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Methods in Molecular Biology 1307 Kursad Turksen Editor Human Embryonic Stem Cell Protocols Third Edition M M B ETHODS IN OLECULAR IOLOGY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire,AL109AB,UK Forfurther volumes: http://www.springer.com/series/7651 . Human Embryonic Stem Cell Protocols Third Edition Edited by Kursad Turksen Ottawa Hospital Research Institute Ottawa, ON, Canada Editor KursadTurksen OttawaHospitalResearchInstitute Ottawa,ON,Canada ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-2667-1 ISBN978-1-4939-2668-8 (eBook) DOI10.1007/978-1-4939-2668-8 LibraryofCongressControlNumber:2015947127 SpringerNewYorkHeidelbergDordrechtLondon #SpringerScience+BusinessMediaNewYork2016 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnot imply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsand regulationsandthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Printedonacid-freepaper HumanaPressisabrandofSpringer SpringerScience+BusinessMediaLLCNewYorkispartofSpringerScience+BusinessMedia(www.springer.com) Preface Inspiteofethicalandpoliticalconcernsregardingtheuseofhumanembryonicstemcells, theirpotentialtoadvancenotonlyregenerativemedicineapplicationsbutalsoour funda- mentalunderstandingofstemcellbiologycontinuestodriveinterestinresearchwiththese cells. In both cases, delineating the steps in and identifying the molecules responsible for thecommitmentanddifferentiationofthesepluripotentcellstodifferentcellfatesremains crucial. With this in mind, I felt that it would be timely to collect some of the most interesting and useful protocols that have emerged during the last several years. I am therefore extremely pleased and thankful to those groups who agreed to share their valuable protocols with others interested in exploring stem cell biology questions in a researchsetting. Onceagain,IwouldliketoacknowledgeJohnWalker,EditorinChiefoftheseries,for continuously fostering my efforts in this series. I am also grateful to Patrick Marton, Executive Editor of the series, for encouraging me throughout the editorial process. In addition, I thank David Casey for keeping me on track and pointing out all those details thatmakecontributionstotheseriesconsistentanddependable. Ottawa,ON,Canada KursadTurksen v Contents Preface.................................................................... v Contributors............................................................... xi DerivationofHumanEmbryonicStemCellLinesfromVitrified HumanBlastocysts......................................................... 1 CaraK.Bradley,JuliaSchaft,TammieK.Roy,BiljanaDumevska, andTeijaT.Peura ® CharacterizingPluripotentStemCellsUsingtheTaqMan hPSC ScorecardTMPanel......................................................... 25 JeffreyFergus,ReneQuintanilla,andUmaLakshmipathy GrowthofHumanPluripotentStemCellsUsingFunctionalHuman ExtracellularMatrix........................................................ 39 AndresSanz-Garcia,MiodragStojkovic,andCarmenEscobedo-Lucea EfficientExpansionofDissociatedHumanPluripotentStemCells UsingaSyntheticSubstrate................................................. 61 EihachiroKawase MicroarrayApproachtoIdentifytheSignalingNetworkResponsible forSelf-RenewalofHumanEmbryonicStemCells ............................ 71 NoboruSatoandAliBrivanlou MonitoringStemnessinLong-TermhESCCulturesbyReal-TimePCR.......... 89 AmparoGalańandCarlosSimoń StudyofGapJunctionsinHumanEmbryonicStemCells ...................... 105 AlicePe´bayandRaymondC.B.Wong ImmunofluorescenceMicroscopyandmRNAAnalysisofHuman EmbryonicStemCells(hESCs)IncludingPrimaryCiliaAssociated SignalingPathways......................................................... 123 MajLineaVestergaard,AashirAwan,CarolineBeckerWarzecha, SørenTvorupChristensen,andClausYdingAndersen AnalysisofIntracellularCalciumSignalinginHumanEmbryonic StemCells ................................................................ 141 AdriennPeńtek,KatalinPa´szty,andA´gotaApa´ti GeneticManipulationofHumanEmbryonicStemCells........................ 149 RachelEiges GeneticModificationinHumanPluripotentStemCells byHomologousRecombinationandCRISPR/Cas9System.................... 173 HaipengXue,JianboWu,ShenglanLi,MahendraS.Rao, andYingLiu UseofMulticolorFlowCytometryforIsolationofSpecificCell PopulationsDerivingfromDifferentiatedHumanEmbryonicStemCells ........ 191 IsabellaMengarelli,AndrewFryga,andTizianoBarberi vii viii Contents DefinitiveEndoderm DifferentiationofHumanEmbryonicStem CellsCombinedwithSelectiveEliminationofUndifferentiatedCells byMethionineDeprivation ................................................. 205 TomonoriTsuyama,NobuakiShiraki,andShoenKume DerivationofEndothelialCellsandPericytesfromHuman PluripotentStemCells ..................................................... 213 SravantiKusumaandSharonGerecht DifferentiationofHumanEmbryonicStemCellsonPeriodontal LigamentFibroblasts....................................................... 223 Y.MuratElçin,Bu¨lendI˙nanç,andA.EserElçin DerivationofEpithelialCellsfromHumanEmbryonicStemCells asanInVitroModelofVocalMucosa........................................ 237 VlastaLungova,CiaraLeydon,andSusanThibeault HumanEmbryonicandHepaticStemCellDifferentiationVisualized inTwoandThreeDimensionsBasedonSerialSections ........................ 245 PeterS.Vestentoft,ChristianB.Brøchner,NielsLynnerup, ClausYdingAndersen,andKjeldMøllga˚rd DerivationofChondrogenicCellsfromHumanEmbryonic StemCellsforCartilageTissueEngineering................................... 263 WeiSeongTohandTongCao MicrogroovedSurfaceModulatesNeuronDifferentiation inHumanEmbryonicStemCells............................................ 281 DavidLu,Chi-ShuoChen,Chao-SungLai,SushantSoni, TaranzeLam,ClarenceLe,EricY.-T.Chen,ThienNguyen, andWei-ChunChin DirectedDifferentiationofHumanEmbryonicStemCells intoNeuralProgenitors .................................................... 289 ErinBandaandLauraGrabel DirectConversionofPluripotentHumanEmbryonicStem CellsUnderDefinedCultureConditionsintoHumanNeuronal orCardiomyocyteCellTherapyDerivatives................................... 299 XuejunH.Parsons GenerationofEpithelialCellPopulationsfromHuman PluripotentStemCellsUsingaSmall-MoleculeInhibitor ofSrcFamilyKinases....................................................... 319 JoshuaA.Selekman,XiaojunLian,andSeanP.Palecek Dual-SMADInhibition/WNTActivation-BasedMethodstoInduce NeuralCrestandDerivativesfromHumanPluripotentStemCells............... 329 StuartM.Chambers,YvonneMica,GabsangLee,LorenzStuder, andMarkJ.Tomishima AcceleratedThree-DimensionalNeuroepitheliumFormation fromHumanEmbryonicStemCellsandItsUseforQuantitative DifferentiationtoHumanRetinalPigmentEpithelium......................... 345 YuZhu,SvenSchreiter,andEllyM.Tanaka Contents ix EfficientProductionofPhotoreceptorPrecursorCellsfromHuman EmbryonicStemCells...................................................... 357 AnatYanai,ChristopherLaver,AaronW.Joe,andKevinGregory-Evans GenerationofMegakaryocytesandPlateletsfromHuman PluripotentStemCells ..................................................... 371 MarjoriePick ASimpleProtocolfor theGenerationofCardiomyocytesfromHuman PluripotentStemCells ..................................................... 379 GlenLesterSequiera,AshishMehta,andWinstonShim Erratumto:MicrogroovedSurfaceModulatesNeuronDifferentiation inHumanEmbryonicStemCells............................................ 385 DavidLu,Chi-ShuoChen,Chao-SungLai,SushantSoni, TaranzeLam,ClarenceLe,EricY.-T.Chen,ThienNguyen, andWei-ChunChin Index..................................................................... 387