JournalofVisualizedExperimentswww.jove.comCopyright©2012CreativeCommonsAttribution-NonCommercialLicenseJune2012|64|e3854|Page1of5VideoArticlePreparationofMouseEmbryonicFibroblastCellsSuitableforCulturingHumanEmbryonicandInducedPluripotentStemCellsJustynaJozefczuk1,KatharinaDrews1,JamesAdjaye11DepartmentofVertebrateGenomics,MaxPlanckInstituteforMolecularGeneticsCorrespondenceto:JustynaJozefczukatjagodzin@molgen.mpg.de,JamesAdjayeatadjaye@molgen.mpg.deURL:http://www.jove.com/video/3854DOI:doi:10.3791/3854Keywords:StemCellBiology,Issue64,MolecularBiology,DevelopmentalBiology,mouseembryonicfibroblasts(MEFs),humanembryonicstemcells(hESCs),humaninducedpluripotentstemcells(hiPSCs),ActivinA-conditionedmedium(CM),cellcultureDatePublished:6/21/2012Citation:Jozefczuk,J.,Drews,K.,Adjaye,J.PreparationofMouseEmbryonicFibroblastCellsSuitableforCulturingHumanEmbryonicandInducedPluripotentStemCells.J.Vis.Exp.(64),e3854,doi:10.3791/3854(2012).AbstractIngeneral,humanembryonicstemcells(hESCs)andhumaninducedpluripotentstemcells(hiPSCs)1canbeculturedundervariableconditions.However,itisnoteasytoestablishaneffectivesystemforculturingthesecells.SincethecultureconditionscaninfluencegeneexpressionthatconferspluripotencyinhESCsandhiPSCs,theoptimizationandstandardizationoftheculturemethodiscrucial.TheestablishmentofhESClineswasfirstdescribedbyusingMEFsasfeedercellsandfetalbovineserum(FBS)-containingculturemedium2.Next,FBSwasreplacedwithknockoutserumreplacement(KSR)andFGF2,whichenhancesproliferationofhESCs3.Finally,feeder-freeculturesystemsenableculturingcellsonMatrigel-coatedplatesinKSR-containingconditionedmedium(mediumconditionedbyMEFs)4.Subsequently,hESCscultureconditionshavemovedtowardsfeeder-freecultureinchemicallydefinedconditions5-7.Moreover,toavoidthepotentialcontaminationbypathogensandanimalproteinsculturemethodsusingxeno-freecomponentshavebeenestablished8.Toobtainimprovedconditionsmousefeedercellshavebeenreplacedwithhumancelllines(e.g.fetalmuscleandskincells9,adultskincells10,fore...
