TheMITCenterforHumanTissueModelsofInfectiousDiseases(MIT.HTMID)willstudyviralinfectionsoffive typesofneuralcellsthataredifferentiatedfromhumanembryonicstem(ES)cellsorhumaninduced pluripotentstem(iPS)cells.Inaddition,MIT.HTMIDresearchwilladdressviralinfectionsofhumanthree dimensional(3D)cerebralorganoidstowardunderstandingZikavirusmicrocephaly.Allcellworkandtissue workfortheCenter?sresearchwillbesupportedbytheHumanCellsandTissuesCorefacility.TheCorewill belocatedattheWhiteheadInstitute,andwillprovideacontinuoussupplyofcellsforbothHT.MIDresearch projects.TopreparecellsandorganoidsfromiPScells,h?umanfibroblastsarereprogrammedintopluripotent iPScells.Next,thepluripotentiPScellsaredifferentiatedintomultipotentneuralprogenitors(NPs).?Neurons, astrocytesandoligodendrocytearedifferentiatedfromNPs.TogenerateahomogenouspopulationofNPs,a SMADinhibitionbasedneuraldifferentiationprotocolisroutinelyused.Adherentculturesofcontrolandmutant ESCs/iPSCswillbedifferentiatedintoneuralrosettesexpressingPax6andNestinasdetectedby immunofluorescence,andfurtherexpandedasneuronalprogenitorsinthepresenceofbFGF.M?ultipotentNPs aredifferentiatedintoneurons.?Togenerateneurons,NPsareculturedinagrowthfactordepletedmediumthat promotesterminaldifferentiationandmaturationofamixedpopulationsofneurons.Theseneuronsare electrophysiologicallyactive,asmeasuredonmultielectrodearrays.?Multipotentneuralprogenitorsare separatelydifferentiatedintoastrocytesoroligodendrocytes?.ToderiveastrocytesfromhumanNPs,wehave developedprotocolsthatallowtheproductionofhighlyhomogeneouspopulations.Specifically,FACSsorted PSANCAM?/A2B5?glialprogenitors,derivedfromNPs,areexpandedinthepresenceofEGFandbFGFto +? deriveS100b+immatureastrocytes.FurthermaturationofthisFACSsortedpopulation,inthepresenceof CNTF,yieldsGFAP+?astrocytes.Thecellscanbesortedtohighpuritybymagneticcellsorting(MACS)against CD44microbeads.Togenerateoligodendrocytes,A2B5+?glialprogenitorswillbeexpandedinbFGFand PDGF?,andfurthersortedbyMACSagainstO4andCD140amarkers.Thesefatedoligodendrocyte precursorsarematuredintoMBP+? oligodendrocytesaftergrowthfactorremovalandexposuretothethyroid hormoneT3.Theprojectinvestigatorsandcorestaffwillmeetregularlytoplanworkflowandanticipatecell needsforProjects1and2.TheHumanCellsandTissuescorewillbeanintegralandessentialpartofthe MIT.HTMIDCenter,anditsoutputwillenhancetheproject?spotentialforsuccess
