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Methods in Molecular Biology 1341 Kursad Turksen Editor 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 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 ISBN978-1-4939-2953-5 ISBN978-1-4939-2954-2 (eBook) DOI10.1007/978-1-4939-2954-2 LibraryofCongressControlNumber:2015951971 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 Research on embryonic stem cells continues to move very quickly, as reflected in the positive responses that I received for the chapters in this volume, which builds upon a relatively recent previous volume. The kinds of studies continue to expand and diversify, andmethodologiesarecontinuouslybeingrefinedandimproved.Amongstareasofintense activity are those related to the very early commitment of stem cells to particular lineages andprogressionofdifferentiationtomaturecellstages.Iamverygratefulonceagaintothe contributors who have shared their insights as well as new and improved methods for othersalreadyinorjustenteringthefield. I would like to acknowledge Dr. John Walker, Editor in Chief of the Methods in MolecularBiologyseries,forhiscontinuoussupportandPatrickMarton,ExecutiveEditor oftheseries,forencouragementduringdevelopmentofthevolume.Iamalsoverygrateful toDavidCaseyforbeingavailabletoanswerallofmyquestionsandforprovidingguidance forshepherdingthevolumethroughproduction. Ottawa,ON,Canada KursadTurksen v Contents Preface.................................................................... v Contributors............................................................... ix MaximizingClonalEmbryonicStemCellDerivationbyERK PathwayInhibition......................................................... 1 JenniferNicholsandThorstenBoroviak ASimpleandEfficientMethodofSlowFreezingforHuman EmbryonicStemCellsandInducedPluripotentStemCells..................... 15 KeitaroImaizumi,MomoeIha,NaokiNishishita, ShinKawamata, ShinichiNishikawa,andTeruoAkuta ResolvingHeterogeneity:Fluorescence-ActivatedCellSorting ofDynamicCellPopulationsfromFeeder-FreeMouseEmbryonic StemCellCulture ......................................................... 25 JurriaanHölzenspies,GeloDelaCruz,andJoshuaM.Brickman AcquiringGroundStatePluripotency:SwitchingMouseEmbryonic StemCellsfromSerum/LIFMediumto2i/LIFMedium ...................... 41 MatteoTosoliniandAliceJouneau GenerationofEmbryonicStemCellsinRabbits............................... 49 MarielleAfanassieff,PierreOsteil,andPierreSavatier GeneTransferintoPluripotentStemCellsviaLentiviralTransduction ........... 67 OrtwinNaujok,UlfDiekmann,andMatthiasElsner ImagingPluripotency:Time-LapseAnalysisofMouseEmbryonic StemCells ................................................................ 87 AnnaPezzarossa,AnaM.V.Guedes,DomingosHenrique, andElsaAbranches ExpandingtheUtilityofFUCCIReportersUsingFACS-Based OmicsAnalysis ............................................................ 101 JamesChappell,BenBoward,andStephenDalton SelectionofAntibodiesInterferingwithCellSurfaceReceptor SignalingUsingEmbryonicStemCellDifferentiation.......................... 111 AnnaN.Melidoni,MichaelR.Dyson,andJohnMcCafferty DeliveringAntisenseMorpholinoOligonucleotidestoTarget TelomeraseSpliceVariantsinHumanEmbryonicStemCells.................... 133 LidaRadan,ChrisS.Hughes,JonathanH.Teichroeb, Lynne-MariePostovit,andDeanH.Betts AnalysisofmRNATranslationRateinMouseEmbryonicStemCells ............ 143 AnisaB.RahimandLeahA.Vardy GenerationandPurificationofDefinitiveEndoderm Cells GeneratedfromPluripotentStemCells....................................... 157 UlfDiekmannandOrtwinNaujok vii viii Contents DefinitiveEndoderm DifferentiationofHumanEmbryonic StemCellsCombinedwithSelectiveElimination of UndifferentiatedCellsbyMethionineDeprivation .......................... 173 TomonoriTsuyama,NobuakiShiraki,andShoenKume SelectiveDifferentiationintoHematopoieticandCardiac CellsfromPluripotentStemCellsBasedontheExpression ofCellSurfaceMarkers..................................................... 181 AtsumasaOkada,KatsuhisaTashiro,TomokoYamaguchi, andKenjiKawabata MethodsforDerivationofMultipotentNeuralCrestCells DerivedfromHumanPluripotentStemCells ................................. 197 JohnAveryandStephenDalton FromNaivetoPrimedPluripotency:InVitroConversion of MouseEmbryonicStemCellsinEpiblastStemCells ........................ 209 MatteoTosoliniandAliceJouneau InVitroDifferentiationofEmbryonicStemCellsinto HematopoieticLineage:TowardsErythroidProgenitor’sProduction ............ 217 IlianaFauzi,NickiPanoskaltsis,andAthanasiosMantalaris DopaminergicDifferentiationofHumanEmbryonicStem CellsonPA6-DerivedAdipocytes............................................ 235 M.OktarGulogluandAnnaLarsen IntracellularCalciumMeasurementsforFunctionalCharacterization of NeuronalPhenotypes.................................................... 245 ´ TalitaGlaser,AnaReginaG.Castillo,AgathaOliveira, andHenningUlrich InVitroDifferentiationofPluripotentStemCellsintoFunctional bIsletsUnder2Dand3DCultureConditionsandInVivo PreclinicalValidationof3DIslets............................................ 257 BipashaBoseandSudheerShenoyP GenerationofCornealKeratocytesfromHumanEmbryonic StemCells ................................................................ 285 AndrewJ.HertsenbergandJamesL.Funderburgh Maintenance,TransgeneDelivery,andPluripotencyMeasurement ofMouseEmbryonicStemCells............................................. 295 TetsuyaS.Tanaka GenerationofaKnockoutMouseEmbryonicStemCellLine UsingaPairedCRISPR/Cas9GenomeEngineeringTool...................... 321 RahelWettstein,MaximeBodak,andConstanceCiaudo DerivationofNeuralPrecursorCellsfromHumanEmbryonic StemCellsforDNAMethylomicAnalysis .................................... 345 IvanRoubal,SunJooPark,andYongKim 26SandPA28-20SProteasomeActivityinCytosolicExtracts fromEmbryonicStemCells................................................. 359 MalinHernebring Contents ix AConciseProtocolforsiRNA-MediatedGeneSuppression inHumanEmbryonicStemCells............................................ 369 PeterF.RenzandTobiasA.Beyer ApplyingShearStresstoPluripotentStemCells............................... 377 RussellP.Wolfe,JuliaB.Guidry,StephanieL.Messina, andTabassumAhsan GeneratingInnerEarOrganoidsfromMouse EmbryonicStemCells...................................................... 391 EmmaLongworth-Mills,KarlR.Koehler,andEriHashino DifferentiationofAdipocytesinMonolayer fromMouse EmbryonicStemCells...................................................... 407 IxcheltCuaranta-Monroy,ZoltanSimandi,andLaszloNagy PancreaticDifferentiationfromMurineEmbryonicStemCells.................. 417 DaisukeSakano,NobuakiShiraki,andShoenKume OsteogenicDifferentiationfromEmbryonicStemCells ........................ 425 YanhongYu,CarlosPilquil,andMichalOpas LimbalStromalTissueSpecificStemCellsandTheir DifferentiationPotentialtoCornealEpithelialCells............................ 437 KishoreReddyKatikireddyandUlaV.Jurkunas Erratum .................................................................. 445 Index..................................................................... 447