h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Goat
umbilical
cord
cells
are
permissive
to
small
ruminant
lentivirus
infection
in
vitro
Gabrielle
R.
Martins
a,∗,
Rebeca
C.
Marinho
a,
Rosivaldo
Q.
Bezerra
Junior
a,
Antoniel
de
O.
Alves
a,
Lilia
M.C.
Câmara
b,
Luiz
C.
Albuquerque-Pinto
b,
Maria
F.
da
S.
Teixeira
aaUniversidadeEstadualdoCeará,ProgramadePós-graduac¸ãoemCiênciasVeterinárias,LaboratóriodeVirologia,Fortaleza,CE,Brazil bUniversidadeFederaldoCeará,LaboratóriodeImunologia,ProgramadePós-graduac¸ãoemMicrobiologiaMédica,Fortaleza,CE,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received6January2016 Accepted16August2016
Availableonline19November2016 AssociateEditor:JoãoPessoaAraújo Junior Keywords: CAEV MVV Cellculture MSC Flowcytometry
a
b
s
t
r
a
c
t
Smallruminantlentivirusesisolatedfromperipheralbloodleukocytesandtargetorgans canbepropagatedinvitroinfibroblastsderivedfromgoatsynovialmembranecells.These cellsareobtainedfromtissuescollectedfromembryosorfetusesandarenecessaryforthe establishmentofthefibroblastprimaryculture.Anewalternativetypeofhostcells,derived fromgoatumbilicalcord,wasisolatedandcharacterizedphenotypicallywithitsmain pur-posebeingtoobtaincellmonolayersthatcouldbeusedforthediagnosisandisolation ofsmallruminantlentivirusesincellculture.Toaccomplishthisgoal,cellswereisolated fromumbilicalcords;characterizedphenotypicallybyflowcytometryanalysis; differenti-ateintoosteogenic,chondrogenicandadipogeniclineage;andsubmittedtoviralchallenge. Theproliferationofgoatumbilicalcordcellswasfastandcellmonolayersformedafter 15days.Thesecellsexhibitedmorphology,immunophenotype,growthcharacteristics,and lineagedifferentiationpotentialsimilartomesenchymalstemcellsofotherorigins.The goatumbilicalcordderivedcellsstainedpositiveforvimentinandCD90,butnegativefor cytokeratin,CD34andCD105markers.Syncytiaandcelllysiswereobservedincell mono-layersinfectedbyCAEV-CorkandMVV-K1514,showingthatthecellsarepermissivetosmall ruminantlentivirusinfectioninvitro.Thesedatademonstratetheproliferativecompetence ofcellsderivedfromgoatumbilicalcordsandprovideasoundbasisforfutureresearchto standardizethiscelllineage.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Thesmall ruminant lentivirus (SRLV) comprises two types of viruses, the Caprine Arthritis Encephalitis virus (CAEV)
∗ Correspondingauthor.
E-mails:[email protected],[email protected](G.R.Martins).
andthe MaediVisnavirus(MVV);bothofwhicharewidely distributed throughout the world. CAEV and MVV share genetic similarities, molecular mechanisms of replication, morphologyandsimilarbiologicalinteractionsintheirhosts. These lentiviruses cause persistent infections, including
http://dx.doi.org/10.1016/j.bjm.2016.11.002
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
encephalitis,arthritis,progressivepneumonia,and mastitis ingoatsandsheep.Likeotherlentiviruses,CAEV andMVV infectcellsofthemonocyte/macrophagelineageanddendritic cells.1,2
CAEVandMVVcanreplicateinprimaryfibroblastsderived fromsynovialmembranesorfromchoroidplexuscultures,3,4 and can also replicate inthe immortalized TIGEF cell line (TImmortalizedGoatEmbryonicFibroblast).5Thesecellsare obtainedfromthetissuescollectedfromembryosorfetuses andarenecessaryfortheestablishmentofthefibroblast pri-maryculture.
Analternative methodwouldinvolve usingcellsderived fromgoat umbilicalcords,which isaninvaluabletoolthat doesnotuseembryosorfetuses.Generally,theumbilicalcord wouldbediscardedaftercalving.However,itisanexcellent sourcematerial, richincells thatare abletooriginate sev-eralcelltypes.Theumbilicalcordisawell-knownsourceof mesenchymalstemcells(MSCs),whichhavebeenisolatedand characterizedinumbilicalcordsamplesfromcanine,6equine7 andovine8species.MSCsareamultipotentadultstemcell.9
UndifferentiatedMSCsexhibitfibroblast-likemorphology andarecharacterizedphenotypicallybytheexpressionof sur-facemarkers;however,thecharacterizationofthesecellshas notyetbeenfully defined.Thereareseveralpositive mark-ersdescribed,andthereisaconsensusthatundifferentiated MSCsshouldbepositiveforCD29,CD44,CD90,CD105,CD73 and negativeforCD34, CD45,CD14and CD3.10,11 MSCs are multipotentstromalcells capableofdifferentiatingto mes-enchymallineages,includingtissuessuchasadiposetissue, bone,cartilageandmuscle.9,12
Theaimsoftheexperimentsinthisstudyweretoisolate, phenotypically characterizeand investigatethe differentia-tionpotentialsofcellsfromgoatumbilicalcords(cGUCs),with themainpurposebeingtoobtaincellmonolayersforusein thediagnosisofsmallruminantlentivirusesviaisolationin cellculture.
Materials
and
methods
Samples
AllexperimentswereapprovedbytheEthicsCommitteefor AnimalUseattheStateUniversity ofCeará,protocol num-ber127.769.79-0.Forthisstudy,mongrelpregnantgoatswere used,agedbetweentwoandthreeyearsandfreeofinfection bySRLV.
Threeumbilicalcordsampleswerecollectedduring calv-ing. Afterbirth, the goat umbilicalcords were clamped at both ends, i.e., next to the fetus and the goat, and then cut and packed in the transport medium. The solutions usedduring transportwere low glucose DMEM(Dulbecco’s Modified Eagle Medium) (Gibco®, Grand Island, NY, USA), MEM-E(MinimumEssentialMediumwithEarle’ssaltand l-glutamine)(Gibco®,GrandIsland,NY,USA),PBS(phosphate bufferedsaline)andsalinesolution0.9%(SS),supplemented with300U/mLpenicillin,300g/mLstreptomycin,0.5mg/mL amphotericinand 10% FBS. The sampleswere transported within 2h to the laboratory, packed in an isothermal box withice.
IsolationandcultureofcGUCs
The tissuesampleswere processed ina laminar flow cab-inet. The umbilical cord was washed with PBS to remove blood. The amniotic epithelium surrounding the umbilical cordwasdissociatedusingsterilescissorsandforceps. Tis-suesamplesofumbilicalcordmatrixwerecutintopiecesof 0.5–1cm(explants)andwereplatedinA25cellcultureflasks and6-wellcultureplatesusingDMEM-lowglucoseorMEM-E supplemented with300U/mL penicillin, 300g/mL strepto-mycin,0.5mg/mLamphotericinand10%FBSandculturedat 37◦CinaCO2incubator(5%CO2).
Thecellcultureswereobservedinaninvertedmicroscope everyday,untilproliferationofthefirstcellsfromtheexplants hadoccurred.Themediawaschangedafter5daystoavoidany mechanical stress,andthereafter, it wasreplenished every third day.At 80%confluence, cells were trypsinized (0.25% Trypsin-EDTA,Gibco®,GrandIsland,NY,USA)andreseeded innewcellcultureflasks.
Virusinfectivityassay
Thecellsfromtheconfluentmonolayerafterthethird pas-sagewereinfectedwitheithertheCAEV-CorkorMVV-K1514 strains,providedbyDr.RobertoSoaresdeCastro(FederalRural UniversityofPernambuco–UFRPE).Thesestrainsoriginated fromtheInstitutNationaldelaRechercheAgronomique, Lyon-France.Thevirusinfectivityassaywasperformedinduplicate foreachofthreeseparateexperiments.Thecellswere incu-batedwithviralsuspension105TCID50ataMOIof1for1h
at37◦C ina5%CO2 incubator.Thereafter,thesupernatant
wasdiscardedandthegrowthmediumwasadded(DMEM sup-plementedwith300U/mLpenicillin,300g/mLstreptomycin, 0.5mg/mLamphotericinand2%FBS).
Thecellmonolayerswereobservedinaninverted micro-scopeeverydayuntilthecytopathiceffectcausedbythevirus wasevident,provingthatthecellswerepermissivefor infec-tionbySRLV.
FACSanalysis
FACS analysiswas performedtoinvestigate theexpression ofthesurfacemarkersCD90,CD105,CD34,andthe intracel-lular markersvimentinand cytokeratin incGUCs afterthe thirdpassage.Thecells were harvested andaliquotedata density of 106cells/mL for each marker assay. First, these
cellswereincubatedwithantibodiesagainstsurfacemarkers [mouseanti-HumanCD90FITC,CD105APCandCD34APC(BD Biosciences®,USA)]for30minat4◦Cinthedark.Then,the cellswerepermeabilizedwithfixation/permeabilization solu-tion (BDCytofix/CytopermFixation/PermeabilizationKit,BD Biosciences®,USA)for20minat4◦Cinthedark. Permeabili-zedcellswereincubatedwithantibodiesagainstintracellular markers[mouseanti-HumanvimentinPEandcytokeratinPE (BDBiosciences®,USA)]for30minat4◦Cinthedark. There-after,thecellswerewashedtwicewithBDperm/washbuffer (BDBiosciences®,USA),fixedwithformaldehydebuffer(PBS with1%formaldehyde)andanalyzedusingaflowcytometer (FACSCaliburBD,BDBiosciences®).Agatewasdefinedand
theminimumnumberofeventsconstitutingwassetto10,000. Thenegativecontrolwasprocessedinasimilarmannerbut withoutantibodies.ThedataobtainedwasanalyzedusingCell QuestProsoftwareandplottedasahistogram.
Adipogenicdifferentiation
For adipogenic differentiation, trypsinized cells (1×104cells/cm2) were seeded into a 24-well plate and
incubated with DMEM supplemented with 300U/mL peni-cillin,300g/mLstreptomycin,0.5mg/mLamphotericinand 10%FBS.After24h,themediumwasdiscarded,andthecells wereincubatedwithStemPro® AdipogenesisDifferentiation BasalMedium(Gibco®,GrandIsland,NY,USA).The differen-tiationmediumwas replacedevery3–4 days.After14days ofculture,the cells were fixedwith 10% formalinatroom temperature,washedwithPBS,andstainedwith0.5%Oil-Red O(Sigma–Aldrich®,St.Louis,MO,USA)for15minandMayer’s Hematoxylin(Sigma–Aldrich®,St.Louis,MO,USA)for5min tovisualizelipiddroplets.
Chondrogenicdifferentiation
Chondrogenicdifferentiationwasinducedinmicromass cul-turesbyseeding5Ldropletsofcellsolution(1.6×107viable
cells/mL)inthecenterofa24-wellplate.Thecellswere incu-bated with DMEM supplemented with 300U/mL penicillin, 300g/mL streptomycin, 0.5mg/mL amphotericin and 10% FBSfor2h.Afterward,thecellswerefedwithaspecific chon-drogenesisdifferentiationmedium(StemPro®,Gibco®,Grand Island,NY,USA).Themediumwasreplacedevery2–3days. After14days,chondrogenicdifferentiationwasconfirmedby stainingforAlcianBlue(Sigma–Aldrich®,St.Louis,MO,USA). Osteogenicdifferentiation
To induce osteogenic differentiation, the cells from the confluentmonolayerofthe thirdpassage(5×103cells/cm2)
wereseededintoa24-wellplateandincubatedwithDMEM supplementedwith 300U/mL penicillin, 300g/mL strepto-mycin,0.5mg/mLamphotericinand10%FBS.After24h,the mediumwasdiscarded,andcellswereculturedinStemPro® OsteocyteDifferentiationBasalMedium(StemPro®, Gibco®, GrandIsland,NY,USA).Themediumwasreplacedevery2–3 days. After 14 days of culture, the osteogenic differentia-tionofstemcells wasconfirmedbyAlizarinRedSstaining (Sigma–Aldrich®,St.Louis,MO,USA).
Results
Growthandculturecharacteristics
Goatumbilicalcordcells startedtomigrateoutofexplants after3–6days ofculture,and thecells graduallygrew into small colonies. In the samples transported in DMEM and MEM-E,cellproliferation started3daysafterbeginningthe incubation,whereassamplestransportedinPBSstarted pro-liferatingafter5daysandthesamplespackedinSSafter6 days.ThesedatademonstratedthatDMEMandMEM-Eenable
betterpreservationofthesamplesand,consequently,enable betteracquisitionofcells.
Duringtheinitialdaysofincubation,cellsdisplayeda non-fibroblastic,rounded,epithelialcell-likephenotype(Fig.1A). Later,cells attained atypicalfibroblast-likespindle-shaped structure (Fig. 1B). As growth continued, adjacent colonies interconnectedwitheach other,andaconfluentmonolayer was obtained(Fig. 1C). Cell monolayers derived from goat umbilical cords were used in viral challenge assays, for immunotypingbyflowcytometry,andinmesodermallineage differentiationassays.
Thegrowthmediausedinthisstudy(DMEMorMEM-E sup-plementedwith10%FBS)werehighlysuitabletosustaining thecells,resultinginhighratesofgrowthandexpansionof thecellstock.After15days,cellshadgrownto80% conflu-ence,atwhichtimethefirstpassagewasperformed.Inlater passages,culturedcellsreached80–90%confluencefaster,in approximately3–4days.
CytopathicityofSRLVincGUCs
ThecGUCsarehighlypermissivetoSRLVinfectioninvitro.The cytopathiceffectsappeared7daysafterinoculation. Classi-calgiantmultinucleatedcells(syncytia)wereobservedinthe monolayersinfectedwithCAEV-Corkvirus(Fig.2B).Thecells infectedwithMVV-K1514virusunderwentsyncytiaformation andcelllysis.Thecytopathiceffectsprogressed,resultingin the progressivedestruction of thecell monolayer (Fig.2A). Thenegativecontrolfortheviralinfectivityassayisshown inFig.2C.
Immunophenotypiccharacterizationbyflowcytometry
Invitrocultured,third-passagecGUCswereanalyzedbyFACS. FACSanalysisrevealedthatthesecellswerepositiveforthe expression of vimentin (91.6%) and CD90 (23%) but were negativewhenstainingforCD105(3.66%),CD34(2.95%)and cytokeratin(1.67%)(Fig.3).
Mesodermallineagedifferentiation
Adipogenic differentiation of cGUCs was confirmed by Oil Red-Ostaining.Afterincubatingthesecellswith adipogenic-inducing medium for 14 days, small lipid droplets were presentinthecytoplasm(Fig.4A).
The chondrogenic potential ofcGUCs was evaluated by invitromicromasscultureofthesecellsinaspecific differenti-ationmedium.After14daysofincubation,theaccumulation ofsulfatedproteoglycanswasvisualizedbyAlcianBlue stain-ing(Fig.4B).
In the osteogenic differentiation assay, the cells prolif-erated andreachedcomplete confluenceafter8–10daysof incubationwithdifferentiationmedium.Thecellular aggre-gateswerethenobservedandwerecharacterizedbycalcium deposits,whichweredemonstratedbypositiveAlizarinRed staining(Fig.4C).
Fig.1–PrimarycultureofcGUCs:morphologicalfeaturesandcellmonolayersformed.(A)Epithelialcell-likephenotype.(B) Typicalfibroblastoidshape.(C)cGUCsexhibitingalarge,flattenedfibroblast-likemorphologyafterthethirdpassage.
Discussion
Theumbilicalcordiscomposedofseveraldifferent compo-nents(amnioticmembrane,umbilicalcordmatrix,umbilical cord vein, and umbilical cord blood) that can be used as sourcesofMSCs.6 In this study,cGUCs were isolated from the umbilical cord matrix and were cultured in DMEM or MEM supplemented with 10% FBS. These conditions were foundtobeverywellsuitedforthemaintenanceofthe cul-turedcells,andcontributedtoquickamplificationofthecell stock.Severalculturemediahavebeentestedforthe main-tenanceofMSCs,suchasMEM,DMEM,RPMI-1640andBasal MediumEagle(BME),supplementedwithFBS,generallyata concentrationof10–20%.Thechoiceoftheculturemediumis importantforthesuccessfulgrowthofMSCsduringinvitrocell culture.13
During the initialdays of incubation, the population of cells washeterogeneous, including bothepithelial-like and fibroblast-likecells.However,afterpassaging,theepithelioid cell population disappeared from the culture and most of thecells exhibited afibroblast-likeappearance. These data are consistent with the observations of Pratheesh et al.14 whoobservedsimilarmorphologicalfeaturesincaprine mes-enchymalstemcellsderivedfromamnioticfluid(cAF-MSCs) andalsowithRenetal.15 intheirstudy withgoat adipose-derivedstemcells(ADSCs).Inaddition,thefibroblast-likecells derivedfromgoatumbilicalcordmatrixaresimilartoMSCs intheirgeneralfeatures.
Theaimofthepresentstudywastoinvestigatethecapacity ofcellsfromgoatumbilicalcordmatrixtogeneratecell mono-layersthat could beusedforviral diagnosisin cellculture assays. Itwasdemonstratedthatthesecellsarehighly per-missivetoSRLVinfectioninvitro.Syncytiaandcelllysiswere observedafter7daysofinoculationwiththeCAEV-Corkstrain, andsimilarresultswereobservedwiththeMVV-K1514strain. These resultsresemble theeffects observedafter infection ofsynovialmembranecells,whicharethecellstraditionally usedforthispurpose.
High amounts ofsmall ruminant lentiviruscan be pro-ducedinvitrousingfibro-epithelialsynovialmembranecells orchoroidplexuscellsfromgoatsorsheep.16,17However,the disadvantageisthatthesetypesofcellscanonlybeobtained fromembryosorfetuses,whereasgoatumbilicalcordsoffera greatalternativeforthegenerationoffibroblastsbecausethe umbilicalcordwouldbediscardedaftercalvingandthus rep-resentsahighlyconvenientsourcematerialforgenerationof cells.
Othercelltypespermissiveforsmallruminantlentivirus infectionaremicroglia18;dendriticcells2;epithelialcellsfrom thelung19;mammarygland20;thirdeyelid21;kidney22;uterus andepididymis23,24andendothelium;smoothmyocytes19,25; granulosacells26; and parenchymacells from theliver and heart.27
ThephenotypiccharacterizationofcGUCswasperformed byflowcytometry.Thegeneralstrategyforidentifyinginvitro cultivatedmesenchymalstemcellswasexecutedasperthe ISCTrecommendation(InternationalSocietyforCytotherapy),
Fig.2–CytopathiceffectscausedbyMVVandCAEV.(A)CelllysisandsyncytiacausedbyMVV.(B)Giantmultinucleated cellscausedbyCAEV.(C)Negativecontrolfortheviralinfectivityassay.
80 70 60 50 40 30 20 10 0 100 101 102 103 104 100 101 102 CD90 FITC Vimentin PE
A
B
Counts Counts 103 104 0 20 40 60 80 100Fig.3–FACSanalysisofVimentinandCD90,invitroculture,thirdpassagecGUCs.(A)VimentinPE.(B)CD90FITC.Calibrated histogramrepresentingthenumberofeventsontheY-axisandfluorescentintensityontheX-axis.Theredareahistogram indicatenegativecontrol.
whichis toanalyze the expressionofcell-surface markers suchasCD-73,CD-44,CD-90andCD-105.28–31Itwas demon-stratedthatthecGUCsare positiveforvimentinandCD90, whereastheywerenegativeforcytokeratin,CD105andCD34. Theseresultscanbecomparedwiththoseofotherstudies. Renetal.15describedMSCsfromgoatadiposethatstained positivelyforvimentin,CD49dandCD13andnegativelyfor CD34andCD106.Caprine mesenchymalstem cells derived fromamnioticfluidwerepositiveforCD90,CD105,CD73,and negativeforCD34.14
Vimentinispresentinnormalandpathological mesenchy-maltissuesand isanimportantmarkerofthe mesoderm. Positive vimentin staining verified that the cGUCs were derivedfrommesodermalstemcells.32Cytokeratin-negative staining demonstrated the predominance offibroblast-like cellsandisconsistentwiththeobservationthatthe epithelial-likecells disappearedfrom cultureand couldno longer be foundfromthirdpassageonward.Additionally, mostofthe cellsexhibitedafibroblast-likeappearance.CD34isasurface markerofhematopoieticstemcells(HSCs)andisexpressed inlymphnodes,bonemarrowHSCs,andvariousendothelial
cells.CD34-negativestainingdemonstratedthatcGUCswere notderivedfromcirculatingstemcells.9,33
The cGUCs from umbilical cordmatrix are negativefor CD105. This marker is usually expressed in mesenchymal stemcells;however,thereisnotyetadefinitivemarkerfor MSCs.Therearealargenumberofpositivemarkersdescribed, buteachgroupofinvestigatorsusesdifferentmarkers,none ofwhicharespecificorusedsingly.Perhapsthedifferences between studies may beattributedto variationsinculture methodsorthestageofcelldifferentiation.34
In this study, FACS analysis revealed low levels of CD90 marker expression (23%) incomparison to Vimentin (91.6%). CD90 is known to be a negative regulator of hematopoieticproliferation.35 Itsexpression, inassociation with CD34-negative staining, confirms that there was no evidence of hematopoietic precursors in this culture. The expression levels of surface markers are also different in other studies. Chang et al.36 isolated cells from umbilical cord blood (UCB) and observed the following two differ-ent morphologic phenotypes: flattened fibroblastic clones andspindle-shapedfibroblasticclones.CD90wasexpressed
Fig.4–Mesodermallineagedifferentiation.Adipogenicdifferentiation(A).Chondrogenicdifferentiation(B).Osteogenic differentiation(C).
differently by these two cell populations. Spindle-shaped, clonogenicMPCsexpressedahighlevelofCD90,while flat-tened, clonogenic MSCs did not expressCD90. These data mightexplaintheinconsistentresultsregardingCD90 expres-sioninUCB-derivedMSCsinvariousreports.37–39
The differentiation potential of cGUCs was investi-gated. These cells showed a capacity to differentiate into adipocytes,chondrocytesorosteoblastswhenincubatedwith the appropriate differentiation medium. Pratheesh et al.14 demonstrated thatMSCs from goatamniotic fluid, suchas their marrow counterparts, were able to differentiate into adipose cells in addition to differentiating into osteogenic andchondrogeniclineages.Goatadipose-derivedstemcells showed the same differentiation potential.15 Goat-derived multipotentMSCshavebeenestablishedfrombonemarrow andsuccessfullyinducedtodifferentiateintoosteogenicand adipogeniclineagesunderspecificcultureconditions.40
The present study establishes that goat umbilical cord cellsare permissiveforsmallruminant lentivirusinfection in vitro, can be successfully used in cell culture diagnosis, andrepresentanewalternativesourceofhostcellsforvirus isolation. These cells exhibit a fibroblast morphology, and immunophenotype,growthcharacteristics andmesodermal lineagedifferentiationpotentialthatareallsimilartoMSCs. ThesecGUCsalsoexpressvimentinandCD90butarenegative forCD34.Thesedatademonstratetheproliferativecapacity ofcellsfromgoatumbilicalcords,andtheyestablishasound basisforfutureresearchaimedatstandardizingthecell lin-eageandusingthesecellsforthediagnosisofseveraldiseases.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Thiswork wassupportedbytheNational Councilof Scien-tificandTechnologicalDevelopment(CNPq),grantnumbers 487425/2012-0and308995/2013-9,andbyPersonnel Improve-mentCoordinationhigherlevel(CAPES),whichprovidedaPhD scholarship.Theauthorsare thankful tothePost Graduate PrograminVeterinaryScience(PPGCV)oftheStateUniversity ofCearáfor supporting the implementation ofthe experi-ments;totheFederalUniversityofCeará,particularlytoLilia Maria CarneiroCâmara, PhD, for unconditional support in performingtheFACSanalysis;andtoMarcosFábioGadelha Rocha, PhD, and Júlio Sidrim, PhD (Post Graduate Medical Microbiology–UFC).Theauthorsalsoexpresstheirgratitude toownersofthefarmsthatallowedtheuseoftheiranimals forthisstudy.
r
e
f
e
r
e
n
c
e
s
1. NarayanO,ClementsJE.Biologyandpathogenesisof
lentiviruses.JGenVirol.1989;70:1617–1639.
2.RyanS,TileyL,McConnellI,BlacklawsB.Infectionof
dendriticcellsbytheMaedi–Visnalentivirus.JVirol.
2000;74:10096–10103.
3.CheblouneY,ShefferD,KarrBM,StephensE,NarayanO.
Restrictivetypeofreplicationofovine/caprinelentivirusesin
ovinefibroblastcellcultures.Virology.1996;222:21–30.
4.SihvonenL,VeijalainenP.KineticsofMaedivirusproduction
insheepchoroidplexuscells.VetMicrobiol.1981;6:1–8.
5.TeixeiraMFS,VeroniqueL,Msebli-LakahlL,ChettabA,
CheblouneY,MornexJF.Imortalizationofcaprinefibroblasts
permissiveforreplicationofsmallruminantlentiviroses.Am
JVetRes.1997;58:579–584.
6.LeeKS,NahJJ,LeeBC,etal.Maintenanceand
characterizationofmultipotentmesenchymalstemcells
isolatedfromcanineumbilicalcordmatrixbycollagenase
digestion.ResVetSci.2013;94:144–151.
7.ReedSA,JohnsonSE.Refinementofcultureconditionsfor
maintenanceofundifferentiatedequineumbilicalcord
bloodstemcells.JEquineVetSci.2012;32:360–366.
8.FadelL,VianaBR,FeitosaMLT,etal.Protocolsfor
obtainmentandisolationoftwomesenchymalstemcell
sourcesinsheep.ActaCirBrasil.2011;26:267–273.
9.PittengerMF,MackayAM,BeckSC,etal.Multilineage
potentialofadulthumanmesenchymalstemcells.Science.
1999;284:143–147.
10.KernS,EichlerH,StoeveJ,KlüterH,BiebackK.Comparative
analysisofmesenchymalstemcellsfrombonemarrow,
umbilicalcordblood,oradiposetissue.StemCells.
2006;24:1294–1301.
11.RebelattoCK,AguiarAM,MoretãoMP,etal.Dissimilar
differentiationofmesenchymalstemcellsfrombone
marrow,umbilicalcordblood,andadiposetissue.ExpBiol
Med(Maywood).2008;233:901–913.
12.ThiemermannC.Mesenchymalstromalcells:current
understandingandclinicalstatus.StemCells.
2010;31:595–596.
13.TappH,HanleyEN,PattJC,GruberHE.Adipose-derivedstem
cells:characterizationandcurrentapplicationin
orthopaedictissuerepair.ExpBiolMed.2009;234:1–9.
14.PratheeshMD,GadeNE,KatiyarAN,etal.Isolation,culture
andcharacterizationofcaprinemesenchymalstemcells
derivedfromamnioticfluid.ResVetSci.2013;94:313–319.
15.RenY,WuH,ZhouX,etal.Isolation,expansion,and
differentiationofgoatadipose-derivedstemcells.ResVetSci.
2012;93:404–411.
16.SigurdssonB,ThormarH,PálssonP.Cultivationofvisna
virusintissueculture.ArchivfürdiegesamteVirusforschung.
1960;10:368–381.
17.CrawfordTB,AdamsS,SandeRD,GorhanJR,HensonJB.The
connectivetissuecomponentofthecaprinearthritis–
encephalitissyndrome.AmJPathol.1980;100:443–454.
18.AdebayoIA,OlaleyeOD,AwoniyiTA.Affinity(tropism)of
caprinearthritisencephalitisvirusforbraincells.AfrJMed
MedSci.2008;39:89–93.
19.CarrozzaML,MazzeiM,BandecchiP,ArispiciM,TolariF.
InsituPCR-associatedimmunohistochemistryidentifiescell
typesharbouringtheMaedi–Visnavirusgenomeintissue
sectionsofsheepinfectednaturally.JVirolMethods.
2003;107:121–127.
20.BoleaR,MonleonE,CarrascoL,etal.Maedi–Visnavirus
infectionofovinemammaryepithelialcells.VetRes.
2006;37:133–144.
21.CapucchioMT,SannaE,SannaMP,FariguS,MinelliR,
GuardaF.Maedi–Visnavirusdetectioninovinethirdeyelids.
JCompPathol.2003;129:37–43.
22.AngelopoulouK,BrellouGD,VlemmasI.Detectionof
Maedi–Visnavirusinthekidneysofnaturallyinfectedsheep.
23.AliAlAhmadMZ,DubreilL,ChatagnonG,etal.Goatuterine
epithelialcellsaresusceptibletoinfectionwithcaprine
arthritisencephalitisvirus(CAEV)invivo.VetRes.2012;43:5.
24.LamaraA,FieniF,ChatagnonG,LarratM,DubreilL,
CheblouneY.Caprinearthritisencephalitisvirus(CAEV)
replicatesproductivelyinculturedepididymalcellsfrom
goats.CompImmunolMicrobiolInfectDis.2013;36:397–404.
25.LerouxC,CordierG,MercierI,etal.Ovineaorticsmooth
musclecellsallowthereplicationofVisna–Maedivirus
invitro.ArchVirol.1995;140:1–11.
26.LamaraA,FieniF,Mselli-LakhalL,TainturierD,CheblouneY.
Efficientreplicationofcaprinearthritis-encephalitisvirusin
goatgranulosacells.VirusRes.2001;79:165–172.
27.BrellouGD,AngelopoulouK,PoutahidisT,VlemmasI.
DetectionofMaedi–VisnaVirusintheliverandheartof
naturallyinfectedsheep.JCompPathol.2007;136:27–35.
28.DominiciM,LeBlancK,MuellerI,Slaper-CortenbachI,
MariniF,KrauseD.Minimalcriteriafordefiningmultipotent
mesenchymalstromalcells.Theinternationalsocietyfor
cellulartherapypositionstatement.Cytotherapy.
2006;4:315–317.
29.DonzelliE,Salvade‘A,MimoP,etal.Mesenchymalstemcells
culturedonacollagenscaffold:Invitroosteogenic
differentiation.ArchOralBiol.2007;52:64–73.
30.YuY,YaoAH,ChenN,etal.Mesenchymalstemcells
over-expressinghepatocytegrowthfactorimprove
small-for-sizelivergraftsregeneration.MolTher.
2007;15:1382–1389.
31.DeMacedoBragaLM,LacchiniS,SchaanBD,etal.Insitu
deliveryofbonemarrowcellsandmesenchymalstemcells
improvescardiovascularfunctioninhypertensiverats
submittedtomyocardialinfarction.JBiomedSci.
2008;15:365–374.
32.WagnerW,WeinF,SeckingerA,etal.Comparative
characteristicsofmesenchymalstemcellsfromhuman
bonemarrow,adiposetissue,andumbilicalcordblood.Exp
Hematol.2005;33:1402–1416.
33.PengH,HuardJ.Muscle-derivedstemcellsfor
musculoskeletaltissueregenerationandrepair.Transpl
Immunol.2004;12:311–319.
34.BydlowskiSP,DebesAA,MaselliLMF,JanzFLL.
Característicasbiológicasdascélulas-troncomesenquimais.
RevBrasHematolHemoter.2009;31:25–35.
35.MayaniH,LansdropPM.Thy-1expressionislinkedto
functionalpropertiesofprimitivehematopoieticprogenitor
cellsfromhumanumbilicalcordblood.Blood.
1994;83:2410–2417.
36.ChangY-J,TsengC-P,HsuL-F,HsiehT-B,HwangS-M.
Characterizationoftwopopulationsofmesenchymal
progenitorcellsinumbilicalcordblood.CellBiolInt.
2006;30:495–499.
37.EricesA,CongetP,MinguellJJ.Mesenchymalprogenitorcells
inhumanumbilicalcordblood.BrJHaematol.
2000;109:235–242.
38.GoodwinHS,BickneseAR,ChienSN,BoguckiBD,QuinnCO,
WallDA.Multilineagedifferentiationactivitybycellsisolated
fromumbilicalcordblood:expressionofbone,fat,and
neuralmarkers.BiolBloodMarrowTransplant.2001;7:581–588.
39.BiebackK,KernS,KluterH,EichlerH.Criticalparametersfor
theisolationofmesenchymalstemcellsfromumbilicalcord
blood.StemCells.2004;22:625–634.
40.ZhangY,FanY,WangZ,etal.Isolation,characterization,and
genemodificationofdairygoatmesenchymalstemcells
frombonemarrow.InVitroCellDevBiolAnim.