ThehumanepisomaliPSCreprogrammingkitfromALSTEMisoneofthebestchoicesforproducingfootprint-freeiPSCs,providingasourceofiPSCsforallstagesofyourpluripotentstemcellresearch.OptimizedbytheALSTEMteam,thisepisomalkithasproventobesuccessfulininducingpluripotencyinanumberofdifferentsomaticcelltypes.
Description
Inducedpluripotentstemcells(iPSCs)aregeneticallyreprogrammedfromadultcells,whicharesimilartonaturalpluripotentstemcells,suchasembryonicstemcells(ESCs).iPSCsexhibitapluripotentstemcell-likestate,suchastheexpressionofcertainstemcellgenesandproteins,chromatinmethylationpatterns,teratomaformation,andpotencyanddifferentiability.Whiletheseartificiallygeneratedcellsarenotknowntoexistinthehumanbody,theyshowqualitiesremarkablysimilartothoseofembryonicstemcells.Therefore,iPSCsareaninvaluableresourcefordrugdiscovery,celltherapy,andbasicresearch.
HumaniPSCswerefirstgeneratedin2007throughretrovirus-orlentivirus-mediatedgenetransduction.However,retroviralorlentiviralvectorsrequireintegrationintohostchromosomestoexpressreprogramminggenes.Integration-freehumaniPSCshavebeengeneratedusingseveralmethods,includingadenovirus,Sendaivirus,thepiggyBacsystem,minicirclevector,episomalvectors,directproteindeliveryandsynthesizedmRNA.Thereprogrammingefficiencyoftheseintegration-freemethodsisimpracticallylowinmostcases.DirectdeliveryofproteinsorRNAislabor-intensive,requiringrepeateddeliveryofthereprogrammingfactors.ModifyingSendaivirusvectorsorpreparingsynthesizedmRNAsaretechnicallydemanding.
TheHumaniPSCellReprogrammingEpisomalKitisanoptimizedmixtureofmultiplevectorsthatcanreprogramsomaticcellstoiPSCswithoutintegration.TheepisomalvectorshavetheoriP/EBNA-1(Epstein-Barrnuclearantigen-1)backbonethatdeliversthereprogrammingfactorsaswellaspuromycinresistantgene.Thissystemhasbeensuccessfullydemonstratedinreprogrammingoffibroblasts,aswellasotheradultcells,toiPSCS.HighexpressionoftransgenesduetooriP/EBNA-1mediatednuclearimportandretentionofvectorDNAallowsiPSCderivationinasingletransfection.Thereprogrammingefficiencyisfurtherenhancedbypuromycinselection.Inaddition,silencingoftheviralpromoterwhichdrivesEBNA-1expressionandthelossoftheepisomesatarateof~5%percellcycleduetodefectsinvectorsynthesisandpartitioningallowstheremovalofepisomalvectorsfromtheiPSCswithoutanyadditionalmanipulation.
ForoptimalreprogrammingwiththeHumaniPSCellReprogrammingEpisomalKit,culturethefibroblastsinFibroblastMediumuntilthedayoftransfection.Aftertransfection,allowthecellstorecoverinFibroblastMediumfor24hours,andthenaddpuromycintoremovetheuntransfectedcells.Reseedthecellsonfeedersabout5daysposttransfection,andswitchtheculturemediumtohESCculturemediumnextday.Thisreprogrammingprotocolisverysimpleandtheeffective.PD0325901,CHIR99021,A-83-01,hLIF,orHA-100isnotrequiredforreprogramming.
CreateYourOwnFootprint-freeiPSCells
HumaniPSCellReprogrammingEpisomalKitisacost-effectiveplasimdmixofmultipleepisomalvectorswhichencodefourreprogrammingfactors.Otherreprogrammingmethods,suchaslentivirusandretrovirus,containtransgenesthatcanintegrateintothehostgenome,potentiallydisruptingthegenomeorcausingunpredictableresults.Thisepisomalmixisabletoefficientlygeneratetransgene-free,virus-freeandfootprint-freeinducedpluripotentstemcells(iPSCs)inbothfeederandfeeder-freeconditions.OptimizedbyALSTEMteam,thisepisomalmixhasproventobesuccessfulininducingpluripotencyforanumberofdifferentsomaticcelltypes.
Theseepisomalvectorsareintroducedintothecellbyelectroporation.AsoriP/EBNA1vectors,theycontainallthereprogrammingfactorsandreplicateextrachromosomallyonlyoncepercellcycle.Atthisreplicationrate,theepisomesarelostatarateofapproximately5%percellgeneration.
GenerateiPSCLinesfromaWideVarietyofSomaticCellTypes
iPScellshavebeengeneratedbyepisomalvectorsfromarangeofsomaticcellsincludingfibroblasts,bonemarrowmononuclearcells,PBMCs,lymphoblastBcells,andvariousdisease-typefibroblastsandPBMCs.Eachkitprovidesenoughmaterialfor10reprogrammingexperiments.
Thefollowingprotocolhasbeenoptimizedforhumandermalfibroblastcells.Werecommendthatyouoptimizetheprotocolforyourcelltype.
I.Feeder-FreeReprogrammingProtocol
Day-1:SeedCells
1.Seed1x106humanfibroblastsinagelatincoatedT75flask.Cellsshouldreachapproximately75–90%confluenceonthedayoftransfection(Day0).
Day0:NucleofectionofEpisomalPlasmidstoHumanFibroblasts
2.PreparetheNeonTransfectionDevicesandkits(using10µltipsinthisprotocol).
3.Preparethegelatin-coated6wellplateandwarmupFibroblastMedium(w/oP/S)intheplate.
4.AspiratethemediumfromthefibroblastsintheT75flask,andwashthecellswithDPBSwithoutcalciumandmagnesium.Add2mlof0.05%Trypsin/EDTAtotheflask,andincubatetheflaskat37°Cfor3minutes.
5.Add5mlofFibroblastMediumtotheflask.Taptheflasktoensurethecellsaredislodgedfromtheflask,andcarefullytransferthecellsintoanew15-mlconicaltube.Spindowncellsat1,000rpmfor5minutesatroomtemperature.
6.ResUSPendthecellsin2mlofDPBS.Countthecells,andthentake3x105cellsintoeach1.5mltubefortwotubes.
Note:Youneed3x105cellsforonetransfection,andthecellnumbercanbemodifiedto1.5–6x105cellspertransfection.
7.Spindownthecellsat2000rpmfor5minatroomtemperature.Inthemeantime,add3mlofsolutionE2tothemicroporationtube,andmix1.5µgofreprogrammingvectorsinone1.5mltubewithe10µlSolutionR.Inanothertube,mix0.5µgforRFPcontrolwith10µlSolutionR.
Note:Ifyouuse100-µltip,mix3µgofreprogrammingvectorsin100µlSolutionRand1µgforRFPcontrolin100µlSolutionR,respectively.
8.Carefullyaspiratemostofthesupernatant.ResuspendthecellpelletinSolutionRwithplasmids.
9.TurnontheNeonunitandentertheelectroporationparametersintheInputwindowto1,650Voltsofpulsevoltage,10msofpulsewidth,and3pulses.
Note:Toincreasetheviability,youmayusetheelectroporationparametersof1,400Voltsofpulsevoltage,30msofpulsewidth,and1pulse.
10.Pressthepush-buttonontheNeon®PipettetothefirststopandimmersetheNeon®Tipintothecell-DNAmixture.Slowlyreleasethepush-buttononthepipettetoaspiratethecell-DNAmixtureintotheNeon®Tip.
Note:Avoidairbubbleswhenpipettingtoavoidarcingduringelectroporation.Ifyounoticeairbubblesinthetip,discardthesampleandcarefullyaspiratefreshsampleintothetipagainwithoutanyairbubbles.
11.InserttheNeon®PipettewiththesampleverticallyintotheNeon®TubeplacedintheNeon®PipetteStationuntilyouhearaclick.EnsurethatyouhaveenteredtheappropriateelectroporationparametersandpressStartontheNeon®touchscreentodelivertheelectricpulse.
12.Immediatelyafterelectroporation,thecellsuspensionsolutionispouredintowarmFibroblastMedium(w/oP/S)inonewellofa6-wellplatepre-coatedwithgelatin.Culturetheelectroporatedcellsin37°C,5%CO2incubator.
Day1,3,5:ChangetheFbroblastMedium
13.ChangemediumtoFibroblastMediumwithP/S,supplementedwith0.5µg/mlofpuromycin.
Note:IfRFPplasmidisusedtomonitorthetransfectionefficiency,theRFPexpressioncanbedetectedduringthesedays.Byaddingpuromycin,nonpuro-resistantcellsdislodge.
Note:DoNOTtreattransfectedcellswithpuromycinmorethanthreedays.
Day6:ReplatingtheTransfectedCells
14.Aspiratethemediumoftransfectedfibroblasts,andwashthecellsinDPBSwithoutcalciumandmagnesium.Add0.5mlof0.05%Trypsin/EDTAtothewell,andIncubatetheplateat37°Cfor2minutes.
15.Add1mlofFibroblastMediumtothewell.Taptheplatetoensurecellsaredislodgedfromtheplate,andcarefullytransferthecellsintoanew15-mlconicaltube.Spindownthecellsat1,000rpmfor5minutesatroomtemperature.
16.Resuspendthecellsin2mlofFibroblastMedium.Countthecells.Seed1x104,2x104,and4x104transfectedfibroblasts,respectively,inthewellsofa6-wellplatecoatedwithMatrigel.
Note:Thecellnumberscanbechangedfrom0.5-3x105cellswhenusinga100-mmdish.Theseedingdaycanvaryaccordingtothecells.
Day7:SwitchtoiPSGenmedium
17.Aspiratethemediumofthereprogrammingfibroblasts,andadd2mlofiPSGenMedium(StemRD).
18.Changethemediumeverydayuptoday14.
Day14:SwitchtoPSGro/mTeSR1medium
19.Aspiratethemediumofthereprogrammingfibroblasts,andadd2mlofPSGro/mTeSR1medium.
20.ChangePSGro/mTeSR1mediumeveryday.
Day24–30:PickingiPS-likecolonies
ByDay24ofpost-transfection,thecellcoloniesintheMatrigel-coatedplatesconsistofiPSCs,whichexhibitahESC-likemorphologycharacterizedbyaflatter,cobblestone-likeappearancewithindividualcellsclearlydemarcatedfromeachotherinthecolonies.
21.Examinetheplatecontainingthereprogrammedcellsunder10Xmagnificationofaninvertedmicroscope,andmarkthecolonytobepickedatthebottomoftheplate.
Note:Werecommendpickingatleast10distinctcoloniesbytheendofeachreprogrammingexperimentandexpandingtheminseparate24-wellMatrigel-coatedplate.
22.Transfertheplatetoabiosafetycabinetequippedwithastereomicroscope.
23.Cutthecolonytobepickedinto5–6piecesinagrid-likepatternusinga25-gauge1½inchneedle.
24.Usinga200-µLpipette,transferthecutpiecestoafreshlypreparedMatrigel-coatedwellofa24-wellplatecontainingmTeSR1mediumsupplementedwith10µMROCKinhibitor(Y-27632,StemRD).
25.IncubatetheMatrigel-coatedplatecontainingthepickedcoloniesina37°C,5%CO2incubator.
26.Allowthecoloniestoattachtothecultureplatefor48hoursbeforereplacingthespentmediumwithfreshmTeSR1medium.Sincethen,changethemediumeveryday.
27.CulturethereprogrammedcolonieslikenormalhumaniPSCcolonies;expandandmaintainthemusingstandardcultureprocedures.
Note:NewlyderivediPSClinesmaycontainafairamountofdifferentiationthroughpassage4.Itisnotnecessarytoremovedifferentiatedmaterialpriortopassaging.Bypropagatingthecellstheoverallculturehealthshouldimprovethroughouttheearlypassages.Otherwise,pickuptheiPS-likecoloniesandcultureinMatrigelcoated24-wellplate.
II.ReprogrammingProtocolUsingMEFFeeders
Day-1:SeedCells
1.Seedhumanfibroblastsat1x106cellsinagelatincoatedT75flask.Cellsshouldreachapproximately75–90%confluenceonthedayoftransfection(Day0).
Day0:NucleofectionofEpisomalPlasmidstoHumanFibroblasts
2.PreparetheNeonTransfectionDevicesandkits
3.Preparethegelatin-coated6wellplatesandwarmupFibroblastMedium(w/oP/S)intheplates.
4.AspiratethemediumfromthefibroblastsintheT75flask,andwashthecellswithDPBSwithoutcalciumandmagnesium.Add2mlof0.05%Trypsin/EDTAtotheflask,andincubatetheflaskat37°Cfor3minutes.
5.Add5mlofFibroblastMediumtotheflask.Taptheflasktoensurethecellsaredislodgedfromtheflask,andthencarefullytransferthecellsintoanew15-mlconicaltube.Spindowncellsat1,000rpmfor5minutesatroomtemperature.
6.Resuspendthecellsin2mlofDPBS.Countthecells,andtake3x105cellsintoeach1.5mltubefortwotubes.
Note:Youneed3x105cellsforonetransfection,andthecellnumbercanbemodifiedto1.5–6x105cellspertransfection.
7.Spindownthecellsat2000rpmfor5minutes.Inthemeantime,add3mlofsolutionE2tothemicroporationtube,andmix1.5µgofreprogrammingvectorsinone1.5mltubewith10µlSolutionR.Inanothertube,mix0.5µgforRFPcontrolwith10µlSolutionR.
Note:Ifyouuse100-µltip,mix3µgofreprogrammingvectorsin100µlSolutionRand1µgforRFPcontrolin100µlSolutionR,respectively.
8.Carefullyaspiratemostofthesupernatant,andresuspendthecellpelletinSolutionRwithplasmids.
9.TurnontheNeonunitandentertheelectroporationparametersintheInputwindowto1,650Voltsofpulsevoltage,10msofpulsewidth,and3pulses.
Note:Toincreasetheviability,youmayusetheelectroporationparametersof1,400Voltsofpulsevoltage,30msofpulsewidth,and1pulse.
10.Pressthepush-buttonontheNeon®PipettetothefirststopandimmersetheNeon®Tipintothecell-DNAmixture.Slowlyreleasethepush-buttononthepipettetoaspiratethecell-DNAmixtureintotheNeon®Tip.
Note:Avoidairbubbleswhenpipettingtoavoidarcingduringelectroporation.Ifyounoticeairbubblesinthetip,discardthesampleandcarefullyaspiratefreshsampleintothetipagainwithoutanyairbubbles.
11.InserttheNeon®PipettewiththesampleverticallyintotheNeon®TubeplacedintheNeon®PipetteStationuntilyouhearaclick.EnsurethatyouhaveenteredtheappropriateelectroporationparametersandpressStartontheNeon®touchscreentodelivertheelectricpulse.
12.Immediatelyafterelectroporation,thecellsuspensionsolutionispouredintowarmFibroblastMedium(w/oP/S)inonewellofa6-wellplatepre-coatedwithgelatin.Culturetheelectroporatedcellsin37°C,5%CO2incubator.
Day1,3,5:ChangetheFibroblastMedium
13.ChangemediumtoFibroblastMediumwithP/S,supplementedwith0.5µg/mlofpuromycin.
Note:IfRFPplasmidisusedtomonitorthetransfectionefficiency,theRFPexpressioncanbedetectedduringthesedays.Byaddingpuromycin,nonpuro-resistantcellsdislodge.
14.Onday5,seedtheMEF/SNLcellsinagelatin-coateddishes(5x105cellsperwellofa6-wellplateor3x106cellsper100-mmdish).
Day6:ReplatingtheTransfectedCells
15.Aspiratethemediumofthetransfectedfibroblasts,andwashthecellsinDPBSwithoutcalciumandmagnesium.Add0.5mlof0.05%Trypsin/EDTAtothewell.Incubatetheplateat37°Cfor2minutes.
16.Add1mlofFibroblastMediumtothewell.Taptheplatetoensurecellsaredislodgedfromtheplate,andcarefullytransferthecellsintoanew15-mlconicaltube.Spindownthecellsat1,000rpmfor5minutesatroomtemperature.
17.Resuspendthecellsin2mlofFibroblastMedium.Countthecells.Seed1x104,2x104,and4x104transfectedfibroblasts,respectively,ontothewellsofa6-wellplatepre-seededMEF/SNLfeedercells.
Note:Thecellnumberscanbechangedfrom5x104to3x105cellswhenusinga100-mmdish.Theseedingdaycanvaryaccordingtothecelltypes.
Day7:SwitchtohESMedium
18.Aspiratethemediumofthereprogrammingfibroblasts,andadd2mlofhESMediumsupplementedwith10ng/mlofbFGF.
19.Changethemediumeverydayuptoday15.
Day16:SwitchtohES/PSGroMedium
20.Aspiratethemediumofthereprogrammingfibroblasts,andadd2mlofhES/PSGroMedium.
Note:YoumayalsousemTeSR1(StemcellTechnologies)orEssential8Medium(LifeTechnologies)toreplacePSGromedium(StemRD).
21.ChangehEC/PSGroMediumeveryday.
Figure2.ExpectedmorphologyofemergingiPSCsduringepisomalreprogrammingofhumandermalfibroblastsastheyundergomorphologicalchangesandiPSCcoloniesbegintoemerge.
Day21–30:PickingiPS-likecolonies
ByDay21ofpost-transfection,thecellcoloniesintheMEFplateconsistofiPSCs,whichexhibitahESC-likemorphologycharacterizedbyaflatter,cobblestone-likeappearancewithindividualcellsclearlydemarcatedfromeachotherinthecolonies(seeFigure2).
22.Examinethecultureplatecontainingthereprogrammedcellsunder10Xmagnificationofinvertedmicroscope,andmarkthecolonytobepickedatthebottomofthecultureplate.
Note:Werecommendpickingatleast10distinctcoloniesbytheendofeachreprogrammingexperimentandexpandingtheminseparate24-wellMEFplate(orMatrigel-coatedplate).
23.Transferthecultureplatetoabiosafetycabinetequippedwithastereomicroscope.
24.Cutthecolonytobepickedinto5–6piecesinagrid-likepatternusinga25-gauge1½inchneedle.
25.Usinga200-µLpipette,transferthecutpiecestoafreshlyprepared24-wellMatrigel-coatedplatecontainingmTeSR1mediumsupplementedwith10µMROCKinhibitor(Y-27632,StemRD).
26.Culturepickedcoloniesina37°C,5%CO2incubator.
27.Allowthecoloniestoattachtothecultureplatefor48hoursbeforereplacingthespentmediumwithfreshmTeSR1medium.Sincethen,changethemediumeveryday.
28.CulturethereprogrammedcolonieslikenormalhumaniPSCcolonies;expand,andmaintainthemusingstandardcultureprocedures.
Note:NewlyderivediPSClinesmaycontainafairamountofdifferentiationthroughpassage4.Itisnotnecessarytoremovedifferentiatedmaterialpriortopassaging.Bypropagatingthecellstheoverallculturehealthshouldimprovethroughouttheearlypassages.Otherwise,pickuptheiPS-likecoloniesandculturein24-wellplatewithfeedercells.
Figure3.ALSTEMhiPSCepisomalreprogrammingkitprovideshighreprogrammingefficiency.Alkalinephosphatase(AP)positiveiPSCcolonieswithtypicalhumanESCmorphologywerecountedonday24-27post-transfection.ThenumberofiPSCcolonieswasfrom1x104transfectedcells.
Figure2.(A)AtypicalimageofthehumandermalfibroblaststransfectedwithRFPusingNeonTransfectionDevices(24hoursafterelectroporation).~30%offibroblastsareRFPpositive.(B)Afterpuromycinselection,morethan90%offibroblastsareexpressingRFP(~5daysafterpuromycinselection).
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