细胞外基质
-
2023年3月19日发(作者：冲击疗法)Anewtechniquetoexpandhumanmesenchymalstemcells
usingbasementmembraneextracellularmatrix
TakehiroMatsubara,a,c,fShinichiTsutsumi,bHaiouPan,aHisatadaHiraoka,cRyoOda,d
MasahiroNishimura,eHiroshiKawaguchi,cKouzouNakamura,candYukioKatoa,f,
*
aJapanScienceandTechnologyCorporation(JST),Chiyoda-ku,Tokyo102-8666,Japan
bDepartmentofOrthopedicSurgery,SchoolofMedicine,GunmaUniversity,Maebashi371-8511,Japan
cDepartmentofOrthopedicSurgery,GraduateSchoolofMedicine,UniversityofTokyo,Tokyo113-8655,Japan
dDepartmentofOperativeDentistry,GraduateSchoolofBiomedicalScience,HiroshimaUniversity,Hiroshima734-8553,Japan
eDepartmentofProstheticDentistry,GraduateSchoolofBiomedicalScience,HiroshimaUniversity,Hiroshima734-8553,Japan
fDepartmentofDentalandMedicalBiochemistry,GraduateSchoolofBiomedicalScience,HiroshimaUniversity,Hiroshima734-8553,Japan
Received12November2003
Abstract
Mesenchymalstemcells(MSC)showaveryshortproliferativelifespanandreadilylosethedifferentiationpotentialinculture.
However,thegrowthrateandtheproliferativelifespanofthestemcellsmarkedlyincreasedusingtissueculturedishescoatedwitha
basementmembrane-likeextracellularmatrix,rmore,the
stemcellsexpandedontheextracellularmatrix,butnotthoseonplastictissueculturedishes,retainedtheosteogenic,chondrogenic,
racellularmatrixhadgreatereffectsontheproliferationof
MSCandthemaintenanceofthemulti-lineagedifferentiationpotentialthanbasicfihymalstemcells
expandedontheextracellularmatrixshouldbeusefulforregenerationoflargetissuedefectsandrepeatedcelltherapies,which
requirealargenumberofstemorprogenitorcells.
Óhtsreserved.
Keywords:Mesenchymalstemcell;Basementmembrane;Extracellularmatrix;Regeneration;Differentiation
Mesenchymalstemcells(MSC)canbeinducedto
differentiateintoavarietyoftissuesincludingbone,
cartilage,tendon,fat,heart,muscle,andbrain,invitro
andinvivo[1,2].AutologousMSChaveadvantages
overEScells:thereisnoteratocarcinomaformation,no
immunerejection,andtherearenoethicalproblems.
However,comparedwithEScells,whichhaveanun-
limitedproliferativelifespan(periodbeforethecells
reachgrowtharrestinculture)andconsistentlyhigh
telomeraseactivity,MSChaveverypoorreplicative
capacityandshortproliferativelongevity[3,4].Thus,an
importantchallengeinregenerativemedicineistoim-
provethereplicativecapacityofMSC,therebytoobtain
anumberofMSCsufficienttorepairlargedefects.
ForcedexpressionoftelomeraseinMSCmarkedly
increasestheirproliferativelifespanandMSCwitha
hightelomeraseactivityshowedosteogenicpotential[5].
However,itisunknownwhetherthesecellscanmaintain
thechondrogenicandadipogenicpotentialorwhether
rthere
thatthegrowthrateandtheproliferativelifespanof
MSCmarkedlyincreasedusingtissueculturedishes
coatedwithabasementmembrane-likeextracellular
matrix(“bmECM”).Furthermore,MSCthatexpanded
106-foldonbmECMretaineditsosteogenic,chondro-
genic,andadipogenicpotential.
Materialsandmethods
-2cells,whichproduce
laminin,typeIVcollagen,andheparansulfateproteoglycans[6–8],
(Riken,Wako,Japan),andbovinecorneal
endothelialcellswereisolatedandmaintainedasdescribed[9].
*:+81-82-257-5629.
E-mailaddress:ykato@().
0006-291X/$-seefrontmatterÓhtsreserved.
doi:10.1016/.2003.11.143
BiochemicalandBiophysicalResearchCommunications313(2004)503–508
BBRC
/locate/ybbrc
BmECM-coateddisheswerepreparedaccordingtothemethodofDr.
Gospodarowicz[10].Thecellswereseededat2Â104cells/cm2on60-
mmtissueculturedishes(Corning,Corning,NY)andmaintainedin
4mlofDulbecco’smodifiedEagle’smedium(DMEM)-Ham’sF12
medium(1:1)(Sigma,,MO)inthepresenceof10%fetalbo-
vineserum(Hyclone,Logan,Utah)andantibiotics(100U/mlpenicillin
Gand100lg/mlstreptomycin)(medium-A).Mediumwaschanged
eculturesbecameconfluent,themediawere
renewedby4mlofmedium-Asupplementedwith5%dextran
(200,000Da,Wako,Osaka,Japan)andthecultureswerefurtherin-
entofthecultureswith20mMNH
3
resulted
incelllysis,exposingtheextracellularmatrixadheringtothesubstrata
stratumwaswashedfivetimeswith
usstudieshaveshownthatbmECMiscomposedoflami-
nin,heparansulfate,entactin,andtypeIVcollagen[11,12].
Preparationoflaminin-coateddishes,typeIVcollagen-coateddishes,
illilitersof10mMNaHCO
3
containing30lg/mltypeIVcollagenor30lg/mllaminin(Koken,
Tokyo,Japan)wasincubatedin60-mmplastictissueculturedishesat
4°dredmicrogramspermilliliterECMgelsolution
(Sigma)il-
lilitersoftheECMgelsolutionwasincubatedin60-mmtissueculture
dishesat4°oncentrationsoflaminin,typeIVcol-
lagen,andECMgelwereoptimalforproliferationofMSC(datanot
shown).
SCwereobtainedfromtheiliumorthe
alveolarboneaccordingtoaprotocolapprovedbyethicalauthorities
nmarrowaspirates(1ml/100-mmdish)
eswereperformed
whencellswereapproachingconflotherwisespecified,
MSCobtainedfromtheprimarycultureswereseededat1Â103cells/
cm2on60-mmoflaminin-,typeIVcollagen-orECMgel-coated
dishes,onbmECM-coateddishesoronplastictissueculturedishes,
andcellswerefedwithDMEM-lowglucosesupplementedwith10%
fetalbovineserumandantibiotics(medium-B)-
thesestudies,weseededMSCatalowdensity(1Â103cells/cm2)to
avoidfrequentpassagesandtheriskofcontaminationconsidering
clinicalapplication,althoughMSCshowedahighergrowthrateanda
longerproliferativelifespanatahighseedingcelldensity(5Â103cells/
cm2)(datanotshown).
Diffogenic,osteogenicoradipogenicconversion
ofMSCwasdeterminedaccordingtotheproceduresreportedby
Pittengeretal.[1],withsomemodifindrogenicdiffer-
entiation,cellswereseededat2.5Â105cellsper15mlplasticcentrifuge
tubeandmaintainedin0.5mlofserum-freea-MEMsupplemented
with3500mg/mlglucose,6.25lg/mlinsulin,6.25lg/mltransferrin,
6.25ng/mlselenite,5.33lg/mllinolate,1.25mg/mlbovineserumal-
bumin,10ng/mltransforminggrowthfactor-b3,100nMdexametha-
sone,and50lg/tureswerefed
fter,the
ere
eogenic
differentiation,cellswereseededat4Â104cellsper16-mmdishand
maintainedfor21–28daysinDMEM-lowglucosesupplementedwith
10lg/mlinsulin,10mMb-glycerophosphate,100nMdexamethasone,
and50lg/pogenicdifferentia-
tion,cellswereseededat2Â105cellsper35-mmdishandgrownto
conflfter,adipogenicdifferentiationwas
inducedbysubjectingconfluentmonolayersto3–4roundsofadipo-
undhadtwosteps;incubationwithadipo-
genicmedium(DMEM-highglucose,10%fetalbovineserum,0.2mM
indomethacin,1lMdexamethasone,0.5mMmethyl-isobutylxanthine,
and10lg/mlinsulin)for72–96handincubationwithmaintenance
medium(DMEM-highglucose,10%fetalbovineserum,and10lg/ml
insulin)for72–ereculturedundertheadipogenicstatusfor
28days.
Glycosaminoglycancontent,alkalinephosphataseactivity,calcium
level,glycerol-3-phosphatedehydrogenaseactivity,andDNAcontent.
Theglycosaminoglycan(GAG)contentwasdeterminedusingasul-
fatedGAGassaykit(Biocolor,Newtownabbey,UK)[13].Thealka-
linephosphatase(ALP)activitywasdeterminedbythemethodof
Bessey[14].ThecalciumlevelwasdeterminedbythemethodofGit-
elman[15].Theglycerol-3-phosphatedehydrogenaseactivitywasde-
terminedusinganassaykit(Hokudo,Sapporo,Japan)[16].TheDNA
contentwasdeterminedusingafluorescentDNAquantificationkit
(Bio-Rad,Chicago,IL).
NAwasextractedusingIsogen(NipponGene,
Tokyo,Japan).Thefirst-strandcDNAwassynthesizedfrom1lgof
totalRNAusingtheSUPERSCRIPTIIRNaseHÀreversetrans-
criptase(LifeTechnologies,Rockville,MD).UsingthecDNAsasa
template,PCRwascarriedoutunderthefollowingconditions:dena-
turationat94°Cfor30sandprimerextensionat65°Cfor1.5minin
fnucleotides,50-TGGTGGAGCAGCAAGAGCAA-
30and50-TGCCCAGTTCAGGTCTCTTA-30fortypeIIcollagen,50-
CCCAACACCAAGACACAGTT-30and50C-ATCACCTTTGATG
CCTGGCT-30fortypeXcollagen,and50-GTCAAGGCCGAGAAT
GGGAA-30and50-GCTTCACCACCTTCTTGATG-30forGAPDH,
50-CATTTTGGGAATGGCCTGTG-30and50-ATTGTCTCCTCCG
CTGCTGC-30forbonesialoprotein,50-CTAGGCATCACCTGTGC
CATACC-30and50-CAGTGACCAGTTCATCAGATTCATC-30for
osteopontin,50-CCACCGAGACACCATGAGAG-30and50-CCATA
GGGCTGGGAGGTCAG-30forosteocalcin,and50-CATTCTGGC
CCACCAACTT-30and50-CCTTGCATCCTTCACAAGCA-30for
edPCRproducts
wereseparatedon1%agarosegelsandstainedwithethidiumbromide.
t’sttestwasused.
Results
TheextracellularmatrixproducedbyPYS-2cellsor
endothelialcellsadheredtothesubstratumofplastic
tissueculturedishesandcouldbeeasilycutwithaneedle
andturnedoverlikeasheetofpaper(Fig.1A).When
cellsinmarrowaspirateswereseededonplasticculture
dishes,adherentcells—MSC—proliferatedinthepres-
enceof10%fetalbovineserumatahighgrowthratein
primarycultures[3],buttheirgrowthraterapidlyde-
creasedinsecondaryandtertiarycultures(Figs.1B–D).
Inculturesonplastictissueculturedishes,non-adherent
cellswereremovedcompletelybythefirstpassage.
However,whencellsinmarrowaspirateswereseeded
directlyonbmECM,bothMSCandmanyothercells
adheredtothesubstratumandtheseadherentcellswere
ingly,we
harvestedMSCwhenthecellswereapproachingcon-
fluenceinprimaryculturesonplastictissueculture
dishes,andseededtheisolatedMSConbmECMor
plastictissueculturedisheswithoutthematrix
(“plastic”)toexaminetheeffectsofbmECMonthe
proliferationofMSC.
ThegrowthrateofhumanMSCisolatedfromthe
ilium(Fig.1B)orthealveolarbone(Fig.1C)on
bmECMwasmuchhigherthanthatonplastic,andthus
thecumulativecellnumberintheculturesonbmECM
was105-foldgreaterthanthatonplasticonday50.
AfterMSCobtainedfromprimarycultureswereseeded
araetal./BiochemicalandBiophysicalResearchCommunications313(2004)503–508
onbmECMorplastic,theproliferativelifespanofMSC
onbmECM(50.3Æ1.5days)wasalsosignificantly
(p<0:0001)longerthanthatofMSConplastic
(29.2Æ4.4days)(Figs.1BandC).TheeffectofbmECM
producedbyPYS-2wassimilartothatofendothelial
cellbmECM(Fig.1).MSCseededatalowdensityand
grownonplasticlosttheirspindle-likeappearance,be-
comingflatwithanincreaseinthepassagenumber
(Fig.1E).Theflatappearanceischaracteristicofse-
r,mostMSCgrownonbmECM
maintainedthespindle-likeappearanceuntilthe5th
passagecultureonday45(Fig.1F),suggestingthat
bmECMsuppressedcellsenescence.
LamininandtypeIVcollagenarethemajorcom-
ponentsofbmECM,butMSConlaminin-ortypeIV
collagen-coateddishesshowedlowergrowthratesthan
onbmECM(Fig.1F).TheECMgelisolatedfrom
Engelbreth–Holm–Swarmmurinesarcomaalsoshowed
lessgrowthstimulationthanbmECM(Fig.1F),sug-
gestingalossofactivesubstancesduringisolationofthe
extracellularmatrixcomponentsorthenecessityofan
intactstructureforgrowthstimulation.
ThechondrogenicpotentialofMSCwasexaminedas
ainedfrom
the2ndandthe5thpassageculturesonday15andday
51(Fig.1B)weremaintainedinpelletculturesfor28
days(Fig.2A).Theamountofcartilageproteoglycan
stainedwithtoluidinebluewasgreaterinthepellets
obtainedfromthe5thpassageculturesgrownon
bmECMthaninthepelletsfromthe2ndandthe5th
ressionsof
typeIIcollagenandtypeXcollagenmRNAswere
higherinpelletsfromthe5thpassagecultureson
bmECMthaninpelletsfromthe5thpassagecultureson
plastic(Fig.2B).GAGcontentandALPactivityinthe
pelletsdecreasedwiththeincreaseinthepassagenum-
ber,irrespectiveofthepresenceorabsenceofbmECM.
However,ateachpassagenumber,theGAGcontent
(Fig.2C)andALPactivity(Fig.2D)werehigherwith
MSCfromculturesonbmECMthanwithMSCfrom
culturesonplastic.
Next,MSCfromthe2ndandthe5thpassagecultures
onbmECMorplasticwereincubatedundertheosteo-
ionofhumanMSConECM-coateddishes.(A)ThegrossappearanceofbmECM.(B)Cellsinmarrowaspirates(1ml/100-mmdish)
fromtheiliumoftwovolunteers(80s)eharvestedwhen
thecellswereapproachingconfluenceinprimarycultures,andtheisolatedcellswereseededonbmECMproducedbyPYS-2cellsoronplastic(the
firstpassagecultures),uently,passageswereperformedontheappropriatesubstrata.*;**Differs
significantlyfromthecellnumberinculturesonplasticdishondays7–8,14–15,23–25,33–37,47–51or68–72(*p<0:05,**p<0:01).Arrows:MSC
obtainedfromthe2ndpassagecultureonday15orMSCfromthe5thpassagecultureonday51weretransferredintochondrogenic,osteogenic,and
adipogenicstatus.(C)MSCobtainedfromthealveolarboneoffourvolunteers(EXP.C,Dintheir20sandEXP.E,Fintheir80s)wereculturedon
plasticorbmECMproducedbybovinecornealendothelialcells,asdescribedabove.*;**;***Differssignificantlyfromthecellnumberincultureson
plasticdishonday(s)7–8,14–15,30,51,47–51or72–74(*p<0:05,**p<0:01,and***p<0:001).(D)MSCobtainedfromtheiliumwerecultured
onlaminin-,typeIVcollagen-,ECMgel-coateddishes,bmECMorplastic,asdescribedabove.*;**;***Differssignificantlyfromthecellnumberin
culturesonlaminin-,typeIVcollagen-orECMgel-coateddishesondays7–8,14–15,23–25,33–37or47–51(*p<0:05,**p<0:01,and
***p<0:001).(E,F)Flatandspindle-likecellshapeonplastic(E)andbmECM(F),(40Â)(onday45inthe5thpassagecultures).
araetal./BiochemicalandBiophysicalResearchCommunications313(2004)503–508505
osteogenesis,wedidnotusebmECMtodiscriminatethe
effectoftheextracellularmatrixonproliferationfromits
directeffectondiffmcultureson
bmECMbecamestainedwithalizarinredmorein-
tenselythanMSCfromculturesonplasticonday21
(Fig.2E).Theexpressionsofbonesialoprotein,osteo-
pontin,andosteocalcinmRNAsonday28werealso
higherinculturesofMSCfromculturesonbmECM
thaninculturesofMSCfromculturesonplasticatthe
5thpassage(Fig.2F).ALPactivityandcalciumlevelon
day28decreasedwiththeincreaseinthepassagenum-
r,MSCfromculturesonbmECMshoweda
higherALPactivity(Fig.2G)andahighercalciumlevel
(Fig.2H)thanMSCfromculturesonplasticatthe2nd
andthe5thpassages.
Toexaminetheadipogenicpotential,MSCfromthe
2ndandthe5thpassageculturesonbmECMorplastic
wereincubatedundertheadipogenicstatusonplastic
mcultureson
bmECMshowedhigheradipogenicdifferentiation,
whichwasindicatedbymoreintensestainingwithoil-
redO(Fig.2I),higherPPAR-c2mRNAexpression
(Fig.2J),andhigherglycerol-3-phosphatedehydroge-
naseactivity(Fig.2K).
Next,humanMSCweregrownonbmECMorplastic
with10%humanserum,sincehumanserummaybe
theseconditions,MSCproliferatedmorerapidlyand
showedalongerproliferativelifespanonbmECMthan
onplastic(Fig.3A).Furthermore,thiseffectofbmECM
wasgreaterthanthatofbasicfibroblastgrowthfactor
(FGF).TheMSCgrownonbmECMfor66daysde-
velopedintoacartilage-liketissue(Fig.3B),even
thoughtheseMSConbmECMhadlostproliferation
rast,scarcelyanycartilage-liketissue
wasformedwithMSCobtainedfrom66-day-oldcul-
turesonplastic(Fig.3C).
Discussion
Theextracellularmatrix(ECM)playsavitalrolein
organmorphogenesis,maintenance,andreconstruction
followinginjury,andactionsofECMcanbeattributed
toitseffectonproliferationofstemcells,sincestemcells
resideonthebasementmembraneintheepitheliumand
someothertissues[17].Inmuscle,satellitecells(stem
cells)—whichcanbeinducedtodifferentiateintomuscle,
ionofchondrogenic,osteogenic,obtainedfromthe2ndandthe5thpassagecultures
onplasticorbmECMproducedbyPYS-2cells(Fig.1B)weretransferredintothechondrogenicstatusinpelletculturesfor28days(A–D)and
stainedwithtoluidineblue(A).(B)ThemRNAlevelsoftypeIIcollagen(typeII)andtypeXcollagen(typeX)
content(C)andALPactivity(D)fromthe2ndandthe5thpassageculturesonplasticorbmECMwere
transferredintotheosteogenicstatus(E–H).(E)Thecelllayerswerestainedwithalizarinredonday21.(F)ThemRNAlevelsofbonesialoprotein
(BSP),osteopontin(OP),andosteocalcin(OC)activity(G)andcalciumlevel(H)ofthecell-matrix
obtainedfromthe2ndandthe5thpassageculturesonplasticorbmECMwereculturedunderthe
adipogenicstatusfor28days(I–K).(I)Thecelllayerswerestainedwithoil-redO.(J)ThemRNAlevelofPPAR-c2wasanalyzedbyRT-PCR.(K)
Glycerol-3-phosphatedehydrogenaseactivitywasdetermined.(C,D,G,H,andK)“)”and“+”representMSCfromcultureonplasticandbmECM,
areaveragesþ=ÀSDforfourcultures.*p<0:05,**p<0:01,and***p<0:001vsplastic.
araetal./BiochemicalandBiophysicalResearchCommunications313(2004)503–508
bone,cartilage,andfat—arealsoinclosecontactwith
thebasementmembrane;satellitecellsbecamemyo-
blastsafterdetachmentfromthebasalmembrane[18].
Theseobservationssuggestthatthebasementmembrane
and/orsomeotherECMsplayacrucialroleinthe
proliferationofstemcellsandthemaintenanceoftheir
undiffstudy,weshowedthat
bmECMmarkedlyincreasedboththegrowthrateand
rmore,MSC
thathadexpanded106-foldonbmECMmaintainedits
multi-lineagediffhanism
bywhichbmECMstimulatesMSCproliferationand
maintainstheirdifferentiationpotentialisnotknown,
butevenMSCtransfectedwithtelomerasegradually
decreaseditsosteogenicpotentialwiththeincreasein
thepassagenumber[5],althoughtheirreplicative
rast,MSConbmECM
maintainedthedifferentiationpotentialevenafterthey
,theextracellular
matrixandtelomerasemayhavecomplementaryeffects
ontheproliferationofMSCandtheirdifferentiation
ase,theremarkableeffectsofbmECM
inMSCculturesdemonstratedheremeettheexpecta-
tionsofdoctorseagertoexpandMSCextensivelyin
vitrofromasmallvolumeofmarrowaspiratesbefore
orth,bmECMwillbeprepared
usinghumanESorhumancelllines,andinthenear
futurebmECM-coateddisheswillprobablyhaveagreat
useinMSCstudiesandregenerationmedicine.
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