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.