HumaniPSCellLine(Episomal,CD34+,ApoE4):#iPS16
ProductDescription
Footprint-freehumaniPS(inducedpluripotentstem)celllinewasderivedfromhumanbonemarrowCD34+mononuclearcellsbyectopicexpressionofOCT4,SOX2,KLF4,c-MYCandLin28genesusingepisomalplasmids.Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescent
Markerordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyofhumaniPScellsareidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersTRA-1-60,SSEA-3andOct4,anddemonstratestrongendogenousalkalinephosphataseactivity.
Figure1.CharacterizationofiPSCsderivedfromhumanbonemarrowCD34+mononuclearcells.ImmunostainingofhiPSCcoloniesexpressingESCspecificmarkersOCT4,Nanog,andTRA-1-60.
Figure2.Thecellshaveanormalkaryotype.
| Neuralrosette(ectoderm) | Bone(mesoderm) | Glandularstructure(endoderm) |
|---|
Figure3.Invivopluripotencytestbyteratomaformation.Variouscelltypessuchasneuralrosette(ectoderm),bone(mesoderm),andglandularstructures(endoderm)arefound.
| ProductSpecifications |
| ProductName | HumaniPSCellLine(Episomal,CD34+,ApoE4) |
| Catalog# | iPS16 |
| Size | >2x105cells/vial |
| Shipping | DryIce |
| StorageandStABIlity | Storeingasphaseofliquidnitrogenimmediatelyuponreceipt.Thisproductisstablefor6monthswhenstoredasdirected. |
| QualityControl | HumaniPScellsweregrowninfeederfreeconditionswithmTeSR1medium.EachlotofhumaniPScellsistestedforgrowthandviabilityfollowingrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofTRA-1-60andOct4,aswellastheactivityofalkalinephosphatase. |
| SafetyPrecaution | ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce. |
| RestrictedUse | ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures. |
| Protocol |  Download |
ThisprotocolcanbeusedforculturinghumaniPScells.Footprint-freehumaniPScellsweregeneratedbytransientlyintroducingepisomalplasmidsencodingthehumantranscriptionfactorsintohumanperipheralbloodmononuclearcells.Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentmarkersordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyoffootprint-freehumaniPScellsisidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersTRA-1-60andOct4,anddemonstrateastrongendogenousAPactivity.
I.Feederfreecultureconditions
Preparationoffeeder-freemedium
- ThawmTeSR15XSupplement(Cat.no.05850,STEMCELLTechnologies)atroomtemperatureorovernightat4°C.
- Addthe100mLofthawed5XSupplementto400mLBasalMediumforatotalvolumeof500mLaseptically.Mixwellandfilterthrougha0.2μm,low-proteinbindingfilter,ifdesired.
- Aliquotintoappropriateamountaccordingtousageandstorethealiquotsat4°C.
CoatingplateswithMatrigel
Matrigel(Cat.no.354277,BD)shouldbealiquotedandstoredat-80°Cforlong-termuse.
- Thawmatrigeloniceuntilliquid.Dilutematrigel1:30to1:50withpre-chilledKODMEM/F12.
- Immediatelyusethedilutedmatrigelsolutiontocoattissueculture-treatedplates.Fora6-wellplate,use1mLofdilutedmatrigelsolutionperwell,andswirltheplatetospreadthematrigelsolutionevenlyacrossthesurface.
- Letthecoatedplatestandfor1hat37°Corovernightat4°C.Ifplatehasbeenstoredat4°C,allowtheplatetoincubateat37°Cforatleast30minutesbeforeremovingthematrigelsolution.
ThawingcryopreservedhumaniPScells
- QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
- Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmmTeSR1dropwisetothetube,gentlymixingasthemediumisadded.
- Centrifugecellsat200xgfor5minutesatroomtemperature.
- Aftercentrifugation,aspiratethemediumfrom15mLtube.GentlyresUSPendthecellpelletin2mLmTeSR1with5µMROCKinhibitor,takingcaretomaintainthecellsassmallcellclumps.
- Removethematrigelsolutionfromacoatedtissueculture6-wellplate.Transferthemediumcontainingthecellclumpstothematrigelcoated6-wellplate.
- Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.
- Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).
PassaginghumaniPScellsgrownunderfeeder-freeconditions
- Useamicroscopetoidentifyregionsofdifferentiation.Markthedifferentiatedcoloniesusinglensmarkeronthebottomoftheplate.
- RemoveregionsofdifferentiationbyscrapingwithaPipettetiporbyaspiration.
- AspiratemediumfromthehumaniPScellcultureandrinsewithDPBS(2mL/well).
- Add1mLperwellofEZStemEnzyme-FreeStemCellDissociationSolution(cat.no.M100,ALSTEM),andincubateat37°Cfor2-3minutes.Oradd0.5mLperwellofaccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse),andincubateat37°Cfor1minute.
- RemoveEZStemEnzyme-FreeStemCellDissociationSolutionoraccutase,gentlyrinseeachwell2-3timeswith2mLofDMEM/F-12perwellandtransferthedetachedcellaggregatestoa15mLconicaltube.
- Add2mL/wellmTeSR1andscrapecoloniesoffwithacelllifter.Transferthedetachedcellaggregatestoa15mLconicaltube.
- Rinsethewellwithanadditional2mLmTeSR1tocollectanyremainingaggregates.Addtherinsetothe15mLtube.
- Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
- Aspiratethesupernatant.ResuspendpelletinmTeSR1containing5µMROCKinhibitorbygentlypipettingandensurethatcellsaremaintainedasaggregates.
- PlatethehumaniPScellaggregateswithmTeSR1inanewplatecoatedwithmatrigel.(Removematrigelsolutionbeforeplating).
Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.
- Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.
- Changemediumdaily.
CryopreservinghumaniPScells
- PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
- Performsteps1-8fromPassaginghumaniPScellsgrownunderfeeder-freeconditions.
- Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
- Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
- Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
- Placevialsintoafreezingcontainerandplacethecontainerat-80°Covernight.
- Transfertoaliquidnitrogentanknextday.
II.Feeder-dependentcultureconditions
PreparationofhumanESmedium
KnockoutDMEM/F12containing20%knockoutserumreplacement,2mMglutamine,0.1mMnonessentialaminoacids,0.1mM2-mercaptoethanol,10ng/mlbFGF,and50Uand50µg/mlpenicillinandstreptomycin.
ThawingcryopreservedhumaniPScells
Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingonthecells.DuetothelowsurvivalrateofcryopreservedhumaniPScells,therecoveryisexpectedtotakeatleastoneweek.
- QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
- Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmhumanESmediumdropwisetothetube,gentlymixingasthemediumisadded.
- Centrifugecellsat200xgfor5minutesatroomtemperature.
- Whilecentrifuging,removeMEFmediumfromthefeedercellplates,andwashthewellstwicewithKnockoutDMEM/F12.Thenadd1mlofhumanESMediumwith5µMROCKinhibitor(Y-27632,StemRD)toonewellof6-wellplate.
- Aftercentrifugation,aspiratethemediumfrom15mLtube.Gentlyresuspendthecellpelletin1mLfreshhumanESmediumcontaining5µMROCKinhibitor(Y-27632),takingcaretomaintainthecellsassmallcellclumps.
- Transferthemediumcontainingthecellclumpstoonewellof6-wellplatewithMEFfeedercells.
- Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.
- Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).
PassaginghumaniPScellsgrownunderfeeder-dependentconditions
- Aspiratethemediumandwashthecellstwicewith1mlofPBS.
- RemovePBScompletely,add0.5mlofAccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse)andincubatefor1-2minat37°C.
- Tapthebottomoftheplatetodislodgethecellsfromthebottomoftheplate.Thenaspiratetheaccutase.
- Add1mlofDMEM/F12totheplateandcarefullywashthefeedercells,andaspiratethemedium.Repeat.
- Add1mlofhumanESmediumcontaining5µMROCKinhibitortotheplateandsuspendthecellcoloniesbygentlypipettingupanddown.Itisimportantnottobreakupthecoloniesintosinglecells.
- RemoveaplateofMEFfeedercellsfromtheincubator.AspiratetheMEFmedium.WashoncewithKODMEM/F12medium.
- Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
- Add1mLhumanESmediumtotheoriginalwellandscrapecoloniesoffwithacelllifter.
- Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
- AddhumanESmediumwithROCKinhibitortoafinalvolumeof2mlperwell.RightafterplatingtheiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.
Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.
- After24hours,removethemediaandreplacewithhumanESmedia(withoutROCKinhibitor).
- ThehumanESmediamustbechangedeverydayandhumaniPScellssubculturedevery5-7days.Trackthepassagenumberofthecells.
CryopreservinghumaniPScells
- PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
- Performsteps1-6fromPassaginghumaniPScellsgrownunderfeeder-dependentconditions.
- Transferthedetachedcellaggregatestoa15mLconicaltube.
- Add1mLhumanESmediumandscrapecoloniesoffwithacelllifter.
- Transferthecellsuspensiontothe15mLconicaltube.
- Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
- Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
- Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
- Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
- Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.
- Transfertoaliquidnitrogentanknextday.
