教育部生物及医学科技人才培育先导型计画教育部...

年類了力不量力行兩勒來年力行年年度料2005年6,8762,382237領度力利列奈利類療領年度六領奈立領領奈領領奈兩力理參力諸力利金六年勵力立類類立里沈寧了都離領李領來精奈奈六領精復臨李李不臨療臨臨療律參理力不參更理立六年李李倫沈寧林龍良理林逸年錄類立TheEstablishmentofHumanEmbryonicStemCells…………………..1CharacterizationofEmbryonicStemCells………………………………..17沈寧林龍CultivationandDifferentiationofEmbryonicStemCells………………27離李Isolation,Expansion,andDifferentiationofAdultStemCells………….47奈倫TheApplicationofNanotechnologyonStemCellBiology……………..63六ApplicationofAnimalCloningTechnologiesinStudiesRelatedtoStemCells………………………………………………………………....75TissueEngineeringTechniqueinStemCellResearch…………………85復CryopreservationandRe-cultivationofStemCells……………………..97臨李ClinicalApplicationofStemCells……………………………………………103療臨ClinicalTrialsandAdvanceResearchforStemCellTherapies………117Commercializationofstemcelltechnology……………………………….129歷…………………………………………………………………………………….143索…………………………………………………………………………………….1491類立TheEstablishmentofHumanEmbryonicStemCellsembryonicstemcellsESCpluripotenttotipotent更數embryonicgermlayergermcell不論來臨都度來類類識來例卵療類1981年立類hESC1998年JamesThomson1度立類立1-3理立理[1]例[2]異[3]立[4]立裡立便來立例不來xeno-free立類便參參2立倫理領不行2行冷56blastocyst來離類類innercellmassICM離來離zonapellucida1.proteasehESmediumorganculturedishOCDFalcon2.錄scoring數精卵精563.PBS0.5%0.22µm濾filter濾1ml4-wellculturedishNUNC0.5mlhESmedium見錄14.370.5%16hESmedium～立MEFOCDhESmedium5.兩度更hESmedium更6.512行陸行hESmediumhESmediumhESmediumprotease4-wellculturedishinnercellmasstrophoblastblastocyst3類來12見3不ABA類量C不DC類immunosurgerytrophoblastanti-humanserumguineapigcomplementA1.10units/ml80µl0.5mlhESmedium2.5~61~23.2~4留420B類anti-humanserumanti-HS錄來1.15例類hESmedium80µl0.5mlhESmedium2.303.2~4留hESmediumproteasehESmediumhESmediumA.4-wellculturedishhESmediumanti-HShESmediumhESmediumB.4-wellculturedish4Ccomplement1.15例guineapigcomplementGPhESmedium80µl0.5mlhESmedium2.anti-humanserum303.2~4留D裡hESmediumOCDA.六B.C.類理D.hESmediumhESmediumhESmediumGPC.4-wellculturedishABCDFeederICM5兩A.切落colony1.Pasteurpipette精拉切利切利切切29~30G2.PBShESmedium3.OCD更bFGFhESmedium4.hESmedium10µg/mldispase1.落57落100µm行2.29~30G行落切切來切輪狀切切落3.切0.5mldispase2~34.見切落離5.20~200µltip來6.OCDPBS+hESmediumhESmediumhESmedium4-wellculturedish1236797.兩落更更落bFGFhESmedium切類1.切2.切3.輪狀切B.量1.PBS類hESmedium2.collagenaseIV200units/ml3.35-mm更bFGFhESmedium1.7類兩2.0.5ml3~63.20~200µltip略離落6mlhESmedium15ml離4.PipetteAid來5~10落來1/10~1/205.離600×g2數量bFGFhESmedium6.35-mm60-mm7.兩更更bFGFhESmedium*若省略來不1237類切1.32.57冷vitrification冷1.冷冷4例DMEMmedia78%FetalBovineSerumHyclone20%A.ES-HEPESmedium1MHEPES2%ES-HEPES80%EthyleneGlycol10%B.10%VitrificationsolutionDMSO10%ES-HEPES30%1MSucroseSolution30%EthyleneGlycol20%C.20%VitrificationsolutionDMSO20%2.冷ABC0.5mlC冷20µl3µl冷3.冷料4.切5.冷A行冷6.BAB17.CBC25123o3µl20µlA.C.B.4-wellculturedish88.C20µlC9.3µlC10.冷straw理立冷*2~3冷冷81.冷4例DMEMmedia78%FetalBovineSerum(Hyclone)20%A.ES-HEPESmedium1MHEPEs2%ES-HEPESmedia80%B.0.2MSucrosemedium1MSucroseSolution20%ES-HEPES90%C.0.1MSucrosemedium1MSucroseSolution10%2.OCD更bFGFhESmedium3.冷4.利冷冷立B.0.2Msucrosesolution20~200µltip冷15.C.0.1Msucrosesolution56.A.ES-HEPESmedium57.hESmediumbFGF8.兩更更*冷類量冷piecesofcoloniessinglecells冷率4-wellculturedishB.1minA.5minA.5minC.5min9六(Feedercells)不狀mouseembryonicfibroblasts,MEFSTO類humanforeskinfibroblast,HFFATCCCRL-1635,Hs68來例humanplacentalfibroblastA.立1.56籠飼2.13PBS3.10cmPBS六4.3ml185.106.MEFmedium見錄375%CO2數1057.更23行8.PBSCa2+/Mg2+-free21ml0.05%trypsin-EDTA372MEFmediumtrypsin-EDTA13行9.2更90%行冷數56六PBS10B.冷1.冷90%FBS10%DMSO冷2.PBSCa2+/Mg2+-free21.5ml0.05%trypsin-EDTA375%CO22MEFmediumtrypsin-EDTA3.15ml離利數haemocytometer行數4.1000rpm離離5min5.量冷冷數1.5×106/0.5ml冷冷6.冷冷NALGENE®Cryo1FreezingContainer-80冷C.1.冷37行2.冷MEFmedium15ml離3.2000rpm離離5min4.MEFmedium75-cm2flaskFalcon5.375%CO22更MEFmedium90%D.a.gelatincoating1.gelatinSigma度0.1%-20冷2.0.1%gelatin3737203.gelatin37b.MitomycinC理1.更mitomycinC10µg/mlMEFmedium372.532.PBSCa2+/Mg2+-free20.05%trypsin-EDTA37210mlMEFmediumtrypsin-EDTA3.15ml離1000rpm離離5min5mlMEFmedium4.利數行數度數1.75×105cells/ml0.1%gelatin理OCD35-mmculturedish5.375%CO2行66.更hESmedium見錄37111行c.理1.了MitomycinC理來裂0.05%trypsin-EDTA37210mlMEFmediumtrypsin-EDTA2.FalconPetridish3000~8000rad度不3.理4.行數例數1.75×105cells/ml0.1%gelatin理OCD35-mmculturedish5.375%CO2行66.更hESmedium371行E.Extracellarmatrixplatesa.Matrigel1.MatrigelBectonDickinson4pipettes-20冷冷Matrigelmatrix22~35易2.冷pipettes離3.serum-freeDMEMMatrigel度度5%4.Matrigel1MatrigelMEFmedium425.Matrigel更conditionedmediumb.1.mitomycinC理MEF3×106cells75-cm2flask20mlMEFmedium2.375%CO224flaskflask73.濾0.22µmfilter-201*類foreskinfibroblastuterineendometriumplacenta類立來4類來不論不都類12不MEF類類不例類類略MEF類異不*類了例CO2度5~7%量pH度CO2O2度21%5%hypoxicculture數5%O2立類狀率5~721%O2不論異5%O2類1.mouseembryonicfibroblasts,MEF2.MEF3.類humanforeskinfibroblast,HFF4.HFF123413類立類立參不數年來類立不4,5來類definedmedium6,7立類來來立類類來例humanforeskinfeederhumanplacentalfibroblast立類8definedmedium立類6TeSR1TGFβPAGABALiClbFGFMEFconditionedmedium更類extracellularmatrixcollagenIVfibronectinlamininvitronectin不Ludwig立了兩類異7行立來類便立來類臨9立類倫理量了數來Klimanskaya1081~2葉blastomere葉葉利類不立來1~2葉不精良類倫理了來臨類數年來nucleartransferNT來量nt-hESC來nucleardonor類DNA來99%粒DNA不來不立類例類立不類立14錄立類率類立立NTU1NTU2NTU3料2Antiserum(dilution)數數立12PN2BAHo