhigherco ncentrationsoftotalglutathioneandGSSG,but

lowerGSH/GSSGratiothancellswiththeshortpromoter(53).F urther,heminwass howntoinducen euronalnecroptosis,which wasrelatedtodepletionofglutathione(29).

Increasedexpressionofglutathionemetabolismgenesin Hmox1^-/-

MSCsw a s a c c o m p a n i e d b y t h e u p r e g u l a t i ono f peroxi redoxin-6.Prdx6istheonly1-Cysperoxiredoxinthat

usesglutathioneinsteadofthioredoxin,andworksasahet- erodimerwithglutathioneS-

transferasep[reviewedin(13)],whichwasalsoupregulatedinhemin- treatedMSCHmox1^-/-.

Peroxiredoxin-

6hasdoubleactivity:peroxidase andphos-

pholipaseA2(1 3) .I n t e r e s t i n g ly,s l i g h t l y h i g h e r l e v e l s o f peroxiredoxin6werereportedinhumanMSCsthanine m-bryonicstemcells(22)andbothperoxiredoxin-6andgluta- thioneS-

transferasepbutalsoperoxiredoxins1and2werehighlyabun dantinhumanMSCs(60).

Expressiono f p e r o x i r e d o x i n -

6 w a s n o t c h a n g e d i n l a t e passageM S C s i n c o m p a r i s o n t o e a r l y p a s s a g e M S C s , a l -

thougha g e d a n d m o r e s e n e s c e n t c e l l s s h o w e d i n c r e a s e d H2O2concentration(19).Inanotherstudy,agedMSCs werecharacterizedratherbyincreasedperoxiredoxin5expression(24).

Surprisingly,hemintreatment,whichisusedtoinduceerythroiddif ferentiationofK562erythroleukemiacells,de-

creasedperoxiredoxin-6levelsinK562cells(25).

Weputforwardthatincreasedexpressionofperoxiredoxin6andotherantioxi dantgenesshouldberatherconsideredasaprotectivem e c h an i sm t h a t a l l o w s c el l st o d eal b et t e r w i th oxidativestressthanBD MC(SupplementaryFig.S7).Al-

thoughlevelsofH2O2infibroblastsandMSCsweresimilar,onlythel attercellswereabletoupregulateperoxiredoxin6anditspartner

—glutathioneS-transferasep.

MSCsisolatedfromHmox1^-/-

m i c e

wereabletoreacttooxidativestressbetterthanfibroblas tsandrecoveredgluta-

thionef a s t e r . O n e m a y s p e c u l a t e t h a t M S C s c a n e x p r e s s

lowerlevelsofKeap1orNrf3,bothofwhichcandecreaseact ivityofNrf2transcriptionfactor.Nevertheless,ourdatash owthatcellssuchasMSCsarebetterequippedwiththeme asurest o d e a l w i t h h a r s h c o n d i t i o n s t h a n o t h e r b o n e marrow-

derivedce l l s , e s p ec i a ll y p r o a n g i o g e n i c c el l s . F u r - ther,wecanspeculatethatcertaincelltypesarelessdepen- dento n h e m e o x y g e n a s e -

1 , w h i c h i s c o n s i d e r e d a c r u c i a l cytoprotectiveenzyme.

TestedfunctionsanddifferentiationpotentialofmurineMSCswere mostlyunaffectedbythelackofHmox1.

MaterialsandMethods Animals

Allp r o c e d u r e s i n v o l v i n g t h e u s e o f a n i m a l s w e r e p e r -

formedaccordingtoapprovedguidelines.Miceweremain- tainedu n d e r t h e s p e c i f i c p a t h o g e n -

f r e e c o n d i t i o n s , i n individuallyvent ilatedcages,wi t hful lac cess to food andwater.Allanimalexperimentsw ereapprovedbytheLocalEthicalCommitteeforAnimalRes earchattheJagiellonianUniversity.

IsolationofMSCs

MSCswereisolatedfromfemursandtibiaofC57Bl6·FVBH mox1^+/

+orC57Bl6·FVBHmox1^-/-.Miceweresacrificedwiththeover doseofketamine/xylazine.Boneswereresected

underthesterilelaminarflowhoodandcutintosmallpieces(ca.1m m²)withabonecutter.Then,bonechipsweredigested

with1mg/mLtypeIIcollagenase(Gibco)for90–

120minin37°Cinarotaryshaker(250rpm).Releasedcellswere washedoncewithPBSandresuspendedinthegrowthmedium[

aMEMsupplementedwith10%FBS(Lonza)a ndpenicillinw ithstreptomycin(Sigma-Aldrich,St.Louis,MO)].

Cellswereseededinsix-wellplates—

bonemarrowfromonemouseperwell.Themediumwaschanged every24hinthefirst3daysandevery2–

3daysafterthat,andcellswerepassagedw h e n c o n f l u e n t . I m p o r t a n t l y , b e f o r e t h e M A C S sorting,cellsweredetac hedwithshorttreatmentwithtrypsin(2min;Gibco)atroomtemperaturetod ecreasethenumberofhighlyadherentmacrophagesinculture.Afterthreepassag es,

MSCswerefurtherpurifiedfrom theCD45+f r a c t i o n

wi thMACSsorting.

MACSsortingofCD45^- murine

bonemarrowstromalcells Bonem a r r o w -

d er i v e d ce l l s w e r e d e t a c h e d w i t h t ry p s in , washedwit hPBS,resuspendedinAutoMACSrunningbuffer(Miltenyi),a n d s t a i n e d f o r 2 5 minw i t h a n t i -

m o u s e C D 4 5 MicroBeads(Miltenyi)in4°C.Then,thecells werewashedwith1 mLo f P B S , r e s u s p e n d e d i n A u t o M A C S R u n n i n g Buffer,andseparatedonMACSMSc olumns(Miltenyi)or

withAutoMACS(Miltenyi).Flow-throughwithCD45^-

cells

wascollected,andcolumnswithCD45^+cellswerediscarded.

PurifiedCD45^-murinebonemarrowMSCswerenext countedandeitheruseddirectlyfortheexperimentsorsee- dedf o r f u r t h e r c u l t u r e ( 1 . 5 –

2 . 0 ·10⁴/1cm²)i na MEMcompletemedium(CM).

Isolationofmurinefibroblasts

MurineadulttailfibroblastswereisolatedfromC57Bl6·FV BHmox1^+/+orC57Bl6·FVBHmox1^-/-

a cc ordi ngtothepreviouslypublishedprotocol(49)andcultu redinDMEM

(Lonza)mediumsupplementedwith10%FBS(Lonza)andpe nicillinwithstreptomycin(Sigma-Aldrich).

IsolationofmurinebonemarrowPAC

Murinebonemarrowproangiogeniccellswereisolatedasdescri bedearlier(14,16)andculturedinEGM2-

MVmedium(Lonza)w i t h 1 0 % F B S a n d p e n i c i l l i n w i t h s t r e p t o m y c i n (Sigma-Aldrich).

Analysisoffibroblast-

orosteoblastcolony-formingunits

Cellsi s o l a t e d f r o m t h e b o n e m a r r o w w e r e c o u n t e d b y usingTu¨rcksolution(Merck)tolyseredbloodcells.

Then,1·10^6ofbonemarrowcellswereseededperwellinsix-well plates.Cellswerecultureduntilcoloniesoffibroblastoidcellswereformed.

Atthatstage,partofthewellswasfixedandstainedwithcry stalvioletandthecoloniesoffibroblastoidcellswerecounte d.Anotherpartofthecellswastreatedwithosteogenicdifferentiation mediumforthenext3weeks,andthenc e l l s w e r e s t a i n e d w i t h A l i z a r i n R e d S a n d p o s i t i v e colonieswerecounted.

MSCsphenotypin g

MSCsculturedforthreepassagesaftertheisolationwerede tachedw itht rypsin,w ashedw ithP BS,a nds tainedf or

25mininAutoMACSRunningBufferat4°Cwiththefol- lowingantibodies:anti-mouseCD45(clone30F- 11;BDBiosciences),anti-mouseCD29(cloneHMb1- 1;BioLegend),anti-

mouseC D31( cloneM EC13.3;BD B iosciences),a nti- mouseCD34(cloneRAM34;BDBiosciences),anti- mouseCD90.2(clone30-H12;BioLegend),anti- mouseCD105(cloneMJ7/18;BioLegend),anti- mouseCD117(c-kit)(clone2B8;eBioscience),anti- mouseCD140a(cloneAPA5;eBioscience),a nda nti- mouseL y-6A/E( Sca-1)

( cloneD 7;eBioscience).Thephenotypeofthecellswasassess edwithBDLSRIIorBDLSRFortessa(BectonDickinson).

MSCs:differentiationtoosteoblasts

MSCsHmox1^{+/+o r}Hmox1^{-/-w e r e}differentiatedtooste- oblastswiththeprotocolbyZhuetal.

(69).Briefly,2.5·10^4ofsortedCD45^-

bonemarrowstromalcellswereseededper1wellof24-

wellplates.Osteoblastdifferentiationwasinducedfor3weekswithaMEMC Msupplementedwith0.1lmol/Ldexamethasone,10mmol/Lb -glycerolphosphate,and50lmol/Lascorb ate-2-

phosphate( all from Sigma-

Aldrich)andv e r i f i e d w i t h A l i z a r i n R e d S s t a i n i n g ( S u p p lementary

Fig.S1A),geneexpressionanalysis,andimmunofluorescentstainingfo rosteopontin(cloneEPR3688;Abcam).Controlcellswerec ulturedinaMEMCM.Lowglucosedifferentia-

tiono r c o n t r o l m e d i u m c o n t a i n e d 5 mmol/Lg l u c o s e , a n d highg l u c o s e d i f f e r e n t i a t i o n o r c o n t r o l m e d i u m c o n t a i n e d 33mmol/Lglucose.

MSCs:differentiationtoadipocytes

MSCsHmox1^{+/+o r}Hmox1^{-/-w e r e}differentiatedtoadi- pocyteswiththeprotocolbyZhuetal.

(69).Briefly,2.5·10^4ofsortedCD45^-

bonemarrowstromalcellswereseededper1wellof24-

wellplates.Adipocytedifferentiationwasinducedfor3weekswithaMEM CMsupplementedwith1.0lmol/Ldexamethasone,50lmol/L3- isobutyl-1-

methylxanthine(IBMX),and10ng/mLinsulin(allfromSigma- Aldrich)andverifiedwithOilRedOstaining,geneexpressionanalysis,and immunofluorescentstainingforFabp4(cloneEPR3579;Abca m).C o n t r o l c e l l s w e r e c u l t u r e d i n aMEMC M . L o w gl ucosedifferentiationorcontrolmediumcontained5mmol/Lglucose,a ndhighglucosedifferentiationor con trolme-

diumcontained33mmol/Lglucose.

MSCs:differentiationtomyofibroblasts

SortedCD45^{-b o n e}marrowstromalcellswereseededin24- wellplates(2.5·10⁴/well).Myofibroblastdifferentiationwasinduce dfor6dayswithaMEMCMsupplementedwith2ng/mLrecombi nanthumanTGFb1(Peprotech)andcon-

firmedw i t h g e n e e x p r e s s i o n a n a l y s i s . C o n t r o l c e l l s w e r e culturedinaMEMCM.

AnalysisofMSCimmunosuppressiveactivityinvivo

C57Bl6·FVBm i c e w e r e i n j e c t e d i n t r a v e n o u s l y w i t h 1 millionofM SCHmox1^+/

+orHmox1^-/-,orsaline(vehiclecontrol)andsacrificed24hlater.N umbersofcirculatingCD3^+Tcells,CD3⁺AnnexinV^+apoptoticT c ells,andCD3⁺CD4^+CD25^highactivatedTcellswereassessedbyflo wcytometryon

anLSRF ortessacytometer( BectonDickinson).Peripheralblood (PB)wascollectedinheparinizedtubes.Redbloodcellswerelysed withammoniumchlorideredbloodcelllysisbuffer

(0.15MNH4Cl,10mMK HCO3,0.1mMEDTA).Obtained totalnucleatedcellswereresuspendedinautoMACSRunning Buffer(Miltenyi

).

Cellswerethenstainedwithanti-

mouseCD3(clone17A2;BDHorizon),anti-mouseCD4(cloneRPA- T4;BDHorizon),anti-

mouseCD25 ( cl oneC 37 ; B DP h arm i nge n) , an dant i- mouseC D 4 5 ( c l o n e 3 0 -

F 1 1 ; B D P h a r m i n g en ) a n t i b o d i e s .

AnnexinV^{+c e l l s}w e r e s t a i n e d w i t h T A C S®A n n e x i n

V (AnV)k i t ( T r e v i g e n ) a c c o r d i n g t o t h e m a n u f a c t u rer’si n -

structions.Thenumberofcellsper1lLofPBwascalculatedbasedo nthetotalleukocytecount(WBC,103c e l l s / 1lLofPB)and thepercentageofeachpopulation withinthecol- lectedevents.WBCwasmeasuredbyusingABCVet(scila nimalcareGmbH).

AnalysisofMSCimmunosuppressiveactivityinvitro MSCH m o x 1^{+/+o r}H m o x 1^-/-

w e r e

s e e d e d o n s i x -

w e l l plates.WhenMSCsreached100%confluency,theywereco- culturedfor24hwith1mlnofprimarymousesplenocytesineachwell .Aftertheco-

culture,splenocyteswereharvestedandstainedwithanti-CD3- AlexaFluor647(clone17A2;BDPharmingen),a n t i - K i 6 7 - A l e x a F l u o r 4 8 8 ( c l o n e B 5 6 ; B D Pharmingen),a ndDAPIfortheanalysisofcellproliferationorw i t h an t i - C D3 - A l ex aF l u o r 6 4 7 ( c l on e 1 7 A 2 ; B D P h a r -

mingen),andAnexinV-

FITC(Trevigen)fortheanalysisofTcellapoptosis.Dataarepresentedasp ercentofproliferating/

apoptoticTcellswithinthepopulationofCD3^+Tcells.

Totalantioxidantcapacityassays

Totala n t i o x i d a n t capacityofconditionedmediaf r o m MSCHmox1^+/+orHmox1^-/-

wasmeasuredwithTACAssay(CellBiolabs)accordingtothe manufacturer’sprotocol,orusingABTS,orFRAPmethod.

ABTSassaywasperformed

accordingtothepreviouslypublishedprotocol(40)basedonthe(12) method.FRAPassaywasbasedontheBenzieandStrainmet hod(4).

Multipleximmunoassays

LevelsoffactorsproducedbyMSCHmox1^+/+orHmox1^-/-

weremeasuredwithMilliplex^®MAPMouseCytokine/Chem okineB e a d P a n e l -

32P l e x (Millipore)o n L u m i n e x FlexMap3Dplatform(Mi llipore)andanalyzedwithMilli-

plexAnalyst3.4software(Millipore).

Proliferationassa y

Thepro liferation o f MSCswa sassessed wi tht h e B rd UmethodbyusingCellProliferationELISA(Roche),accord- ingt o t h e m a n u f a c t u r e r ’ s p r o t o c o l . L o w g l u c o s e m e d i u m contained5mmol/Lglucose,andhighglucoseme diumcontained33mmol/Lglucose.Cellswereculturedinhighorlo wglucosemediumandBrdUlabelingsolutionfor24h.

Lactatedehydrogenaseactivityassay

CytotoxicityofheminorH2O2inMSCswasevaluatedwithCyt oTox96^®NonRadioactiveCytotoxicityAssay(Promega),accor dingtothemanufacturer’sprotocol.Lowglucoseme-

diumcontained5mmol/Lglucose,andhighglucosemediumcon tained33mmol/Lglucose.Cellsweretreatedwithhemin,H2O2,a nd/orhighglucosefor6hbeforetheanalysis.

7- aminoactinomycinD-basedcellviabilityassay

TheviabilityofMSCsstimulatedfor6hwithhighdosesofhe minwasassessedby7-aminoactinomycinD(7-

AAD)staining.S t i m u l a t e d a n d c o n t r o l c e l l s w e r e d e t a c h e d w i t h trypsin,washedwithPBS,andresuspendedin AutoMACSRunningB u f f e r (Miltenyi).T h e n , c e l l s w e r e s t a i n e d f o r 10minwith7-

AAD(BDPharmingen)accordingtothemanufacturer’sprotoco landanalyzedonaBDLSRFortessacytometer.

MeasurementofMSCviabilityinresponsetoTNFa

Analysisofgeneexpression TotalRNAwasisolatedbyphenol-

chloroformextraction,anditwasreversetranscribedwiththeoligo(dT)pri mersandRevertAidR e v e r s e t r a n s c r i p t a s e ( F e r m e n t a s ) o r w i t h t h e NCode™VILO™miRNAcDNAsynthesi skit(Invitrogen).Theexpressionofgeneswasassessedbyqua ntitativereal-timePCR(qRT-

PCR),whichwasperformedintheStepO-

nePlussystem(AppliedBiosystems,FosterCity,CA)witht hes p e c i f i c p r i m e r s ( S u p p l e m e n t a r y T a b l e s S 1 a n d S 2 ) , cDNAandSYBRGreenQuantitativeRT- PCRkit(Sigma-

Aldrich),u n d e r c o n d i t i o n s s u m m a r i z e d i n S u p p l e m e n tary

MSCHmox1^+/+orHmox1^-/-

wereincubatedwith10ng/mLTNFafo r24 h.T h e n , n um berso f ea rl yan d lat ea p o pt otic

TableS3.

Expressiono f l i p i d m e t a b o l i s m g e n e s i n H m o x 1_-/-

+/+or cellswereassessedbyflowcytometrybyusingthestaining Hmox1 MSCsdifferentiated t oadipocytesw asassessed_® withHoechst33342and7-AADaccordingtotheprotocolby withTaqMan ArrayMouseLipid-

RegulatedGenes(Ap- Schmidetal.(45).

MeasurementofcellularH2O2l e v e l s

LevelsofcellularH2O2w e r emeasuredwithH2DCFDAa ssay.H m o x 1^+/+orH m o x 1^-/-

M SC s

a n d f i b r o b l a s t s w e r e stimulatedfor6,24,or48hwith 50lmol/Lhemin(FrontiersScientific).A f t e r e a c h t i m e p o i n t , t h e s t i m u l a t e d c e l l s a n d

nonstimulatedcontrolswereharvestedwithtrypsin,washedwithP BS,andstainedfor30minwith0.1lmol/LH2DCFDA(Sigma- Aldrich)inPBS.Then,cellswerewashedtwicewithPBS,andDCFDAf luorescencewasassessedwithaBDLSRFortessacytometer(BectonDic kinson).

Hemecellularcontentassay

MSCsH m o x 1^+/+orH m o x 1^-/-werec u l t u r e d i n 2 4 - w e l l plates.HemecontentwasassessedwiththemethodbyFor -estietal.(15)innonstimulatedcells(T0),after2hofstim- ulationw i t h 5 0 lmol/Lh e m i n ( T 2 ) a n d a f t e r 2 a d d i t i o n a l hoursofcultureinfreshaMEMCM(T4).Cellswerelyse dwith8 0 % f o r m i c a c i d ( P O C H S . A . ) , a n d t h e l y s a t e w a s transferredtoclearplastic96-

wellplates.Absorbancewasmeasureda t l =398nmw i t h a G E N i o s m i c r o p l a t e r e a d e r (Tecan).

SnPPbindingassay

Cellss t i m u l a t e d w i t h S n P P ( F r o n t i e r s S c i e n t i f i c ) s h o w fluorescencei n A P C c h a n n e l ( lex=640nm,e m i s s i o n d e -

tectedwith670/25bandpassfilter).Therefore,westimulatedHmox1^+/

+andH m o x 1^-/-

MSCsw i t h 1 0 , 2 5 , o r 5 0 lmol/LSnPPfor6handanalyzedth eirfluorescencewithflowcy-

tometry.Cellsweredetachedwithtrypsin,washed,andre- suspendedinAutoMACSRunningBuffer(Miltenyi).Flowcyt ometryanalysis w asp er f o r m ed on aB DLS R F ortessac ytometer(BectonDickinson).

GSH/GSSGassay

LevelsoftotalGSH,totalGSSG,andGSH/GSSGratioinMSCsa ndfibroblastswereassessedwithGSH/GSSG-

Glo™Assay(Promega)accordingtothemanufacturer’sprotoc ol.

Then,1.0·10^4cellswereseededperwellin96-

wellplates.Cellswerestimulatedfor4hwith50lmol/LhemininaMEM CMandculturedfor2hinaMEMCMtoletthemrecoverGSHl evels.TotalGSHandtotalGSSGdataareshownasaratiotothecontr olnonstimulatedcells.

pliedBiosystems)andTaqManUniversalPCRMasterMix (AppliedB io syst ems)w ith theP CRprogram d escrib edin SupplementaryTableS3.

Statisticalanalysis

Statisticala n a l y s i s o f t h e d a t a w a s p e r f o r m e d w i t h GraphPadPrismsoftware.Resultsareexpressedasmea n–

SDunlessotherwisestated.Statisticalsignificancewasa c-

ceptedatp<0.05.Dataobtainedininvitroexperimentswereanalyzedw ithStudent’st-

testwhentwogroupsofsampleswereuse d. In a no t h er c a s e , w e us ed o n e- w ay o r tw o -

w ay analysisofvariancewithBonferronipost-

test.Thekindofstatisticaltestappliedtoanalyzegivensetsofdataispro videdinthedescriptionoffigures.

Acknowledgments

ThisworkwassupportedbythePolishNationalScience Centre(grants2013/11/N/NZ3/00958,2013/11/N/NZ1/02 399,2015/18/NZ3/00387),theEuropeanUnionundertheEurope anR e g i o n a l D e v e l o p m e n t FundO p e r a t i o n a l Pro-grammeInnovativeEconomy2007–2013(POIG- 01.02.01-

109/09),andthegrantfromtheNationalCentreforResearchandD e v e l o p m e n t ( S T R A T E G M E D ( 2 / 2 6 9 4 1 5 / 1 1 / N C B R / 2015).W N N w a ss u p p o r t e d b y t h e F o u n d at i o n f o r P o l i s h Science(FNP).TheFacultyofBiochemistry,Biophysics andBiotechnologyofJagiellonianUniversityisapartnerofthe LeadingNationalResearchCenter(KNOW)thatissupportedbyth eMini str yof Science andHigherEd ucation. S ervier Medic alArtimagebankwasusedtoprepareFigure5A.

AuthorDisclosureStatement

Nocompetingfinancialinterestsexist.

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