胎兒心臟黏附細胞研究論文

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【摘要】為了觀察人心臟是否含具有間充質祖細胞特性的細胞，從胎兒心臟分離、培養單個核細胞并從形態、表型和功能3個方面與骨髓間充質祖細胞進行比較和鑒定。結果表明，從心臟分離培養的細胞為成纖維樣，表面抗原為CD73,CD105,CD29,CD44,HLA-ABC,CD166陽性，而CD45,CD34,CD86,HLA-DR陰性。在不同的分化體系中，細胞能分化為脂肪細胞、成骨細胞和軟骨細胞。細胞擴增迅速，具有低免疫原性特性。結論：從心臟分離培養的細胞具有間充質祖細胞特性。

【關鍵詞】胎兒心臟；心臟黏附細胞；間充質祖細胞

HumanFetalHeart-derivedAdherentCellswithCharacteristicsSimilartoMesenchymalProgenitorCells

AbstractThisstudywasaimedtoinvestigateifhumanheartharboredapopulationofprimitiveundifferentiatedcellswiththecharacteristicsofMPC.Cellswereisolatedfromhumanfetalheartandwereculturedunderconditionsappropriateforbonemarrow-derivedMPCs.Theirmorphology,phenotypesandfunctionsweretestedbymethodsdevelopedforMPCfromothersources.Theresultsshowedthatmorphologically,cellswerespindleshapedandresembledfibroblasts.Intheirundifferentiatedstate,cellswereCD73,CD105,CD29,CD44,HLA-ABC,CD166positiveandCD45,CD34,CD86,HLA-DRnegative.Whenculturedinadipogenic,osteogenicorchondrogenicmedia,cellsdifferentiatedintoadipocytes,osteocytesandchondrocytesrespectively.TheycouldbeextensivelyexpandedinvitroandexhibitedverylowimmunogenicityasevaluatedbyTcellproliferationassays.Itisconcludedthatcellsisolatedfromfetalheartpossesssimi-laritytotheiradultandfetalbonemarrowcounterpartsinmorphologic,immunophenotypic,andfunctionalcharacteristics.

Keywordsfetalheart;heartderivedadherentcell;mesenchymalprogenitorcell

Bonemarrow-derivedmesenchymalprogenitorcells(MPC)haveattractedgreatattentionbecauseoftheircapabilityforrenewalanddifferentiationintovariouslineagesofmesenchymaltissues［1］andtheirpotentialplatformsforthesystemicdeliveryoftherapeuticproteinsinvivofollowinggenetransferusingoncogenicretroviruses［2］.Studiesinvolvingavarietyofanimalmodelshaveshownthatadultbonemarrow-derivedMPCcanmigrateandengraftinnumerousorgansanddifferentiatealongtissue-specificlineagesunderthestimulationoflocalfactors,andmaybeusefulintherepairorregenerationofdamagedormutatedbone,cartilage,ormyocardialtissues［3-5］.RecentworkhasshownthatMPCarepresentinmanytissues,includingumbilicalcord［6］,umbilicalcordblood［7］,bonemarrow,fetalblood,andfetalli-ver［8］.ThoughtheuseofMPCinthetreatmentofacutemyocardialinfarctionhasbecomeanoveltherapeuticoption,theknowledgeoftheirpresenceinheartislimited［9］.OuraimwastoinvestigatewhethertherewerecellswiththecharacteristicsofMPCinhumanfetalheart.

MaterialsandMethods

Isolationandcultureofadultandfetalbonemar-row,fetalheartmesenchymalprogenitorcells

TheResearchEthicsCommitteesofXuanwuHospital

approvedhumantissueforresearchpurposes.Humanfetalheartsampleswereobtainedfromaccidentalabortusof4.0-5.0monthsunderconsent.Single-cellsuspensionsoffetalheartmesenchymaltissuewerepreparedbycarefullyrinsingoutofthebloodandmincingmyocardialtissue,awayfromthebloodvessel,througha70-μm-nylonfilter.Thencellsfromadultandfetalbonemarrowsamplesandfetalheartsamplesweredilutedbyusing10%fetalbovineserum(FBS;Hyclone,Logan,UT,USA)inDulbecco''''sModifiedEagle''''sMedium-LowGlucose(DMEM-LG;GibcoBRL,LifeTechnologies,Paisley,UnitedKongdom)with50U/mlpenicillin,50μg/mlstreptomycin,and2mmol/LL-glutamine.Cellswereplatedinto6-wellplateatadensityof100000cells/cm2andincubatedat37℃in5%CO2.After36hours,nonadherentcellswereremoved,andthemediumwasreplaced.At80%confluence,cellswereharvestedwith0.25%trypsinand2mmol/LEDTAfor5minutesat37℃andwerereplatedin75-cm2flasks.Toexpandthecellsthroughsuccessivepassages,theywereplatedat104cells/cm2,growntonearconfluence,andharvestedwiththesameprotocol.

Fluorescence-activatedcellsorting(FACS)analysisofculturedcells

Monolayeradherentcellsfromadultbonemarrow(n=4),fetalbonemarrow(n=4),andfetalheart(n=4)weretrypsinizedandlabbledwithanti-CD73-phycoerythrin(PE;PharMingen,USA),CD105-fluoresceinisothiocyanate(FITC;Serotec,Oxford,UnitedKingdom),HLA-ABC-FITC,CD44-PE,CD29-PE,CD166-PE,CD45-FITC,CD34-PE,HLA-DR-FITC,CD86-PE(BectonDickinson,USA)andwereanalyzedbyflowcytometry(BeckmanCoulter,USA).

Adipogenic,osteogenic,andchondrogenicdiffe-rentiation

AdipogenicdifferentiationwasassessedbyincubationwithDMEMwith10%FBSsupplementedwith1μmol/Ldexamethasone,10μg/mlinsulin,0.5mmol/Lisobutylmethylxanthine,and200μmol/Lindomethacin(Sigma,St.Louis,MO,USA)for2weeks.ThepresenceofadipocyteswasassessedbythecellularaccumulationofneutrallipidvacuolesthatstainedwithOilredO(Sigma).Osteogenicdifferentiationwasassessedbyculturingcellsinanosteogenicmedium(DMEMwith10%FBSsupplementedwith0.1μmol/Ldexamethasone,50μmol/Lascorbicacid,and10mmol/Lβ-glycerolphosphate).Theonsetofosteoblastswasevaluatedbycalciumaccumulation(vonKossastaining).MediumwithDMEMcontaining2.5%FBS,50ng/mLtransforminggrowthfactor-β1(Peprotech,RockyHill,NJ,USA),50μg/mlascorbicacid,1mmol/Lsodiumpyruvate,6.25μg/mlbovineinsulin,6.25μg/mltransferrin,6.25μg/mlseleniousacid,and1.25μg/mlbovineserumalbuminwasusedforchondrogenicdifferentiation.Extracellularmatrixusedtoassesschondrogenicdifferentiation,wasdetectedbyAlcianbluestaining.

Mixedlymphocytereactions(MLR)

MPCsandstimulators(peripheralbloodmononuclearcells,PBMNC)wereirradiated(30Gy)beforebeingculturedwithTlymphocytes.CD3TcellspurifiedfromPBMNCbyusingtheMACSCD3isolationkit(MiltenyiBiotec)in5×104/wellweremixedatdiffe-rentratiowithMPCsin96-wellcultureplatestoensureefficientcell-cellcontactfor4daysin0.2mlRPMI1640medium(GibcoBRL)containing20%heat-inactivatedFBS.T-cellproliferationwasmeasuredonday3bymeansofan16-hourpulsewith3H-thymidine(3H-TdR)1μCi/well.3H-TdRincorporationwasmeasuredbyusingaliquidscintillationcounter.Theexperimentswereperformedforatleast3times.

Results

Morphologyandimmunophenotypecharacteristicsofcellsfromhumanfetalheart

Nucleatedcellsfromhumanfetalheartplatedatlow-densityformedindividualcoloniesdisplayingfibroblast-likemorphology.Aftersubcultivation,nucleatedcellsproliferatedwithapopulation-doublingtimeof23hoursandreachedaconfluentcondition.Adherentcellscouldbereadilyexpandedinvitrobymeansofsuccessivecyclesoftrypsinization,seeding.Andcultureevery3daysfor20passagesdisplayednovisiblechangesintermsoftheirmorphologyunderlightmicroscopy.Theimmunophenotypeofcellsfromadultandfetalbonemarrowandfetalheartwasdeterminedbyflowcytometry.Thestainingpatternofthecellswassimilar.AsshowninFigure1,adherentcellsisolatedfromadultandfetalbonemarrowandfetalheartwerepositiveforCD73,CD105,HLA-ABC,CD44,CD29,CD166andlackedofexpressionofCD45,CD34,CD86,HLA-DR.Thephenotypicprofileofadult,fetalbonemarrowandfetalheartadherentcellsdidnotchangeafter12passagesinculture.

DifferentiationabilityofcellsfromhumanfetalheartAdipogenicdifferentiationwasapparentafter1weekofincubation;twoweekslaterintracellularlipidaccumulationwasvisualizedusingOilRedOstaining(Figure2.A,B,C).DepositionofmineralizedmatrixontheculturevesselswasshownbyvonKossastaining(Figure2.E,F,G).Thestainingresultsindicatedthedifferentiationofculturedcellsintoosteocyticlineage.Thepositivealcianbluestaining(Figure2.I,J,K)suggestedtheexpressionoftypeIIcollagenofchondrocyte.Controlcellsdidnotshowspontaneousadipocyte,osteoblastorchondrocyteformationevenafter3-4weeksofcultivation(Figure2.D,H,L)

ImmunosuppressiveeffectofcellsfromhumanfetalheartinvitroTheMLRdatasuggestedanonspecificimmunosuppressiveeffectofcellsfrombonemarrowandfetalheartonhumanCD3Tcellproliferationinadosedependentmanner(Figure3).

Discussion

InthisstudywedemonstratedthattherewereadherentcellswiththecharacteristicsofMPCresidedinhumanfetalheart.Theyshowedfibroblastlikemorphologyand,likeMPCfrombonemarrow,werepositiveforsomemesenchymalmarkers,suchasCD73(SH2,SH3),CD105(SH4)andnegativefortheendothelial/hematopoieticprogenitormarkerCD34andthepanleukocytemarkerCD45.Thismeantthattherewerenotendothelialprogenitorcells(EPC),whichexpressedCD45andCD34.Inaddition,theadherentcellshadsimilarabilitytodifferentiateintoadipocyte,osteoblastandchondrocyte.Finallyandmostimportantly,asMPC,theadherentcellsexertedanimmunosuppressiveeffectonTcellsthatwasbeneficialforclinicalapplication.Theout-datedviewwasthattheheartlackedapoolofstemcellscapableofself-renewalanddifferentiation.Butmoreandmoreevidencesshowthattheadultheart,likethebrain,iscomposedofmainlyterminallydifferentiatedparenchymacellsnotreenteringthecellcycle,isnodoubtaterminallydifferentiatedorganbutcontainingadultstemcellssupportingitsregeneration.Excitingnewevidencehasemergedthatthetransplantedhumanheartharborsapopulationofprimitiveundifferentiatedcellsderivedfromboththerecipientandthedonor.Theseprimitivecellsmaybecardiacstemcellsandmayplayapivotalroleintheremodelingprocessfollowingtransplantation［10］.Andrecently,BeltramiAPetal［11］haveisolatedcardiacstemcellsfromadultrat,whichshowedinvitroandinvivoself-renewingandmultipotent.Whatismore,invivomanipulationofthesestemcellscouldregeneratelargeamountsoffunctionalmyocardium,showntobeoneofthemostextensivesolidorgantissueregenerationsbyusingstemcellsreportedsofar.Maybeautologouscardiac-specificstemcellsaremorebeneficialtoclinicalcelltherapyforcardiacdiseases.Theadherentcellsweisolatedarenotthesameasthecardiacstemcell.ThoughtheybotharenegativeforCD34,CD45,cardiacstemcellsdonotexpressfibronectinandvimentin,whichisdifferentfromtheadherentcells(datanoshown).Theheartandthebonemarrow,cardiomyocytesaswellasbonemarrowMPC,areofmesodermalorigin,sowepostulatethattherewillbeMPCinadultheart.Butitneedsfurtherstudytomakethingsclearer.

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