The lower in vitro chondrogenic potential of canine adipose tissue-derived mesenchymal stromal cells (MSC) compared to bone marrow-derived MSC is not improved by BMP-2 or BMP-6

Original

Article

The

lower

in

vitro

chondrogenic

potential

of

canine

adipose

tissue-derived

mesenchymal

stromal

cells

(MSC)

compared

to

bone

marrow-derived

MSC

is

not

improved

by

BMP-2

or

BMP-6

M.

Teunissen

a,

*

,

F.

Verseijden

a

,

F.M.

Riemers

a

,

G.J.V.M.

van

Osch

b

,

M.A.

Tryfonidou

a a

DepartmentofClinicalSciences,FacultyofVeterinaryMedicine,UtrechtUniversity,Yalelaan108,3584CM,Utrecht,TheNetherlands

b

DepartmentofOrthopaedicsandDepartmentofOtorhinolaryngology,ErasmusMC,UniversityMedicalCenterRotterdam,3015GD,Rotterdam,The Netherlands ARTICLE INFO Articlehistory: Accepted22December2020 Keywords: BMP Cartilage Pelletculture Regenerativemedicine Surfacemarkerexpression

ABSTRACT

Mesenchymal stromalcells (MSC)are usedforcell-based treatmentforcanineosteoarthritis(OA).

ComparedwithhumanMSCs,detailedinformationonthefunctionalcharacterisationofcanineMSCsis

limited.Inparticular,thechondrogenicdifferentiationofcanineadiposetissue-derivedMSCs(cAT-MSCs)

ischallenging.Inthisstudy,weaimedtocomparecAT-MSCswithbonemarrow-derivedMSCs

(cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone

morphogenetic proteins(BMP).cBM-MSCs andcAT-MSCswerecharacterisedusing flowcytometry

and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic

differentiationpotentialofallcMSCpreparationsinthepresenceofTGF-β1aloneorwhensupplemented

with 10,100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical,

histochemicalandimmunohistologicalanalyses.

BothcBM-MSCs andcAT-MSCsexpressedthesurfacemarkers CD90, CD73,and CD29,and were

negativeforCD45andCD34,althoughtheexpressionofCD73andCD271variedwithdonorandtissue

origin.Interestingly,expressionofACANandSOX9washigherincBM-MSCsthancAT-MSCs.Incontrast

withcBM-MSCs,cAT-MSCscouldnotdifferentiatetowardthechondrogeniclineagewithoutBMP-2/-6,

andtheirinvitrochondrogenesiswasinferiortocBM-MSCswithBMP-2/-6.Thus,cAT-MSCshavelower

invitrochondrogeniccapacitythancBM-MSCunderthestudiedcultureconditionswith10,100,or250

ng/mLBMP-2orBMP-6.Therefore,furthercharacterisationisnecessarytoexplorethepotentialof

cAT-MSCsforcell-basedOAtreatments.

©2021TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Osteoarthritis (OA) is a common progressive joint disease, characterisedbycartilagedegradation,subchondralbonechanges, and synovial inflammation. OA affects 2.5%–20% of the canine population, which increases to 80% in dogs aged >8 years (Anderson et al., 2018).Mesenchymal stromalcells (MSCs) are promisingcandidatesforcell-basedOAtreatmentsbecauseoftheir immunomodulatorypropertiesandchondrogeniccapacity( Whit-worthandBanks,2014;Im,2018).HumanMSCsisolatedfromthe bonemarrow(hBM-MSCs)possessinvitrochondrogenicpotential (Johnstoneetal.,1998;Pelttarietal.,2008).Comparedto hBM-MSCs, humanMSCsisolatedfromtheadiposetissue(hAT-MSC) possess several advantages, including ease of isolation with

minimalmorbidity,relativelyhigherMSCconcentration( Oedayr-ajsingh-Varmaetal.,2006),higherproliferationrate(Schäfflerand Büchler, 2007), and stronger immunosuppressive capabilities (Mattar and Bieback, 2015). While hAT-MSCs can differentiate towardmultiplelineagesinvitroandinvivo,theirosteogenicand chondrogenicpotentialareinferiortothoseofhBM-MSCs(Strioga etal.,2012).

CanineMSCs(cMSCs)havebeenlessextensivelycharacterised thanhMSCs(deBakkeretal.,2013).Particularly,thechondrogenic differentiation of cAT-MSCs is challenging; chondrogenesis in earlier studies was either unsuccessful (Russell et al., 2016), inferiortothatofcBM-MSCs (Reichetal., 2012;Beardenetal., 2017),orlackedrobustevidence(Robey,2017).

Therefore,weaimedtoenhancethechondrogeniccapacityof cAT-MSCstothelevelexhibitedbycBM-MSCsbysupplementing differentdosesofbonemorphogeneticprotein(BMP)-2orBMP-6. BothBMPsandtransforminggrowthfactor(TGF)-β1areknownto synergistically affect chondrogenesis (Sekiya et al., 2005).

*Correspondingauthor.

E-mailaddress:m.teunissen@uu.nl(M. Teunissen).

http://dx.doi.org/10.1016/j.tvjl.2020.105605

1090-0233/©2021TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

The

Veterinary

Journal

Furthermore, BMP-6 may specifically induce TGFβ receptor 1 expression, which is otherwise downregulated in hAT-MSCs comparedtothatinhBM-MSCs,therebyrecoveringtheeffectof TGF-β1 on hAT-MSC chondrogenesis (Hennig et al., 2007). Additionally,(surface)markerexpressionlevelsincAT-MSCsand cBM-MSCswerecomparedforobtainingmoreinsightsregarding differencesincellpopulation.

Materialsandmethods Animalsamples

Bonemarrowandadiposetissuesampleswerecollectedfrom healthydogs(n=14)aftereuthanasiaforunrelatedexperiments (AppendixA:Supplementarymaterial).Theprotocolwasapproved bytheEthicsCommitteeonAnimalExperimentation ofUtrecht University(2012.III.07.068,2013.III.08.254,2013.III.08.054). IsolationofcMSCsfromthebonemarrowandadiposetissue

cBM-MSCsand cAT-MSCswere isolatedand expandedasper methodsdescribedpreviously (Tryfonidou etal.,2014;Malagola etal.,2016).Briefly,afterisolation,thecellswereplated(cBM-MSCs; 1.3–2  106 _cells/cm2_{, cAT-MSCs: 4  10}4 _cells/cm2_{) in}

MSC-expansion medium (α-MEM, Invitrogen) containing 10% foetal bovine serum (FBS; Gibco, high performance), 1% penicillin/ streptomycin(P/S;GEHealthcareLifeSciences),0.1mMascorbic acid2-phosphate(Sigma), and1ng/mL basicfibroblastgrowthfactor (bFGF;AbDSerotec)untilconfluencywasreachedandthecellswere cryopreserveduntilfurtheruse.Detaileddescriptionisprovidedin AppendixA:Supplementarymaterial.

Colony-formingunit-fibroblast(CFU-F)assay

Cryopreservedcellswerethawedoniceandplatedinexpansion medium atadensityof17cells/cm2 _{(correspondingto}

approxi-mately 1000cells/Petridish(CellStar,664160,GreinerBio-One). After10–14days,thecellswerefixedwithmethanolandstained withCrystalViolet(0.5%inmethanol(100%),Sigma)for15minat roomtemperature(RT).Afterrinsing,thecolonies(consistingof >50cells)werecounted.

FACSanalysis

Sevencryopreserveddonor-matchedcAT-MSCsandcBM-MSCs (2106_{cells/donor)atpassage2wereanalysed.Thecellswere}

thawedonice,washedinHanks’bufferedsalinesolution(HBBS) andresuspendedinstainbuffer(FSB,BDPharmingen)atadensity of 0.5–1 105 _{cells per reaction. Subsequently, the cells were}

incubatedoniceinthedarkforatleast15minwithcanine-specific phycoerythrin (PE), _fluoresceinisothiocyanate (FITC),orisotype

controlantibodies(Table1).Afterwashing,thecellswerestained with7-aminoactinomycinD(7AAD,0.25mg/test;BDBiosciences) todifferentiatebetweendeadandlivecells.Datawerecollected usingtheFACSDivasoftware(v8.0)onaCANTOII(BDBiosciences) andanalysedusingtheFlowJosoftware(v10.0).

Chondrogenicdifferentiation

TheprotocolusedforchondrogenicdifferentiationofthecMSCs wasadaptedfromJohnstoneetal.(1998)andwasfirstperformed usingTGF-β1(10ng/mL,RandDSystems)andBMP-6(10ng/mL, Peprotech;Hennig etal., 2007)in chondrogenic differentiation medium (Dulbecco’s modified Eagle medium [DMEM] high glucose, Invitrogen) containing 1% P/S, 1% ITS + premix (BD), 0.04mg/mLproline(Sigma),0.1mMascorbicacid2-phosphate, and10 7_{Mdexamethasone(Sigma;AppendixA:Supplementary}

material).Pelletswerecollectedforreverse transcription-quanti-tative polymerasechain reaction (RT-qPCR), glycosaminoglycan (GAG),and DNAanalyses,and immunohistochemicalevaluation after35days.

Asthisdidnotresultinchondrogenicdifferentiationofthe cAT-MSCs, follow-up experiments were performed with higher concentrations of BMP-6 and BMP-2 (10,100, and 250 ng/mL). RT-qPCRwasperformedafter7daystodetectearlytranscriptional changesthatprecedethechangesattheproteinlevel.Additionally, cultureperiodof21dayswasusedbasedonmethodsreportedby

Mackayetal.(1998;AppendixA:Supplementarymaterial). GAGandDNAcontent

Threepelletsper donorwerepooled foreach conditionand digestedovernightat60Cusing600

m

Lpapaindigestionsolution (11.9

m

L/mLpapain[Sigma]and15.7

m

g/mLcysteineHCl[Sigma] inpapainbuffer[13mg/mLH2NaPO4
2H2Oand3.26mg/mLEDTA,

pH6]). GAGcontentwas determinedusingdimethyl methylene blue(DMMB,Sigma)assayasperprotocolsdescribedpreviously (Bach et al., 2015). DNA content was measured according to manufacturer’s instructions with the QubitTM _{dsDNA HS assay}

(ThermoFisherScientific). Immunohistochemicalevaluation

Eachpelletwasfixedin4%neutralbufferedformalin(Klinipath B.V.)with0.1%eosin(BoomBVMemmel)andembeddedin2.4% alginateandparaffin.Five-micrometresectionswerestainedfor SafraninO/FastGreenstaining(0.125%SafraninO,Sigma;0.4%Fast Green, Sigma) and collagen type II (COL-II) and I (COL-I) immunohistochemistry as per methods described previously (Bach et al., 2015) using COL-I mouse monoclonal antibody (0.07

m

g/mL, Abcam, ab6308) and COL-II mouse monoclonal antibody (0.02

m

g/mL, DSSHB, II-II6B3), respectively. Normal

Table1

TheantibodiesusedforFACSanalysis.

Target Host Reactivity Clone Fluorochrome Manufacturer Catalognumber

CD90 Rat Dog YKIX337.217 PE eBioscience 12-5900-42

CD73 Rabbit Human,Mouse,Rat,Dog,Chicken Unknown FITC Biossantibodies bs-4834R

CD29 Mouse Human,Cow,Dog TS2/16 PE BioLegend 303004

CD271 Mouse Dog,Human,Mouse ME20.4 PE Invitrogen 12-9400-42 CD146 Mouse Dog,Human,Mouse,Rabbit P1H12 FITC Invitrogen 11-1469-42

CD45 Rat Dog Unknown PE LSBio LS-C127720

CD34 Mouse Dog IH6 PE BDPharmingen 559369

RatIgGK Rat Isotypecontrol eB149/10H5 PE eBioscience 12-4031-82 RabbitIgGk Rabbit Isotypecontrol Unknown PE Antibodiesonline ABIN376422 MouseIgGk Mouse Isotypecontrol MOPC-21 PE BioLegend 400112 IgGk,immunoglobulinkappa;PE,phycoerythrin;FITC,fluoresceinisothiocyanate.

mouseisotypeIgG(IgG1(RandD,HAF007)wasusedascontrol.

Sections werestained for 5 minwith Toluidine Blue O (0.04%, Sigma;BasicBlue17,86%dye,dissolvedin0.2Macetatebuffer). RT-qPCRanalysis

Thesurfacemarkerandchondrogenicgeneexpressionprofiles were investigated in expansion passage 2 of five matched, undifferentiatedcAT-MSCandcBM-MSCdonors(0.1106_cells/

donor); the bone marrow was used as the control. The chondrogenic gene expressionof the differentiated cMSCs was determinedinthreepooledpelletsperdonorforeachcondition. The pelletsweresnap-frozenand crushedwitha pestle (Argos technologies Inc, 9951-901). Total RNA was isolated using the RNeasyminikit(Qiagen,74134)ormicrokit(Qiagen,74004),which includedanon-columnDNasestep.RNAwasquantifiedusingthe NanoDropND-1000spectophotometer(IsogenLifeScience).cDNA wassynthesisedusingtheiScriptTMcDNAsynthesiskit(Bio-Rad) accordingtothemanufacturer’sinstructions.

RT-qPCRwasperformedusingtheBioRadCFX-384cyclerandIQ SYBRGreen supermix (Bio-Rad) and surface marker-and chon-drogenic lineage-specificprimers (Table 2).Relative expression wasestimatedusingtheefficiency-correcteddelta-deltaCt(

DD

Ct)

methodandsixreferencegenes;HNRPH,RPL8,GUSB,SRPR,YWHAZ, andsDHA(Table2).

Statisticalanalysis

DetaileddescriptionofthestatisticalanalysesusingRsoftware (version3.0.2)isprovidedinAppendixA:Supplementarymaterial. Inshort,linearmixedmodelswereusedfornormallydistributed dataandtheKruskal-Wallisranksumtestfordatathatwerenot distributednormally.DifferenceswereconsideredsignificantifP values were <0.05 after multiple comparison correction. Addi-tionally,theeffectsize(ES)wascalculatedusingCohen_’sd(Cohen, 1977).Effectsizeof>0.8wasconsideredtobelarge,and>2.0was consideredtobehuge(Cohen,1977;Sawilowsky,2009). Results

cBM-MSCsandcAT-MSCswereplastic-adherentandformcolonies Alldonorsdemonstratedthetypicalfibroblast-likephenotype (Fig.1).Approximately80%confluencywasreachedwithin5days forcAT-MSCs(range;3–7days)andwithin8daysforcBM-MSCs (range;7–9days).Onaverage,cAT-MSCsandcBM-MSCsproduced

Table2

PrimersusedinRT-qPCR.

Marker Gene PrimerSequence AmpliconSize(bp) Annealingtemperature(_{C) NCBIaccessionno.}

Surfacemarkerexpression CD90 F:CAGCATGACCCGGGAGAAAAAG 134 63.5 NM_001287129 R:TGGTGGTGAAGCCGGATAAGTAGA CD146 F:GGGAATGCTGAAGGAAGG 99 63 XM_022418207 R:CTTGGTGCTGAGGTTCTG CD166 F:AAGCGTCATAAACCAAACAG 150 61 NM_001313804 R:TATAGCAGAGACATTCAAGGAG CD73 F:CTCCAACACATTCCTTTACAC 150 61 XM_532221 R:ACTCAACCTTCAAATAGCCT CD105 F:CATCCTTCACCACCAAGAG 139 60 XM_005625330 R:CAGATTGCAGAAGGACGG CD44 F:CTTCTGCAGATCCGAACACA 147 60 NM_001197022 R:GAGTAGAAGCCGTTGGATGG CD14 F:CCCGGCGCTCACCACCTTAGAC 98 60 XM_843653 R:CCTGGAGGGCCGGGAACTTTTG CD45 F:GACCATGGGGTGCCTGAAGAT 90 58 64 XM_005622282 R:CACAATGGGGCCACTGAAGAAG CD31 F:GTTCTGCGTGTCAAGGTG 85 65 XM_022422841 R:TGTCCTTCCCAAACTCCA CD34 F:TCAGGGCCCCCGACATCTC 115 66 NM_001003341 R:TCTCTGCTCACCCCTCTGGAAAAA

Chondrogenesis ACAN F:GGACACTCCTTGCAATTTGAG 110 61 62 NM_001113455 R:GTCATTCCACTCTCCCTTCTC COL2A1 F:GCAGCAAGAGCAAGGAC 150 60.5 65 NM_001006951 R:TTCTGAGAGCCCTCGGT SOX9 F:CGCTCGCAGTACGACTACAC 105 62 63 NM_001002978 R:GGGGTTCATGTAGGTGAAGG COL1A1 F:GTGTGTACAGAACGGCCTCA 109 61 NM_001003090 R:TCGCAAATCACGTCATCG COL10A1 F:CCAACACCAAGACACAG 80 61 XM_849417 R:CAGGAATACCTTGCTCTC TGFβR1 F:CAGTCACCGAGACCACAGACAAAGT 100 54 XM_014117881 R:TGAAGATGGTGCACAAACAAATGG

Housekeepinggenes HNRPH F:CTCACTATGATCCACCACG 151 61,2 XM_538576 R:TAGCCTCCATAACCTCCAC RPL8 F:CCATGAATCCTGTGGAGC 64 55 XM_532360 R:GTAGAGGGTTTGCCGATG GUSB F:AGACGCTTCCAAGTACCCC 103 62 NM_001003191 R:AGGTGTGGTGTAGAGGAGCAC SRPR F:GCTTCAGGATCTGGACTGC 81 61,2 XM_546411 R:GTTCCCTTGGTAGCACTGG YWHAZ F:CGAAGTTGCTGCTGGTGA 94 58 XM_843951 R:TTGCATTTCCTTTTTGCTGA sDHA F:GCCTTGGATCTCTTGATGGA 92 61 DQ402985 R:TTCTTGGCTCTTATGCGATG F,Forward;R,Reverse;bp,basepair;no,number.

10132and11149coloniesper1000platedcells,respectively (nosignificantdifference).

Passage2cAT-MSCshadlowCD73,ACAN,andSOX9expressionlevels The expressionprofile, investigatedusingFACSanalysis, was similar for passage 2 cBM-MSCs and cAT-MSCs, with high frequencies ofCD90+_{and CD29}+ _{cellsand negligiblenumber of}

CD271+,CD146+,CD34+,andCD45+cells(Fig.2a).Thenumberof CD271+_{cells weresignificantlylowerin cAT-MSCsthan that in}

cBM-MSCs(P<0.001).LowbutvariablefrequenciesofCD73+_cells

wereobserved(cBM-MSC:17.7%18.7%;cAT-MSC:14.7%8.8%). RT-qPCRwasperformedtoinvestigatetheexpressionofsurface markers,againstwhichcanine-specificFACSantibodieswerenot available.HighCD90,CD105,andCD166expressionandlowCD146 expression were observed in both cell types (Fig. 2b). CD44 expressionwas lowincBM-MSCs andwasundetectable in cAT-MSCs(P=0.26).CD73expressionwassignificantlyhigherin cBM-MSCthanthatincAT-MSC(P=0.0088).CD34,CD45,CD14,andCD31 were undetectablein both cell types.In undifferentiated cBM-MSCs,theexpressionlevelsofSOX9(P=0.02)andACAN(P=0.055) werehigherthanthoseincAT-MSC(Fig.2c).COL2A1andCOL1A1 weresimilarlyexpressedinbothcelltypes.

cAT-MSCsdidnotundergochondrogenesiswithouthigh concentrationsofBMP-2orBMP-6

DifferentiationwithTGF-β1and10ng/mLBMP-6assessedatday35 Afteradditionof TGF-_β1,DNAcontentincreasedin bothcell types(P=0.00015)comparedtothatinthecontrolgroup,without additionaleffectofBMP-6(Fig.3a).Thiswasalsoreflectedinthe increaseinpelletsizeafterTGF-β1addition(Fig.3c).Theincrease

in GAG deposition, observed only in cBM-MSCs (Fig. 3a), was confirmedbyincreaseinACANexpression(P=0.007,Fig.3b)and positivity to Safranin O and Toluidine Blue staining (Fig. 3c). DepositionofCOL-IIbycBM-MSCswasconfirmedusing immuno-histochemistryandwasinagreementwiththeincreaseinCOL2A1 expression(Fig.3band3c).COL1A1expressionwassignificantly upregulatedincBM-MSCsandcAT-MSCsuponadditionofTGF-β1 alone(P=0.00003)orwithBMP-6(P=0.00003).Further,all cBM-MSCandcAT-MSCdonorsexpressedCOL-I.ExpressionofTGFβR1 wassignificantlylowerincAT-MSCsthanthatincBM-MSCs(P= 0.0008),anditsexpressiondidnotincreasewithTGF-β1orBMP-6 treatment.COL10A1expressionwasnotdetected,irrespectiveof theconditionanddonor.

DifferentiationwithTGF-β1anddifferentconcentrationsofBMP-2 andBMP-6

RT-qPCRwasperformedafter7daystoassessearly transcrip-tional changes (Fig. 4a). A BMP dose-dependent effect was observed in cBM-MSCs, with signi_ficant increase in ACAN expressionuponadditionof100or250ng/mLBMP-6(P=0.014 and0.049,respectively),and100or250ng/mLBMP-2(P=0.0017 and 0.0059, respectively), compared to that in the control. Additionally,comparedtothatinthecontrol,COL2A1expression significantlyincreasedwith100and250ng/mLBMP-2(P=0.046 and0.042,respectively),and250ng/mLBMP-6(P=0.049).In cAT-MSCs,amoderatedose-dependentincreasewasobservedonlyfor SOX9,ACAN,andCOL1A1afterstimulationwithBMP-2orBMP-6. TGFBR1expressionwassignificantlylowerincBM-MSCstreated with100or250ng/mLBMP-6(P_<0.001)thaninthosetreated withTGF-β1alone.Thisdownregulationwasalsosignificantfor cAT-MSCs in the presence of 250 ng/mL of BMP-6 (P = 0.02). Expressionof TGFBR1in cAT-MSCswas significantlylowerthan

Fig.1.MSCmorphologyandCFU-Fassays.(a)Brightlightphasecontrastimagesofcaninebonemarrow(BM)-andadiposetissue(AT)-derivedmesenchymalstromalcells (MSC)demonstrateatypicalfibroblast-likephenotype.Scalebars,400mM(b)Representativeimagesofthecolony-forming-units(CFU)assayafterstainingwithCrystal Violet;cAT-MSCs,10132colonies;cBM-MSCs,11149coloniesper1000platedcells.

Fig.2. Surfaceandchondrogenicmarkerexpressionincaninebonemarrow(BM)-andadiposetissue(AT)-derivedmesenchymalstromalcells(MSC)inpassage2.(a)Mean standarddeviation(SD)ofthepercentageofcellspositiveforCDmarkersincBM-MSCs(blackbars)andcAT-MSCs(greybars),assessedusingFACSanalysis.(b)MeanSDof thenormalisedrelativeexpressionofCDmarkersinthebonemarrow(triangle),cBM-MSCs(circles),andcAT-MSCs(squares)comparedtothemeangeneexpression(dotted line).(c)MeanSDofthenormalisedrelativeexpressionofACAN,COL2A1,SOX9,andCOL1A1ofcBM-MSCs(circles)andcAT-MSCs(squares)comparedtothemeangene expression(dottedline).Individualdonorsareshownindifferentcolours.*,Significantdifferencebetweencelltypeswithinageneormarker(*P<0.05;**P<0.01).s, DifferencebetweenthetwocelltypeswithP<0.1andaneffectsize>0.8.

Fig.3.Biochemical,histological,andRT-qPCRanalysesofcaninebonemarrow(cBM,circles)-andcanineadiposetissue(cAT,squares)-derivedmesenchymalstromalcells (MSC),differentiatedtowardsthechondrogeniclineagefor35daysinpelletculturewithorwithout10ng/mLTGF-β1and10ng/mLBMP-6.(a)Biochemicalanalysisofthe glycosaminoglycan(GAG)content,DNAcontent,andGAGcontentcorrectedforDNAcontent(meanstandarddeviation(SD)).(b)MeanSDofthenormalisedrelative expressionofACAN,COL2A1,SOX9,COL1A1,andTGFBR1comparedtothemeanexpressionofthecontrolsamplesofbothcelltypes(dottedline).Individualdonorsareshownin differentcolours.##,Significantdifferenceinbothcelltypesinthisconditionvs.thecontrolgroup(P<0.01);**,significantdifferenceinthecBM-MSCgroupcomparedtothat inthecBM-MSCcontrolgroupandthecorrespondingcAT-MSCgroup;P<0.01.(c)Histologicalanalysisofdepositedglycosaminoglycans(GAG),usingSafraninO/FastGreen andToluidineBlue,andimmunohistochemicalanalysisofcollagentypeIandcollagentypeII.Scalebars,200mm.

thatincBM-MSCsinthepresenceof250ng/mLBMP-6(P=0.049). COL10A1expressionwasnotdetectedunderanyconditionorwith anydonor.

After 21days, GAG content and GAG/DNA increased signi fi-cantlyonlyafteradditionof250ng/mLBMP-2tocBM-MSCs(P< 0.01)andcAT-MSCs(P=0.07;Fig.4b).BMP-2(100ng/mL)induced chondrogenesisonly in a subsetof thecBM-MSCand cAT-MSC donors.GAGproductionanddepositionwasnotobservedafterthe additionofTGF-β1or10ng/mLBMP-2/-6.Uponsupplementation with100or250ng/mLBMP-6,chondrogenesiswaslimitedtoonly one cBM-MSCdonor. Irrespective of the growth factor supple-mented, cBM-MSCs deposited more GAGsthan cAT-MSCs. DNA contentsignificantlyincreaseduponadditionof100and250ng/ mLBMP-2(P<0.01),demonstratedbytheincreaseinpelletsize (Fig.4c).PositiveSafraninO,ToluidineBlue,andCOL-IIstaining wasobservedincBM-MSCpelletsfromdonorsthatdemonstrated GAGdepositionatthebiochemicallevel(Fig.4c),whileCOL-IIwas absentinallcAT-MSCs(Fig.4c).COL-Iwasdepositedinpelletsofall donorsaftergrowthfactorstimulation(Fig.4c).

Discussion

AT-MSCsarecommonlyusedindogsfortreatingOA(Hoffman and Dow, 2016; Gugjoo et al., 2019) owing to their immune-modulatory and regenerative abilities. However,their chondro-genic potential remains unclear. Here, wefurthercharacterised and comparedthesurfacemarkerexpressionandchondrogenic potential of cAT-MSCs with those of cBM-MSCs, a reference standard.

Adonor-matchedcomparisonofMSC-relatedsurfacemarkers demonstratedthattheexpressionpatternsofcBM-MSCsand cAT-MSCs were similar at passage 2. Differential expression was observedforCD73andCD271,bothpoorlyexpressedincAT-MSCs. CD73 positivity in hAT-MSCs has been associated with high chondrogeniccapacity andlowosteogenic capacity(Radaetal., 2011).CD271isconsideredahighlyselectivesurfacemarkerfor hBM-MSCs; CD271+ _{cells are considered to possess higher}

chondrogenic potential(Lvet al.,2014;Somozaet al.,2014;Lu

etal.,2020).TheoverallCD271+_{frequencywaslowerinbothcell}

typesthanthefrequenciesreportedinhumans(Lvetal.,2014).The cAT-MSCpopulationcontainsa relativelylowerfractionofcells withchondrogenicpotential, which possiblycontributed tothe poorchondrogenesisobservedinthisstudy.However,CDmarker expressionvariesbetweenreportsandspecies(Uderetal.,2018); moreimportantly,markerprofileschangeduringpassaging(Bara et al., 2014), thereby rendering their predictive capacity of chondrogenesisdebatable.Forexample,CD34expressionishighly affectedbycellculture,which,whilefoundtobehighincAT-MSCs immediately after isolation, disappearsafter culture(Lin et al., 2012).Furthermore,althoughCD34expressionhasbeenreported incAT-MSCs(Russelletal.,2016;Kriston-Páletal.,2017),ourstudy andstudiesreportedbyothers(Ivanovskaetal.,2017)havenot beenabletodetectit.InterpretationoftheCDpro_fileswasfurther complicatedbythepreviousfindingthat FACS-sorted subpopu-lationsofMSCshad similarCD markerexpressionprofilesafter culturing,whileretainingtheirfunctionaldifferences( Sivasubra-maniyan et al., 2018). In this complicated landscape, surface marker characterisation in the veterinary field is further chal-lenged by the lack of species-specific antibodies. Although RT-qPCRanalysisofCDmarkerscanbeused,discrepanciesbetween mRNAandproteinexpressionshouldbeconsidered.Forexample, thecanineMSCswerereportedlypositiveforCD44,whilethegene expressionlevelofthisCDmarkerwaslow.Altogether,thisimplies thattheapplicabilityofthemarkersproposedbytheInternational SocietyforCellularTherapy(ISCT;Dominicietal.,2006)indogs remainstobedetermined.AlthoughCDmarkerexpressionwas largelysimilar,higherexpressionofACANandSOX9inpassage2 undifferentiated cBM-MSCs was detected. We speculated that thesedifferencesingeneexpressionwereobservedbecause cBM-MSCsweremoreprimedtowardchondrogenesisthancAT-MSCs, which was influenced by thetissue origin (Rasi Ghaemiet al., 2013).However,furtherinvestigationregardingthechondrogenic potential of specific subpopulations within cBM-MSCs or cAT-MSCsiswarranted.

Ideally, upon chondrogenic differentiation, cMSCs should primarily produce COL-II rather than COL-I. Our results

Fig.4. Biochemical,histological,andRT-qPCRanalysesofthechondrogenicdifferentiationwithorwithoutdifferentdosesofbonemorphogeneticprotein(BMP)-2orBMP-6 ofcaninebonemarrow(cBM,circles)-andcanineadiposetissue(cAT,squares)-derivedmesenchymalstromalcells(MSC).(a)Meanstandarddeviation(SD)ofthe normalisedrelativeexpressionofACAN,COL2A1,SOX9,COL1A1,andTGFBR1ofcBM-MSCsandcAT-MSCsdifferentiatedtowardsthechondrogeniclineagefor7daysinpellet culturewithorwithoutTGF-β1and10,100,or250ng/mLBMP-2orBMP-6.Geneexpressionhasbeenpresentedastheexpressioncomparedtothemeangeneexpressionof thecontrolsamplesofbothcelltypes(dottedline).Individualdonorsareshownindifferentcolours.#,significantdifferencebetweenallcelltypesinthisconditionandthe controlgroup(#,P<0.05;##,P<0.01);*,significantdifferencevs.thecontrolgroup,butonlyinthiscelltype(*,P<0.05;**,P<0.01);$,significantdifferencevs.thegroup treatedwithonlyTGF-β1forcBM-MSCs(COL1a1,TGFβR1),cAT-MSCs(ACAN),orindependentofcelltype(DNAcontent;P<0.05);&,significantdifferencebetweencelltypes withinthesamecondition(P<0.05).(b)Biochemicalanalysisoftheglycosaminoglycan(GAG)content,DNAcontent,andGAGcontentcorrectedforDNAcontent(meanSD) ofcBM-MSCandcAT-MSC,differentiatedtowardthechondrogeniclineagefor21daysinpelletculturewithorwithoutTGF-β1and10,100,or250ng/mLBMP-2orBMP-6.(c) Histologicalanalysisofglycosaminoglycans(GAG),usingSafraninO/FastGreenandToluidineBlue,andimmunohistochemicalanalysisofcollagenIandcollagenII.

demonstratedthatcBM-MSCsproducedbothcollagens,with COL-II being primarily produced under effective chondrogenic con-ditions,whichwasinagreementwiththeobservationsreportedby otherstudies(Hodgkiss-Geereetal.,2012),whilecAT-MSCsonly deposited COL-Iunder the same culture conditions. Contrarily, COL-II depositionincAT-MSCpelletshasbeendemonstratedby others(Neupaneetal.,2008;Vieiraetal.,2010).However,afew studiesthatdirectlycomparedcBM-andcAT-MSCsreportedthat cAT-MSCsexpressedlessCOL2A1(Reichetal.,2012)andexhibited limitedCOL-IIand ToluidineBluestaining(Beardenetal.,2017) withoutBMPsupplementation.COL-IdepositionbybothcMSCsis not remarkable, as TGF-_β1 is known to exert _fibrotic effects (Cutroneo, 2007). In the absence of sufficient chondrogenic stimuli, cMSCsproduceamorefibrousinsteadofhyaline-based cartilaginousmatrix, explainingtheincreaseinpelletsizeinall TGF-β1-stimulated pellets. Furthermore, within the course of chondrogenic differentiation, hMSCs follow the endochondral ossificationpathway,therebyexpressingmarkersofhypertrophy (COL-X,ALP,andMMP13;Pelttarietal.,2008).Inthisstudy,COL-X wasundetectableatthegeneandproteinlevels.WhethercMSCs eventually also undergohypertrophic differentiation when cul-turedforextendedperiodsoftimeorwhetheradditionalstimuli arenecessaryremainstobedetermined.

ThechondrogenicpotentialofhumanAT-MSCsmaydependon growthfactorsotherthanTGF-β1(Hennigetal.,2007).However, cAT-MSCsdidnotundergochondrogenesiswiththeadditionof10 ng/mLBMP-6,norwasthereCOL-IIdepositionwithhigherdosesof BMP-2/-6.Additionally,TGFβR1,whichwasreportedtorestorethe chondrogenic potential of hAT-MSCs, was significantly down-regulatedincAT-MSCssupplementedwithhighdosesofBMP.This downregulationisprobablytheresultofnegativefeedbackloops initiatedbythehighBMPconcentrationthatactatthereceptor level(Yanetal.,2018).Thediscrepancyinoutcomebetweenthis studyandotherstudiesmightbeduetocultureprotocoland/or speciesdifferences(Martínez-Lorenzoetal.,2009).Incontrastto sheep or human chondrocytes, canine chondrocytes lose their abilitytore-differentiateunderchondrogenicinductionafterfew passages(Giannonietal.,2005).Alternativegrowthfactorsmight thereforeberequiredtorestorethechondrogenicpotentialof cAT-MSCs(BoeufandRichter,2010).It isalsopossiblethat environ-mental factors arising during culture, such as mechanical stimulation, oxygen tension, and nutritional supplementation, mayexplainwhytheresultsofthisstudydifferfromthoseofother studies(Henniget al.,2007; Neupaneetal.,2008;Vieiraetal., 2010),andtheireffectoncAT-MSCsshouldbefurtherinvestigated. Conclusions

Thisstudydemonstratedthatthechondrogeniccapacityof cAT-MSCs under standardculture conditionswas inferiortothat of cBM-MSCs,asdemonstratedbythedepositionofGAGsandCOL-II, and that chondrogenic capacity could not be restored with differentdosesofBMP-2 orBMP-6.Furtherinvestigationofthe stimulinecessaryforthechondrogenicdifferentiationofcAT-MSCs iswarrantedbeforeconsideringcAT-MSCsforcell-basedtreatment strategiesforOA.

Conflictofintereststatement

Noneoftheauthorshasanyfinancialorpersonalrelationships thatcouldinappropriatelyinfluenceorbiasthecontentofthepaper. Acknowledgements

ThisworkwassupportedbytheAOBone(S-10-48T)andthe DutchArthritisSociety(LLP22).WethankHarryvanEngelenfor

providingassistancewiththecollectionofthetissuesamples,Ger ArkensteinforaidingwiththeFACSanalysis,andKarinBenzfor giftingBMP-2generously.Preliminaryresultswerepresentedasa poster at the first MBE (Matrix Biology Europe) conference, Rotterdam,21–24June2014.

AppendixA.Supplementarydata

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