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1、©2000NatureAmericaInc.•http://biotech.nature.comTECHNICALREPORTSgenerationofembryoidbodies(EBs;stage2),theuseofadefinedEfficientgenerationofmediumtoselectforCNSstemcells(stage3),theproliferationofCNSstemcellsinthepresenceofmitogen,basicfibroblastgrowthfactor(bFGF;stage4),andthedifferent
2、iationofthestemcellsbymidbrainandhindbrainremovalofthemitogen(stage5;Fig.1A).ThesuccessfulconversionofEScellsintomidbraindopamin-ergic(DA)neuronsinvitroshouldbedependentontheinductionneuronsfrommouseofthesamegenesthatareexpressedinCNSstemcellsandneuronsinvivo.TheOTXhomeoboxgenes(OTX1and
3、OTX2)arewidelyexpressedatearlystagesofneurectodermdifferentiation16.OTX2embryonicstemcellsisexpressedthroughouttheepiblastandsubsequentlyrestrictedtoanteriorneurectoderm,whereitisrequiredfordevelopmentoftheforebrainandmidbrain.ThehomologOTX1isfirstexpressedinSang-HunLee1†,NadyaLumelsky1
4、†,theneurectoderminthedorsaltelencephalon,andinteractionsLorenzStuder1,2,JonathanM.Auerbach1,betweenthesetwoOTXgenesarethoughttospecifythedevelop-andRonD.McKay1*mentofthemidbrainandhindbrain17.OTX2isexpressedintheundifferentiated(stage1)EScellsandpresentatlowerlevelsat1LaboratoryofMolec
5、ularBiology,NINDS,NIH,Bethesda,MD20892.stage2and3ofdifferentiation.OTX1isnotexpressedathighlev-2Currentaddress:LaboratoryofStemCellandTumorBiology,Neurosurgeryelsuntilstage3(Fig.1B).andCellularBiochemistryandBiophysics,MemorialSloanKetteringCancerCenter,NewYork,NY,10021.†Bothauthorscont
6、ributedequallytothiswork.Severalgenes(Pax2,Pax5,Wnt1,En1,Nurr1)havebeenidenti-.com*Correspondingauthor(mckay@codon.nih.gov).fiedthatcontroldifferentiationofdopaminergicandserotonergicneuronsinthemidbrainandhindbrain18,19.PCRanalysisdetectedReceived15April,2000;accepted24April,2000expres
7、sionofthesegenesatstages3and4ofEScelldifferentiation.h.natureAfterstage4,thebFGF-expandedcellsaremorphologicallyuniformEmbryonicstem(ES)cellsareclonalcelllinesderivedfromtheandexpressnestin,anintermediatefilamentproteincharacteristicinnercellmassofthedevelopingblastocystthatcan
