adipose是什么意思pose在线翻译读音例句

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NeurospheresFromHumanAdiposeTissue

TransplantedIntoCulturedMouseEmbryos

canContributetoCraniofacial

Morphogenesis:APreliminaryReport

TakashiNagase,MD,PhD,*DaisukeMatsumoto,MD,

1

MikiNagase,MD,PhD,

2

KotaroYoshimura,MD,PhD,

1

TomokuniShigeura,MS,

1

MakotoInoue,PhD,

4

MamoruHasegawa,PhD,

4

MasaakiYamagishi,MD,PhD,*MasafumiMachida,MD,PhD*

Tokyo,Japan

Adipose-derivedstromalcells(ASCs)areoneofthe

mostpromisingstemcellpopulationsthatdiffer-

entiateintothemesodermalaswellasneural

study,weexaminedthe

neuraldifferentiatingpotentialofhumanASCsby

pheres

derivedfromhumanASCsexpressedNestinand

Musashi-1genes,whicharemar春风250sr官方报价 kergenesfor

esecellswerelabeled

withgreenfluorescentproteingenetransfectionby

Sendaivirusvectorandtransplantedintothehead

regionofmouseembryosusingawholeembryo

culturesystem,thesecellswereincorporatedinto

ansplanted

cellsappearedtomigratealongthesecondbran-

chialarches,implicatingsomesimilaritytothe

ghpreliminary,our

resultssupportanideathatASC-derivedneuro-

sphereshavepropertiesofneuralprogenitorsin

vitroandinvivo.

KeyWords:Adipose-derivedstromalcells,neuro-

sphere,neuralstemcells,embryo,stemcellsAdipose-derivedstromalcells(ASCs)were

originallyreportedasasubtypeofthe

mesenchymalstemcells(MSCs)isolated

fromliposuctionaspiratesdifferentiating

intothemesodermaltissuessuchasbone,cartilage,

andadiposetissue.1CharacterizationofASCshas

recentlybeenstudiedworldwidebymanygroups,

includingours.2Y4ASCsarenowregardedasoneof

themostpromisingadultstemcellsforregenerative

medicinebecausetheycanbeharvestedsafelyby

liposuction,andagoodyieldcanbeanticipated.

Advancesinstemcellresearchhaveresultedin

anovelconceptofcellularplasticityofdifferentiation

d

ASCscandifferentiateintoneuronal(andthus

ectodermal)derivatives,althoughthesecellsare

primarilymesodermal.5,6Recentreportsfurther

indicatethatstemcellswithneuralcharacteristics

canbeisolatedfromthemesodermaltissuessuchas

thedermisandtheheart.7Y9Inthesecases,thecells

wereharvestedbyaneurospheremethod,which

wasoriginallydevelopedasaculturemethodof

isolatingspheresofneuralstemcellsfromthe

embryonicandadultbrain.10Y12However,itistobe

elucidatedwhetherthismethodisalsoapplicable

forobtainingneuralstemcellsfromtheadipose

tissueorASCs.

Inthisstudy,theneurospheresexpressing

neuralstemcellmarkergeneswereobtainedfrom

transplantedthesecellsinto

mouseembryosculturedinvitrotoexaminewhether

thesecellsbehavesimilartoneuronalcellsinvivo.

49

Fromthe*ClinicalResearchCenter,NationalHospital

OrganizationMurayamaMedicalCenter,Tokyo,Japan;

1

Depart-

mentofPlasticandReconstructiveSurgery,

2

Departmentof

NephrologyandEndocrinology,UniversityofTokyoGraduate

SchoolofMedicine,Tokyo,Japan;and

4

DNAVECCorporation,

Tsukuba,Ibaraki,Japan.

i

Nagase,Head,DivisionofAdvancedMedicalResearch,Clinical

ResearchCenter,NationalHospitalOrganizationMurayama

MedicalCenter,2-37-1Gakuen,Musashimurayama-City,Tokyo

208-0011,Japan;E-mail:tnagase@

Thefirsttwoauthorscontributedequallytothiswork.

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MATERIALSANDMETHODS

IsolationofHumanASCsandNeurosphere

CellCultureASCswereisolatedfromthehumanliposuction

aspiratesasreportedpreviously.3Thesuctioned

fatwasdigestedwith0.075%collagenaseinphosphate-

bufferedsaline(PBS)for30minutesonashakerat

adipocytesandconnectivetissueswere

ellswerealso

eliminatedbytreatingwitherythrocytelysisbuffer,

atively,

ASCscouldbeisolatedfromthefluidportionsof

liposuctionaspiratesbytreatingwitherythrocytelysis

bufferanddensitygradientcentrifugationwithFicoll

(GEHealthcareBio-sciences,Piscataway,NJ).

Neurosphereculturewasperformedasdescribed

previouslywithslightmodification.12Freshlyisolated

ASCswereplatedatadensityof2107cellsin10cm

unc着组词 oateddishesandculturedintheneurosphere

culturemediumat37-Cinanatmosphereof5%CO

2

in

rospheremediumwasaDulbecco’s

ModifiedEagle’sMedium/F12(1:1)-basedmedium

supplementedwithhumanrecombinantepidermal

growthfactor(EGF,20ng/mL,PeproTech,RockyHill,

NJ),humanrecombinantbasicfibroblastgrowthfactor

(FGF,20ng/mL,KakenPharmaceutical,Tokyo,

Japan),2%B27supplement(Gibco,Carlsbad,CA),

100U/mLpenicillin,and100mg/mLstreptomycin.

Halfofthemediumwasreplacedwithafreshmedium

onthefourthtofifthday,andthepassagingwas

performedontheeighthday.

QuantitativeReal-TimeReverse-Transcriptase

PolymeraseChainReaction

TotalmRNAwasextractedusingRNeasy-minikit

(Qiagen,Hilden,Germany)fromtheneurosphere

cellsderivedfrompassageoneASCs,whichwere

preculturedinthenormalmediumcontainingM199

mediumand10%fetalbovineserum(FBS).The

preculturingwasnecessaryforreducingthecontam-

lmRNAwasalso

extractedfromthepassageoneundifferentiated

ASCsculturedinM199plus10%FBS.

Expressionsofundifferentiatedneuralstemcell

markergenesNestinandMusashi-113andadipogenic

differentiationmarkerLeptinwereanalyzedbyreal-

timequantitativereverse-transcriptionpolymerase

chainreaction(RT-PCR)usinganABIPRISM7700

Fig1Neurosp一挥而就 hereformationofadipose-derivedstromalcellsculturedinneurospheremediumfor5days(A),6days(B),

and7days(C)(magnification200).

Fig2Quantitativereal-timereverse-transcriptionpolymerasechainreactionanalysisofgeneexpressionsofneuralstem

cellmarkerNestin(A),Musashi-1(B),andadipogenicdifferentiationmarkerLeptin(C).Control=undifferentiatedadipose-

derivedstromalcells;Sphere=wereperformedintriplicate,andstandarderrorsareindicatedby

errorbars.

THEJOURNALOFCRANIOFACIALSURGERY/VOLUME18,NUMBER1January2007

50

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(AppliedBiosystems,FosterCity,CA),asreported

pressionofthetargetsequence

wasnormalizedtothatofthehousekeepinggene

riptlevelinthecontrol(undifferen-

tiatedASC)groupwasarbitrarilyexpressedas1.

TaqManchemistryandassaybydesignprimersand

probesetswereusedforhumanNestin,Musashi-1,

Leptin,primersandprobesets

werepurchasedfromAppliedBiosystems.

MouseWholeEmbryoCultureand

TransplantationofNeurosphere-LikeCells

NeurospheresderivedfromhumanASCswere

transfectedwithgreenfluorescentprotein(GFP)

geneusingtheSendaivirusvector(DNAVEC

a,Japan),asreportedpreviously.14,15

TheoriginalvectorSeV/DFlackstheFgene

encodingfusionproteinnecessaryforpenetration

ofribonucleoproteincomplexintoinfectedcells,

andisthusnontransmissibleandnonpatho-

genic.14ThemodifiedSeV/DFvectorhasaddi-

tionalmutationstoreduceitscytotoxicity,15and

weusedthemodifiedvectorinthepresentstudy.

Neurosphereswereincubatedfor1hourinthe

mediumwiththemodifiedSeV/DFcarryingthe

GFPgeneatamultiplicityofinfectionof250and

rinsedwithPBS.

Mousewholeembryoculturewasperformed

asreportedpreviously.16Y19Ninemouseembryos

atembryonicday(E)8weredissectedoutwithout

damagingyolksacs,andtheGFP-transfectedneuro-

spherecellsweretransplantedusingmicropipettes

ryos

wereculturedforapproximately40古文在线翻译 hours,and

presenceorabsenceoftheGFP-positivetransplanted

cellswasinvestigatedunderafluorescentdissecting

erimentalprocedureswereper-

formedattheUniversityofTokyounderapprovalof

theethicalcommittee.

RESULTSWefirstculturedhumanASCsintheneuro-

sphereculturemediumcontainingEGFand

hirddayofculture

offreshlypreparedASCs,thefloatingASCsstarted

toformsmallmasses(datanotshown).Theneuro-

sphere-likecellularaggregateswereclearlyobserved

onthefifthday(Fig1A).Thenumberandthesizeof

thespheresbecameincreasinglylargerwithinthe

next2days(Fig1,BandC).Thepassagingwas

performedontheeighthdaywhenthesphereswere

dissociatedandresuspendedinthenewmedium.

Thespheroidswerenewlyformedafterculturing

againforseveraldays(datanotshown),suggesting

selfrenewaloftheneurospherecells.

Tocharacterizetheneurospherecells,wenext

examinedexpressionsofneuralstemcellmarker节日古诗大全100首

NestinandMusashi-1genesandadipocytemarker

sions

ofNestinandMusashi-1geneswereremarkablyup-

regulatedintheneurospherecellscomparedwiththe

controlASCswithoutculturingintheneurosphere

medium(Fig2,AandB),suggestingcharacteristics

sely,Leptinexpression

Fig3Neuralcrest-likemigrationsofgreenfluorescent

protein(GFP)-transfected,adipose-derivedstromalcell-

derivedneurospheresgraftedintomouseembryocultured

invitro.(A)Appearancesofmouseembryosculturedfor40

hoursfromembryonicday8.(BandC)Fluorescentviews

-positiveneurospherecellswerearranged

inarow(arrows),suggestingtheirmigrationalongsecond

=500mm.

ADIPOSE-DERIVEDNEUROSPHERE/Nagaseetal

51

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wasdramaticallyreducedintheneurospheres

(Fig2C),indicatinglossofadipogenicpotential.

Toinvestigatefunctionsoftheneurospherecells

invivo,welabeledthesecellsbythemodifiedSendai

virusvectorcarryingtheGFPgeneandtransplanted

themintotheheadregionoftheE8mouseembryos.

Aftertheembryoswereculturedforapproximately

40hoursinvitro,thetransplantedGFP-positivecells

wereclearlyobservedandappearedviableinonly

GFP-positivecellswereincorporatedintothecranio-

facialregionaswellastheheartandthetrunkin

thesetwoembryos(Fig3).Notably,thetransplanted

cellswerearrangedinarowalongthesecond

branchialarch(arrowsinFig3,BandC)inaquite

similarpatterntotheneuralcrestcellsmigrating

ghnot

confirmatory,thisresultsuggestsaintriguingpossi-

bilitythatneurospherecellsderivedfromASCshave

neuralcrest-likeproperties.

DISCUSSIONASCsareprobablyoneofthemostwell-known

re

originallyreportedbyZuketal1fromtheclinical

ingtotheir

broadspectrumofdifferentiationpotential,ASCs

havebeenusedinanumberofpreclinicalanimal

studiesofinvivoregenerationofavarioustissues

suchasbone,20,21cartilage,22vessels,4,23,24soft

tissue,4bonemarrow,linical

casewasreported,inwhichacalvarialdefectwas

repairedbyASCscombinedwithscaffold.26Several

groupsreportedneuraldifferentiationofASCsin

vitro,5,6,27andKangetal28reportedfunctional

recoveryoftheratmodelwithcerebralinfarction

afterASCtransplantationinvivo.

Theneurospheremethodwasoriginally

reportedbyReynoldsetal10,11andisoneofthe

mostfrequentlyusedmethodsforisolatingneural

stemcellsfromtheembryoorfromtheadultcentral

r,thismethodhasnotyet

beenappliedforobtainingneuralstemcellsfrom

preliminarystudy,weobtainedneurospheresfrom

era-

tionofthesecellswasquiterapid,possiblyfaster

thanotherneurospheresfromvarioustissueorigins

suchasthedermisandtheheart,7Y9suggesting

advantagesofASCsasaoriginofneuronalprogeni-

eurosphere

cellsexpressedNestinandMusashi-1,markergenes

forneuralstemcells,probablyreflectingtheir

tendencyofdifferentiatingintoneuronalprogeni-

ewisfurthersupportedbyinhibitionof

theirexpressionofLeptin,amarkerforadipogenic

differentiationandmaturation.

DotheASC-derivedneurospherecellsbehaveas

neuronalprogenitorsinvivo?Ourattemptofgrafting

thesecellsintotheculturedmouseembryorevealed

thatsomeofthecellsmigratealongthesecond

branchialarchandcontributetocraniofacialmor-

igratorypatternisquitesimilarto

thatofcranialneuralcrestcells,aswereported

previously.16Theneuralcrestcellsareanembryonic

cellularpopulationcharacterizedbyextensivemigra-

tionandauniquerepertoireofdifferentiation.29The

neuralcrestcellsareoftenregardedasstemor

progenitorcellsforperipheralneuronsandSchwann

cells,andthecraniofacialskeletalmesenchymeisalso

neural-crestderived.17,19,29,30Recentstudiesindicate

thattheneuralcreststemcellscanbeharvestedfrom

theseemingly‘‘mesodermal’’tissuesofadultani-

mals,suchasthedermis,7thehairfolliculardermal

papilla,8ortheheart,9bymeansoftheneurosphere

method,implicatingthatitisalsothecaseinthe

eourdataarepreliminaryand

wehaveasmallsamplesize,furtherstudiessuchas

thosewithdetailedexpressionanalysisofneural/

neuralcrestmarkergenesandlarge-scaleinvivo

graftingarenecessarytoconfirmthisinterestingidea.

REFERENCES

,ZhuM,MizunoH,ineagecellsfrom

humanadiposetissue:implicationsforcell-basedtherapies.

TissueEng2001;7:211Y228

,ZhuM,AshjianP,diposetissueisa

lCell2002;13:

4279Y4295

uraK,ShigeuraT,MatsumotoD,teriza-

tionoffreshlyisolatedandculturedcellsderivedfromthefatty

hysiol

2006;208:64Y76

otoD,SatoK,GondaK,-assistedlipotransfer

(CAL):supportiveuseofhumanadipose-derivedcellsforsoft

s.

nPH,ElbarbaryAS,EdmondsB,odiffer-

entiationofhumanprocessedlipoaspiratecellsintoearlyneural

econstrSurg2003;111:1922Y1931

E,RubinJP,entialofadipose-

derivedadultstemcellsasasourceofneuronalprogenitor

econstrSurg2005;116:1453Y1460

,AkhavanM,FernandesKJ,ionof

multipotentadultstemcellsfromthedermisofmammalian

lBiol2001;3:778Y784

desKJ,McKenzieIA,MillP,lnichefor

lBiol

2004;6:1082Y1093

Y,MatsumuraK,WakamatsuY,cneural

crestcellscontributetothedormantmultipotentstemcellin

iol2005;170:1135Y1146

dsBA,TetzlaffW,potentEGF-responsive

THEJOURNALOFCRANIOFACIALSURGERY/VOLUME18,NUMBER1January2007

52

Copyright@,orizedreproductionofthisarticleisprohibited.

striatalembryonicprogenitorcellproducesneuronsandastro-

sci1992;12:4565Y4574

dsBA,tionofneuronsandastrocytes

fromisolatedcellsoftheadultmammaliancentralnervous

e1992;255:1707Y1710

raY,MoriH,KobayashiS,tionofinvitro

proliferativeactivityofhumanfetalneuralstem/progenitor

cellsusingindirectmeasurementsofviablecellsbasedon

sciRes2002;69:869Y879

Y,SakakibaraS,ImaiT,i-1:an

evolutionallyconservedmarkerforCNSprogenitorcells

rosci2000;22:139Y153

,ZhuYF,AsakawaM,lasmicRNAvector

derivedfromnontransmissiblesendaiviruswithefficientgene

2000;74:6564Y6569

,TokusumiY,BanH,nsmissiblevirus-

likeparticleformationbyF-deficientsendaivirusistempera-

turesensitiveandreducedbymutationsinMandHN

2003;77:3238Y3246

T,SanaiY,NakamuraS,fHNK-1

carbohydrateepitopeanditssyntheticglucuronyltransferase

2003;203:

77Y88

T,NagaseM,OsumiN,facialanomaliesof

theculturedmouseembryoinducedbyinhibitionofsonic

hedgehogsignaling:ananimalmodelofholoprosencephaly.

JCraniofacSurg2005;16:80Y88

T,NagaseM,YoshimuraK,enesiswithin

thedevelopingmouseneuraltubeisdependentonsonic

hedgehogsignaling:

Ce登高杜甫原文翻译赏析 lls2005;10:595Y604

Y,NagaseT,NagaseM,pression

changesofsonichedgehogsignalingcascadeinamousemodel

ofacSurg2005;16:1055Y1061

M,ShiYY,AalamiOO,e-derivedadult

Biotechnol2004;22:560Y567

JL,LiebermanJR,LeeRS,-engineeredbone

frombmp-2-transducedstemcellsderivedfromhumanfat.

PlastReconstrSurg2005;115:1665Y1673

JL,SamimiB,ZhuM,-engineeredcartilage

andboneusingstemcellsfromhumaninfrapatellarfatpads.

JBoneJointSurgBr2003;85:740Y747

-BenardV,SilvestreJS,CousinB,cityof

humanadiposelineagecellstowardendothelialcells:physio-

ation2004;109:

656Y663

illeA,HeeschenC,SengenesC,ementof

postnatalneovascularizationbyhumanadiposetissue-derived

ation2004;110:349Y355

B,AndreM,ArnaudE,titutionoflethally

m

BiophysResCommun2003;301:1016Y1022

kelS,JodickeA,ChristophisP,gousstem

cells(adipose)andfibringlueusedtotreatwidespread

traumaticcalvarialdefects:omaxillofac

Surg2004;32:370Y373

raJ,OgawaR,MizunoH,differentiationof

adipose-derivedstemcellsisolatedfromGFPtransgenicmice.

BiochemBiophysResCommun2005;333:116Y121

,LeeDH,BaeYC,ementofneurological

deficitsbyintracerebraltransplantationofhumanadipose

Neurol2003;183:355Y366

rinN,ralCrest,dge:

CambridgeUniversityPress,1999

T,NakamuraS,HariiK,callylocalized

HNK-1epitopeperturbsmigrationofthemidbrainneural

wthDiffer2001;43:

683Y692

ADIPOSE-DERIVEDNEUROSPHERE/Nagaseetal

53