Download Mouse embryonic stem cells can differentiate via multiple

bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Mouseembryonicstemcellscandifferentiateviamultiplepathstothesame
state
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Authors:JamesA.Briggs1,*,VictorC.Li1,*,SeungkyuLee2,CliffordJ.Woolf2,AllonM.Klein1,+,andMarcW.
Kirschner1,+
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Affiliations:1DepartmentofSystemsBiology,HarvardMedicalSchool,Boston,MA02115.2Department,
ofNeurobiology,HarvardMedicalSchool,andFMKirbyNeurobiologyCenterBostonChildren’sHospital,
Boston,MA02115.
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Abstract
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Inembryonicdevelopment,cellsmustdifferentiatethroughstereotypicalsequencesofintermediate
statestogeneratematurestatesofaparticularfate.Bycontrast,directprogrammingcangenerate
similarfatesthroughalternativeroutes,bydirectlyexpressingterminaltranscriptionfactors.Yetthecell
statetransitionsdefiningthesenewroutesareunclear.Weappliedsingle-cellRNAsequencingto
comparetwomousemotorneurondifferentiationprotocols:astandardprotocolapproximatingthe
embryoniclineage,andadirectprogrammingmethod.Bothundergosimilarearlyneuralcommitment.
Then,ratherthantransitioningthroughspinalintermediateslikethestandardprotocol,thedirect
programmingpathdivergesintoanoveltransitionalstate.Thisstatehasspecificandabnormalgene
expression.Itopensa‘loop’or‘wormhole’ingeneexpressionthatconvergesseparatelyontothefinal
motorneuronstateofthestandardpath.Despitetheirdifferentdevelopmentalhistories,motor
neuronsfrombothprotocolsstructurally,functionally,andtranscriptionallyresemblemotorneurons
fromembryos.
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Introduction
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Embryonicdevelopmentproceedsthroughdefinedintermediatestates,suchasgermlayer
intermediates,andlineage-specificprogenitors.Intermediatesbifurcateintomultiplestatesovertime,
andspecializetheirbehaviors,ultimatelyproducingalineagetreethatdefineseachmaturecelltypeby
aparticularsequenceofintermediates.Thiswasfirstappreciatedthroughclassicallineagetracingand
cellablationstudies.Thesestudiesshowedthatspecificallylabeledintermediatestatesgenerate
stereotypedsetsofdownstreamcelltypes,andthatthesedownstreamcelltypesfailtoformifan
intermediatethatisupstreamintheirlineageisablated1,2.Furthermore,embryosingeneraldonot
produceamaturecelltypethroughmultipledifferentiationpaths.
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Incontrasttothisrigidandhierarchicalprocess,recentprotocolsthatexperimentallydirectly
programcellfatesuggestthattheexactsequenceofintermediatesdefiningalineagemaybemore
flexible3-11.Thesestudiesrevealthatmaturecellstatescanbereachedthroughpathsthatdonotinvolve
activationoftheintermediateprogenitorgenesthatareessentialinembryos.Mouseembryonicstem
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Theseauthorscontributedequallytothiswork.
Correspondence:marc@hms.harvard.edu;Allon_Klein@hms.harvard.edu
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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cells(mESCs),forexample,canbeconvertedintomotorneurons(MNs)byaprocessthatinvolves
overexpressionofthreetranscriptionfactors,Ngn2+Isl1+Lhx33,12,andthatneverexpressestheneural
progenitortranscriptionfactorsSox1andOlig23.mESCscanalsobedrivenrapidlyintoaterminalmuscle
phenotypewithoutnormalupregulationofintermediategenessuchasPax7andMyf5,througha
combinationofcell-cycleinhibitionandMyoDoverexpression4.Thisplasticityofdifferentiationextends
further,totheinterconversionofmaturecellstates.Fibroblastscanbeconvertedintomatureneuron
phenotypes6,11,includingMNs5,seeminglywithoutcompletelydedifferentiatingandretracingthe
embryoniclineage,asindicatedbylackofexpressionofspecificcorepluripotency(Oct4,Sox2and
Nanog)andneuralprogenitorgenes(Nestin)5.
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Althoughthesedirectprogramming(DP)experimentsimplytheexistenceofdifferentiation
pathsthatdifferfromthoseinembryos,muchofwhatactuallyoccursinthesenewprogramsremains
mysterious.DoesDPbypassnormalintermediatesbyshort-circuitingthenaturallineage,ordoesit
transitionthroughalternativeintermediates(Fig.1a)?Doesitdivergeonlybrieflytobypassspecificearly
orlatestates,ordoesitutilizeanentirelydistinctpath(Fig.1b)?AndcanDPconvergefullytothesame
finalstatethatisproducedinembryosdespitetakinganalternativepath(Fig.1c)?Thesequestionshave
beenchallengingtoanswerinpartduetothehighdegreeofheterogeneityindirectprogramming
experiments,whereunbiasedbulkmeasurementsofglobalgeneexpressionobscurechanges,andalso
becausemarkergenesallowingtheisolationofnewbutpotentiallyimportantDP-specificintermediates
arenotknowninadvance.HereweaimedtoovercometheseissuesbyapplyingsinglecellRNA
sequencingtocomparethegeneexpressiontrajectoriesofDPandgrowthfactorguideddifferentiation
ofmESCsovertimeintoMNs.OurcoreresearchquestionsaresummarizedinFigure1.
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Results
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DissectionoftwoMNdifferentiationprotocolsusingInDropssinglecellRNAsequencing
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WecomparedtwoinvitrodifferentiationprotocolsthatconvertmESCsintoMNs.SpinalMNswere
chosenforstudybecausetheseprotocolshavebeenhighlyoptimized.Thefirst,standardprotocol(SP)is
awidelyusedmethodthatattemptstorecapitulatetheknownembryonicintermediatesthrough
sequentialexposureofdevelopmentalsignals(Fgfs,RetinoicAcid,andSonichedgehog)(Fig.1A) 13,14.It
providesanapproximationofthelineagethroughwhichmotorneuronsdevelopintheembryo.The
second,directprogramming(DP)protocolinvolvesdrivingtheexpressionoftranscriptionfactors
(Ngn2+Isl1+Lhx3)3,12thatcharacterizethematuremotorneuronstateandatthesametimefavoringand
stabilizingtheG1statebyincubatinginagrowthfactorfreemedium4.
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Weusedsingle-cellRNAsequencing(InDrops)15totrackthedifferentiationtrajectoriesofboth
protocolsovertime(Figs.1Band1C).Single-celldatahasemergedasapowerfulwaytotrace
differentiationprocesses,particularlyinpopulationsthatarenotpureandthatcontainrare
intermediates11,16-20.Weprofiledatotalof4,590cellssampledfromearly(day4/5)andlate(day11/12)
timepointsforeachprotocol,andalsousedourpreviouslypublisheddatafrom975mEScells15.To
visualizethesinglecelldataandidentifycellstatesweappliedt-distributedstochasticneighbor
embedding(tSNE)toreducedimensionality21,22,definedcellstatesusinganunsuperviseddensity
gradientclusteringapproach,andthenfoundspecificmarkergeneswithknownannotationstoreveal
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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theidentityofeachstate(Fig.2B-2E;Supp.Figs.S1andS2;Supplementarymethods).Foreach
protocol,thedominantfeaturewasacontinuousgeneexpressiontrajectorysweepingacrossthe2D
plot.Thesetrajectoriescorrelatewithchronology:theybeginwithmESCs,passthroughneural
progenitorstatesandterminateinmatureMNstates.Inbothprotocolswealsoobservedamixtureof
off-targetdifferentiationbyproductsfromallthreegermlayers.
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OursinglecelldataallowedustodefinetheefficiencyofMNproductionforeachmethod.For
DP,MNproductionwasobservedasearlyasday4(19%),andincreasedovertimeto66%byday11(Fig.
2F).Aminorityofoff-targetneuronsubtypes,glia,andmiscellaneouscellswerealsoidentified.IntheSP
23%and9%ofthepopulationresembledMNsatdays5and12respectively(Fig.2G).Thislower
efficiencywasaccompaniedbyafarlargerfractionofoff-targetproductsincludingoligodendrocytes
(7.2%),astrocytes(8.6%),muscle(30.6%),andstroma(8.9%)thattogetheraccountedfor55.3%ofthe
populationbyday12.
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TheDPdifferentiationtrajectorylacksintermediatesexpressingOlig2andNkx6-1
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Whatarethedifferentiationpathstakenbyeachprotocol?IntheSPdifferentiationpath,cellstransit
throughsevenstates(Fig.2Cand2E).Thesestatetransitionsparallelpatterningeventsinthe
embryo13,23,24:cellsfirstcommittotheneurallineage(Sox1+/Sox3+),thenareposteriorized
(Hoxb8+/Hoxd4+),ventralized(Nkx6-1+/Olig2+),enterthecommittedMNprogenitorstate(Mnx1+),and
thenmatureintoaneuronalphenotype(Tubb3+/Map2+).Thisisnotasurpriseasthegrowthfactor
cocktaildefiningthismethodwasdesignedtoreflectthesignalingeventstakingplaceintheembryo.By
contrast,wefoundthatthepathproducedbyDPwascondensedrelativetotheSPpath(Fig.2Band2D),
consistingofonlyfourstatesasopposedtoseven.Afterneuralcommitment(Sox1+),cellsimmediately
beganexpressingcommittedMNmarkers(Mnx1+/Tubb3+),seeminglywithoutthetypicalspinal
embryonicintermediates(Olig2-/Nkx6-1-).AlackofOlig2expressionduringDPhasbeenobserved
previously3,andourresultsconfirmatthesinglecelllevelthatintermediatesexpressingOlig2andNkx61appearentirelyabsent.Olig2isnecessaryforMNdevelopmentinembryos1,indicatingtheDPdrives
differentiationfromESCintoMNsthroughanewroute.
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Weconfirmedthatourinferreddynamicsfromsnapshotsingle-celldatacorrespondtothe
actualunderlyingdifferentiationdynamicsbyperformingadenseqPCRtimecourseforapanelofMN
genes(Supp.Fig.S3).Thesebulkmeasurementsconfirmedthat,forDP,committedMNmarkersare
upregulatedimmediatelyfollowingearlyneuralprogenitorgenesinrealtime.
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TheDPandSPtrajectoriesbifurcateafterearlyneuralcommitmentandconvergeseparatelytothe
MNstate
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SincetheDPomitsspinalembryonicintermediatescharacteristicoftheSPpath,theremustbeoneof
twopossibletrajectories.EitherDPmustdiscontinuouslytransitionfromanearlyneuralprogenitorinto
aMN,oritmusttransitthroughalternateintermediatestate(s).Todeterminewhichofthese
possibilitieswasthecase,weemployedadatavisualizationtechniquecalledSPRING25todirectly
comparethetopologyofbothpaths.WhiletSNEisapowerfulmethodforidentifyingdiscretecellstates,
SPRINGprovidesacomplementarydescriptionemphasizingcontinuumgeneexpressiontopologies.
SPRINGbuildsak-nearest-neighborgraphovercellsinhigh-dimensionalgeneexpressionspace,and
thenrendersaninteractive2Dvisualizationofthecellgraphusingaforcedirectedlayout.This
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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representationrevealedthattheDPandSPtrajectoriesoverlapduringearlyneuralcommitment,but
thattheythenbifurcateandtransitdistinctpathsthatconvergeindependentlytothesameMNstate
(Fig.3A).Thedynamicsofgeneexpressionoverthesetrajectoriesresembledthebehaviorinferredusing
tSNE,withDPomittingintermediateprogenitorgenesfollowingitsbifurcationfromtheSPpath(Fig.3B).
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Thebifurcationandsubsequentconvergenceofthetwodifferentiationpathscanalsobe
appreciatedbytwoothercomplementaryanalyses.Pairwisecosinesimilaritiesbetweenthecellstates
frombothtrajectories(Fig.3C;Supplementarymethods)indicatesimilaritiesbetweentheearlystates
(ESCandNP;cosinesimilarity>0.64)andlatestates(LMN;cosinesimilarity=0.55),butnotthe
intermediatestates(PNP,PVNP,andMNP;cosinesimilarity<-0.28,-0.09,and0.06respectively).We
alsoassignedeveryindividualcellalongtheDPpathtoitsmostsimilarclusterintheSPpathusinga
maximumlikelihoodmethod(Fig.3D;Supplementarymethods).Thisshowedthatitwasvirtually
impossibletofindasinglecellresemblingtheSPintermediateprogenitorsintheDPapproach.Similarity
wasagainseenonlyattheearlyandlatestates.
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DPtransitionsthroughanabnormalintermediatestatewithforebraingeneexpression
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ThebifurcationoftheSPandDPtrajectoriesleadstodifferentintermediatecellstatesineachcase.A
totalof26transcriptionfactors(TFs)aredifferentiallyexpressedbetweentheDPandSPintermediate
states(Fig.4A).Amajorityofthese(61%)wereinvolvedinananterior-posteriorpositionalgene
expressionaxis.TheSPintermediateswereenrichedmorethan6-foldfornineposteriorandspinalTFs
includingOlig2,Nkx6-1,Lhx3,andsixHoxgeneswithacorrectedp-value<0.001.EachoftheseTFsis
expressedinembryonicMNs.Bycontrast,theDPintermediateswereenrichedforsevenforebrainTFs
includingOtx2,Otx1,Crx,Six1,Dmrta2,Zic1,andZic3atthesamestringency,despitetheabsenceof
MNsintheforebrainofembryos.Anteriorgeneexpressionwaspreviouslyobservedthroughbulk
measurementsofDP3,andourresultsrevealthatitoccurswithinaspecificsubpopulationofcellsinthe
processofdifferentiatingintoMNs.Wevalidatedtheseexpressiondifferencesbyisolatingintermediate
populationsofeachdifferentiationpathusingaMnx1::GFPreportercellline,sinceMnx1expressionis
localizedpreciselytothedistinctintermediatepopulationsofeachpath(Fig.3B).Bulkcomparisonsof
thesetwopopulationsconfirmedtheenrichmentofforebrainTFs,anddepletionofspinalprogenitor
andpositionalgenesintheDPintermediateswithjustoneexception–Zic1wasenrichedinDPbyour
singlecellcomparisonbutinSPbymicroarray(Fig.4A).
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TheabnormalpositionalgeneexpressionsignaturethatcharacterizestheDPintermediatestate
appearstransient.ForebraingeneexpressionisupregulatedalongtheDPdifferentiationpathascells
exittheearlyNPstateintotheEMNintermediatestate(Fig.4B).Thistransitionisalsoaccompaniedby
thedownregulationofproliferation-associatedgenes(Fig.4B;Supp.Fig.S4).Bythetimecellsexitthe
EMNstateandtransitionintothemoredifferentiatedLMNstate,theydownregulateforebraingenes
andreplacethisabnormalpositionalsignaturewithaspinalHoxexpressionsignaturecharacteristicof
normalMNs(Fig.4B).ThuscellsconvergetotheMNstateinpositionalaswellasneuronalidentitygene
expressioninthefinalstagesofDP.
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BothDPandSPapproachatranscriptionalstatesimilartobona-fideMNsinembryos
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Giventhatthetwoprotocolsinducedistinct–andinthecaseofDP,unnatural–differentiationpaths,
wewerecurioushowtheirfinalproductscomparedwithprimaryMNs.WeharvestedMNsfromthe
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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embryoofaMnx1::GFPreportermouseandperformedinDropsmeasurementson874E13.5MNsafter
FACSpurification.ThoughthemajorityofHb9+sortedcellswereMNs(73.8%),thispopulationalso
containedglia(20.1%),fibroblast-likecells(1.8%),andimmune-typecells(1.2%;Fig.5A;Supp.Fig.S5).
Usingonlythecellsidentifiedasbona-fideMNs,weprobedthesimilarityoftheinvitroderivedcellsto
theprimaryMNs,usingthreemeasures:globalsimilarityofthetranscriptomes(cosinesimilarity);coclusteringfrequency;anddifferentialgeneexpressionanalysis.Inbothpaths,neuronsbecomemore
similartoprimaryMNsovertime(Fig.5B).TheclustersmosthighlycorrelatedwithprimaryMNswere
theLMNstatefromtheDPprotocol(cosinesimilarity=0.62),andtheLMNstatefromtheSP(cosine
similarity=0.37).Notably,only2.7%ofoutputcellsfromtheSPwereintheLMNstate,comparedto
62.7%forDP.Thus,theratiooftheefficiencyofformingLMNsbytheDPprotocoltotheSPprotocolis
23fold,seven-foldhigherthanwhatwecalculatedbasedonacomparisonofmarkergenesalone.Atthe
levelofsinglecells,co-clusteringofthedifferentexperimentsshowedthat95%ofDPMNsrobustlycoclusterwithprimaryMNscomparedwith26%forSPderivedMNs(Supp.Fig.S6).However,differential
geneexpressionanalysisrevealedthatneitherprotocolperfectlyrecapitulatesthegeneexpression
profileofMNsisolatedfromthemouse.BothprotocolsshowedadepletionofthemostposteriorHox
genes,perhapsindicatingananteriorspinalcordidentityofinvitroMNs,andasmallenrichmentof
severalgenesrelatedtomicrotubulefunctionandcellcycleexitthatmayindicatesubtledifferencesin
neuronalmaturation(Supp.Fig.S7).
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DPMNshavestructuralandfunctionalpropertiesoftrueMNs
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HavingestablishedthatMNsderivedviabothgeneexpressiontrajectoriesreachroughlythesameMN
transcriptionalstate,wewishedtovalidatethattheirfunctionandstructuralorganizationwasalso
independentoftheirdistinctdevelopmentalhistories.TheSPhasbeencharacterizedextensivelyas
givingrisetofunctionalMNs13,sohereweexaminedstructuralandfunctionalcharacteristicsfollowing
DP.WeconfirmedthatselectedproteincontentmatchesthemRNAmarkersbyimmunostainingfor
Tubb3,Map2,VACht,Isl1,andHb9(Fig.5C).Tubb3andMap2werepresent,andVAChtwasseenat
discretepunctaontheaxons(suggestinglocalizationtoacetylcholinesecretoryvesicles).TFsIsl1and
Hb9werelocalizedinthenucleus.Finally,theGFPfromtheMnx1::GFPreporterwasactivatedand
expressedinthecytoplasm.TotestthefunctionalpropertiesoftheDPMNs,weperformedwhole-cell
patchclamprecordings.Depolarizationinducedsingleormultipleactionpotentialsincurrent-clamp
experiments(Fig.5D).Depolarizingvoltagestepsinducedfastinwardcurrentsandslowoutward
currentscharacteristicofsodiumandpotassiumchannels,respectively(Fig.5E).Exposureto500nM
Tetrodotoxin(TTX)blockedtheinwardcurrent,indicatingsodiumchannelinvolvement.Wethentested
whetherourDPneuronswouldrespondtoneurotransmittersthatactonMN(Fig.4F).Exposureofthe
neuronstoAMPA,kainate,GABA,andglycine(100µMeach)inducedineachcaseinwardcurrents
similartothatseeninprimaryembryonicMNs.ToseeiftheDPneuronscouldalsoformneuromuscular
junctions,weco-culturedtheneuronswithdifferentiatedC2C12skeletalmusclemyotubesand
incubatedthemfor7days.WeobservedclusteringofacetylcholinereceptorsontheC2C12myotubes
nearcontactpointswiththeDPneurons,whichcanbeseenwithalpha-bungarotoxin(α-BGT),which
bindstoacetylcholinereceptors(Fig.4G).WethenobservedregularcontractionsofsomeC2C12
myotubesthatbeganafterseveraldaysinco-culture(Fig.4G,Supp.Video1).Thesecontractionscould
bestoppedbytheadditionof300µMTubocurarine(curare),anantagonistofacetylcholinereceptors,
indicatingthatthecontractionswereinducedbytheacetylcholinereleasefromtheMNs.Similarly,we
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
is the dauthor/funder. It is made available
a CC-BY
4.0 International
license.
E13.5 HB9+ primarypeer-reviewed)
cells
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5 second interval
Fig. 5. Validation that DP and SP trajectories both approach a bona-fide MN state. a) tSNE visualization of 874 single cell
transciptomes from FACS purified Mnx1+ MNs from embryos reveals heterogeneity within this population. b) Comparison of cell
states along both trajectories with true primary MNs (orange population in a). In both DP and SP similarity increases as differentiation proceeds. Late DP states are the most similar to embryonic MNs. c) Immunostaining of MN markers in DP MNs confirming expression and correct subcellular localization of Tubb3, Map2, VACht, Isl1, and Hb9. DP MNs also: d) can fire single or
multiple action potentials upon stimulation, e) show sodium and potassium channel currents, and f) are responsive to multiple
MN neurotransmitters - AMPA, kainate, GABA, and glycine. g) Co-culture experiments show that DP MNs can induce clustering
of acetylcholine receptors on muscle myotubes (indicated by α-BGT staining) and induce their contractions. The induced
contractions are dependent on MN activity as they can be blocked by addition of the acetylcholine antagonist curare.
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noticedthattheDPMNscouldinducecontractionsinDPmusclemyotubesthatwepreviously
generatedwithMyoD(Supp.Video2)4.TheseresultsconfirmthatDPMNshavetheexpectedfunctional
propertiesofbona-fideMNs.
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Discussion
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Theresultswehavedescribedprovideevidenceatthesinglecelllevelthatdifferentiationcanproceed
bymultipleroutesyetconvergeontosimilartranscriptionalstates.WeshowthatwhileusingtheSPcells
aredriventoretracetheembryoniclineage,DPinducescellstodifferentiatethroughadramatically
differentpath.TheDPpathbypassesmultipleintermediateprogenitor-statesthatevolvedinthe
embryo,andyetstillconvergestothesamediscreteandrecognizableMNphenotype.Thisconvergence
occursfromanabnormalintermediatestate,anddoesnotappeartoinvolveasharedsetofterminal
cellstatetransitions;itishighlyorthogonal.Moreover,ascellsconvergetheymanagetonotonly
establishgeneexpressionrelatedtoMNfunctions,buttheyalsocorrectpositionalgeneexpression
defects(exchangingforebrainforspinalgeneexpression)intheabsenceofexternalsignals.Relativeto
ourinitialresearchquestions(Fig.1),weconcludethatDPofmESCsintoMNsoccursviaalatebypass
thatinvolvesalternativeintermediatestatesnotseenintheembryo,andthatthisnewrouteconverges
nearperfectlytothesamefinalstate.ConvergenceintoaMNthereforedoesnotappeartodepend
rigidlyontheprecisehistoryofintermediatestatesthroughwhichcellsdifferentiate.
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This‘historyindependence’ofthefinalstateisconsistentwithadynamicalviewofgene
regulationinwhichcellstatescorrespondto‘attractorbasins’,i.e.stablestatesofgeneexpressionthat
arerobusttomodestperturbations.Ifattractorbasinsdonotexist,theprecisionoftheobserved
overlapbetweenDPandSPMNswouldrequireaspecialcoincidence,likefindinganeedleinahigh
dimensionalhaystack.Theconceptofcellstatesbehavingasattractorshasbeenproposedpreviouslyto
explainseveralpropertiesofbloodcelltypes26-28.Thereareatleasttwoimportantcorollariesofthis
behaviorapplyingindevelopment.Fromapracticalperspective,itisacommonconcernthatDP
methodsmaygeneratecelltypeswithsubtledefectsduetotheirunusualdevelopmentalhistories9.
Attractorswouldberobusttothisvulnerabilityandindeedourresultsshowthatitisnotnecessaryto
recreatetheprecisesequencesofstepstakeninembryostogeneratebona-fideMNs.Itcouldalsohint
atamechanismthatmighthelpanimalbodyplansevolveflexibly.Specifically,bydecouplingthe
identityofmaturecellstateattractorsfromtheirdevelopmentalhistoriesevolutionwouldbeabletoact
oneachindependently.Inprinciplethiscouldcontributetoevolvabilitybyallowingmaturecellstatesto
betransposedontonewlineagesinnewbodylocations.
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ThemechanismsthatdefinetheMNattractorbasinandallowtheartificialDPtrajectoryto
convergeontothecorrectfinalstatearelargelyunclear.TheMNstateisthoughttobestabilizedbya
networkofself-reinforcingTFs24,involvingMnr2,Mnx1,Lim3,Isl1,Isl2,andLhx3,Ngn2,Myt1l,Nefl,and
Nefm.DPaimstokick-startthisnetworkbyactivatingasubsetofimportantcomponents.Yet,farfrom
immediatelyactivatingthisnetwork,ourdatashowthatDPinitiallydrivescellstodifferentiateintoan
earlyNPstatethroughthesamepathwayastheSPtrajectory,seeminglyoblivioustotheDPTFs,and
thenevenactivatesnon-MNgenesinthetransitionalstate.UnderstandingwhytheactivationoftheMN
programlagsbehindTFinductionmayprovideimportantcluesintohowtheDPfactorsact.Onepossible
sourceforalagisthatactivatingacompleteneuronalprogramrequiresfirstactivatingadditionalcore
TFs(so-called‘feed-forward’circuitry).Indeed,recentstudieshaveshownthatEbfandOnecutare
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
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activatedbyNgn2(oneoftheDPTFs),andthatbotharerequiredtosubsequentlydirectbindingofIsl1
andLhx3(theothertwoDPTFs)toMNtargetgenesacrossthegenomeduringDP12,29.Asecondpossible
sourceoflagisthatextracellularsignalingprovidesinputsthatimmediatelyaffectcellstate,buttake
timetosensitizecellstotheDPfactors.Forexample,signalingchangesmightactivateDPTFsthrough
post-translationalmodifications,byactivatingco-factors,orbyinducingchromatinstatechanges.We
haveindeedobservedthatMNsarenotgeneratedifDPTFsareinducedincellsculturedinpluripotency
media,indicatingarequirementforchangesinsignaling(notshown).Conversely,whenmEScellsare
transferredtominimalmediawithoutinducingDPfactors,theyacquireaforebrainneuralprogenitor
identitybydefault30-32.Thissuggeststhattheearlydynamicsandabnormalforebrain/MNexpressionof
theDPtransitionalstatemightinfactbedrivenbythesignalingenvironmentandnottheDPTFs.These
alternativessuggestfutureexperimentstobetterresolvethemechanismsdrivingtheDP,byre-mapping
thetrajectoryinducedduringDPafterchangingsignalingconditions,orthechoiceofDPTFs.
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Asamethodology,DPissignificantlymoreefficientthantheSPwithoutlossofqualityintheMN
populationsproduced(Fig.2F-G;Fig.5).Thehigh-efficiencyofDPlikelyderivesfrombothitsmore
uniformexperimentalconditionsaswellasitsmoredirectdifferentiationpath.Experimentally,DP:
relieson2Dratherthan3Dtissueculture(asintheSP),minimizinguncontrolledcell-cellcommunication;
forceseveryindividualcelltoexpressMNTFsfromageneticallyintegratedconstruct,increasing
uniformity;andemploysadefined-mediawithoutgrowthfactorsthatmayminimizeproliferationof
progenitorstates.ThemoredirectdifferentiationpathinducedbyDPshouldalsoitselfincreaseMN
conversionefficiencybyminimizingerrorpropagationthroughchainedopportunitiesforoff-targetfate
choices.Duringsequencesofintermediatecellstatetransitions,eachtransitioncanhavecompetingofftargetfates.Thus,differentiationprocessesthatinvolvemanysequentialintermediatetransitionssuffer
frommultiplicativeefficiencylosses.Indeed,thelongersequenceofintermediatestatestheinSP
generatesafarlargerfractionofoff-targetpopulationsthatincreaseswithtime,suggestinga
progressivelossofefficiency.Targetingterminalattractorbasinsthroughtheshortestpossible
differentiationpathsmayprovetobeagenerallyeffectivestrategytogeneratedesiredcellstates.
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Acknowledgements
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30
WearegratefultoEstebanMazzoniforprovidingthetranscriptionfactorcassettecontainingEScellline
usedintheDPexperimentspresentedhere.
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32
Competinginterests
33
V.L.andM.K.arecofoundersofStemCellerant,LLC.A.K.andM.K.arecofoundersof1CellBio,Inc.
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Authorcontributions
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Allauthorshelpeddesignthestudyanditsexperimentalquestions.V.L.establishedtheMN
differentiationprotocolsandperformedtheirmolecularcharacterization.J.B.performedthesingle-cell
datacollectionandanalysis.S.L.didtheelectrophysiologyrecordings.M.K.,A.K.andC.W.assistedwith
12
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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analysisandinterpretationofexperimentalresults.Allauthorscontributedtothewritingofthe
manuscriptandpreparationoffigures.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
19
bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
1
SupplementaryMethods
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ESCculture
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ThemouseEScelllinecontainingdoxycycline-inducibleNgn2+Isl1+Lhx3(NIL)andtheHb9::GFPreporter
wasgraciouslyprovidedbyEstebanMazzoni.ESCswereculturedinstandardmedia(DMEMwithLIF+
15%fetalbovineserum)on0.2%gelatin-coateddishes.
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Differentiationintomotorneurons:directandstandardprogramming
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Twenty-fourhoursbeforestartingdifferentiation,ESCsweretrypsinizedandseededontoplatesprecoatedwithamixofpoly-d-lysine(100μg/ml)andlaminin(50μg/ml)insteadofgelatinforadherence.
ESCswerecountedbyaBeckmanCoulterCounterandseededatadensityofapproximately200,000
cellsperwellofa6-welldish.Atday0(ESCs),themediawasswitchedfromstandardESmediatoN2B27
media(Invitrogen).Doxycyclinewasalsoaddedat3μg/mlstartingtoinduceexpressionoftheNIL
transcriptionfactors.Mediawaschangeddaily.Forthestandardprogrammingprotocol,thesteps
describedinWuetal.werefollowedstrictly.
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Mouseembryonicmotorneuroncultures
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TheB6.Cg-Tg(Hlxb9-GFP)1Tmj/Jmice(JAX#005029)werebredwithC57BL/6J(JAX#000664)for
embryonicmotorneuronsdissection.AllanimalprotocolswereapprovedtheInstitutionalAnimalCare
andUseCommitteeatBostonChildren’sHospital.Ongestationalday13(E13),thefemalemicewere
anesthetizedandallembryoswerecollectedbycaesariansection.OnlyGFPembryoswereusedfor
furtherdissection.Thespinalcordswereisolatedandtheirmeningeswereremoved.Eachisolated
spinalcordwasdissociatedbytrypsinandmechanicaltrituration.Afterfilteringthecellswith100μm
strainers,thecellswerespundownandre-suspendedinPBS,andsubjectedtoflowcytometry.Cells
wererunthrougha100μmnozzleatlowpressure(20p.s.i.)onaBDFACSariaIImachine(Becton
Dickinson,USA).Aneuraldensityfilter(2.0setting)wasusedtoallowvisualizationoflargecells.
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SinglecelltranscriptomicsusingInDrops
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WedissociateddifferentiatingmESCculturesusinga0.25%Trypsin2mMEDTAsolution(Gibco).Primary
HB9+sortedmotorneuronsweredissociatedasabove.Dissociatedcellsuspensionswereverifiedtobe
monodisperseandofviability>95%usingacoultercounterwithtrypanbluestaining(BioRad).Wethen
performeddroplet-basedbarcodingreversetranscription(RT)reactionsandpreparedmassively
multiplexedsequencinglibrariesusingInDropsasdescribedinKleinetal.Briefly:cells,lysisandRT
reagents,andbarcodingprimersattachedtohydrogelbeadsarecombinedinnanolitersizeddroplets
suspendedinanoilemulsionusingtheInDropsmicrofluidicdevice.AbarcodingRTreactionisthen
carriedoutinthedropletemulsion,uniquelylabelingtheRNAcontentsofeverycellusingacellbarcode
anduniquemolecularidentifier(UMI).FollowingRT,barcodedemulsionsaresplitintobatches,andthe
emulsionisbroken.CombinedmaterialisamplifiedintoananomolarIlluminasequencinglibrary
throughaseriesofbulkreactions:secondstrandsynthesis,invitrotranscription(IVT),fragmentation,
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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RT2,andafinallowcyclenumberPCR.ThemajorityoftheamplificationtakesplaceduringIVTensuring
uniformlibrarycoverage.SinglecelllibrarieswerethensequencedoneithertheIlluminaHiSeqor
NextSeqplatforms.Readsweredemultiplexedusinganupdatedversionofthecustombioinformatics
pipelinedescribedinKleinetal..Apythonimplementationofthispipelineisnowpublicallyavailableon
GitHub.Briefly,itfiltersforreadswiththeexpectedbarcodestructure,splitsreadsaccordingtotheir
cellbarcode,alignsthemtoareferencetranscriptome(weusedGRCm38withsomeadded
mitochondrialgenometranscripts),andthencountsthenumberofdifferentUMIsappearingforeach
geneineachcell.Thefinaloutputisacountsmatrixofcellsvs.genesthatweloadedintoMATLABfor
furtheranalysis.
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Single-celldatacleanup:minimumexpressionthreshold,totalcountnormalization,stressedcell
removal
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Beforeperformingtheanalysesdescribedbelow,threestepsweretakentoensurethatthedatawasof
highquality.First,werequiredallcellstohaveatleast1000UMIsdetected.Thisremovedanysignal
potentiallycomingfromemptydroplets.Second,dataweretotalcountnormalizedtoensure
differencesbetweencellswerenotduetotechnicalvariationinmRNAcaptureefficiencyorcellsize.
Finally,cellsthathadahighstressgenesignaturewereexcludedfromanalysis.Stressedcellswere
initiallyrecognizedasasmallpercentageofcells(<10%)thatclusteredapartfromeverythingelseand
specificallyexpressedveryhighlevelsofamitochondrialgenesetthatisassociatedwithcellularstress.
Masksthatconvertrawcountsintoourfilteredsetareprovidedonline.
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Visualizationofsingle-celldatausingtSNE
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Tovisualizehigh-dimensionalsinglecelldata,dimensionalityreductionisessential.Wechoseto
implementtSNEasdescribedinKleinetal.Thecorestepsaresummarizedasfollows.Steps1-2preceed
tSNE,andfocusthealgorithmongenesthatbestdescribedifferencesbetweencellpopulations.
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1. Extractthetop1000highlyvariablegenes.Wedothisusingastatisticaltestderivedspecifically
forInDropsdata(Kleinetal.).
2. Extractprincipalvariablegenes.Principalvariablegenesareasubsetofthehighlyvariablegenes
fromstep2thatwefindbestdescribesthecellpopulationstructure.Thestepstofindprincipal
variablegenesare:
a. PerformPCAusingthetop1000biologicallyvariablegenes.
b. Identifythenumberofnon-trivialprincipalcomponents.Thisisdonebycomparingthe
eigenvalueofeachprincipalcomponentfroma.totheeigenvaluedistributionforthe
samedataafterbeingrandomized.Onlyprincipalcomponentswitheigenvalueshigher
thanthoseobservedonrandomdataareretained.
c. Extractgenesthatcontributemosthighlytotheseprincipalcomponentsbyimposinga
thresholdonthegeneloadingsforeachnon-trivialPC.
3. PerformtSNE.WeusedtheMATLABimplementationoftSNEfromVandeMarteenetal.As
input,wesuppliedz-scorenormalizedprincipalvariablegenes,andaskedtSNEtoperforman
initialPCAtoanumberofdimensionsequaltothenumberofnon-trivialprincipalcomponents
instep4.tSNEthentakescellsembeddedinthisPCAspaceandnonlinearlyprojectsthemonto
twodimensionsforvisualization.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Subpopulationanalysis
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Thefirstgoalofoursinglecellanalysiswastodescribetheidentityandproportionsofcellstates
generatedbyeachoftwomotorneurondifferentiationstrategies.Forthispurposewefoundthatgood
resultswereachievedusinglocal-densitygradientclusteringonthe2DtSNErepresentationofthedata.
Thisapproachprovidedaclearandnaturalcellstateclassificationthatwaswellalignedwithprior
knowledgeaboutmarkergeneexpressiondomains.Thestepswetookaresummarizedasfollows:
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1. PerformtSNE(asabove)oncellspooledfromalltimepointsforeachprotocol(e.g.Fig.2B).
2. Applylocaldensitygradientclusteringtodefinecellstates.
3. Identifygenesspecificallyexpressedbyeachcellstate,andusepriorknowledgeontheir
expressiondomainstogenerateacelltypeannotation(e.g.Fig.2D).
4. Quantifythefractionofcellsineachstateateachtimepoint(e.g.Fig.2F).
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Weidentifiedgenesthatwerespecificallyexpressedbyeachsubpopulationthroughpairwiset-testsand
visuallyinspectedtheirexpressionoverthetSNEembedding.InFigure2D-Eofthemaintext,weshow
thez-scorenormalizedexpressionofaselectionofmarkergenesthatweusedasaheatmap.Z-score
normalizationpreservesdifferencesbetweenpopulationswhileputtingtheexpressionlevelofevery
geneonthesamescale.InSupplementaryFigs.S1andS2,weshowtheun-normalizedexpressionof
eachofthesegenesindividuallysothatthereadermaycompare.
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Initialidentificationofdifferentiationtrajectories
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Duringoursubpopulationanalysisweobserved,forbothprotocols,acontinuousprogressionofcells
thatspannedtheinitialEScellstatethroughtheearlyandlatedifferentiationtimepoints.This
progressionwaspunctuatedbyfamiliarprogenitorstatesthatwereorderedinawaythatwas
consistentwithknowneventsinmotorneurondifferentiation(Figure2D-E),andwascorrelatedwith
chronology(Figure2B-Cinset).Weinterpretedtheseprogressionsasdifferentiationtrajectories.Eachis
reconstructedfromthreepopulationsnapshots(day0,day4/5,andday11/12).Becausedifferentiation
invitroisasynchronous,thesinglecelldataoverlappedfromonetimepointtothenext.Wededuce
fromthisthatintermediatestateswerenotmissedduetothespacingofourtimepoints.Wealso
validatedthatimportantintermediategeneswerenotdetectedoveradenselysampledtimecourse
usingqPCR(seebelow).
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Differentialgeneexpressionanalysisbetweencellstatesfromsingle-celldata
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Weidentifieddifferentiallyexpressedgenesbetweencellstatesbyusingtwo-tailedt-testswitha
multiplehypothesistestingcorrection.Wedefineddifferentiallyexpressedgenesconservativelyata
FDRof5%andasignificancelevelofp<0.0001.Weonlyconsideredgeneswhereatleastoneofthe
statesbeingcomparedhad>=10cellswithnon-zeroexpression.Tofindmarkergenesofapopulation
weaskedforgenesthatwereenrichedinthatpopulationversuseverythingelse.Forseveral
comparisonswerestrictedouranalysistoRikentranscriptionfactors.Thislistcontains~1,500genes
withmanuallyannotatedtranscriptionfactoractivity.Werepresenteddifferentialexpressiondatausing
25
bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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volcanoplots,andcoloredtheexpressionintensityofeachgeneusingacolorbar;themeanofthe
higherexpressingstatewasused.
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ComparisonofdifferentiationpathsI:Visualizationofalternatedifferentiationtrajectoriesusing
SPRING
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Afterourinitialidentificationofthedifferentiationpathsforthestandardprotocolandfordirect
programmingwewishedcomparetheirroutes.Oneofthemostpowerfulwaystobeginaddressingsuch
aproblemistosimplyvisualizethedata.Yet,wefoundtSNEgaveunclearresultsforadirectcomparison
ofthepaths;visualizingbothprotocolstogetherresultedinsomemixedclusters,andsomedistinct
clusters,butnooverallcoherencetotherepresentationaswehadfoundlookingatoneortheother
trajectoryseparately.ThelimitationsoftSNEforanalyzingcontinuousprocessesarewellknown.
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Wethereforeturnedtoanewmethoddevelopedinparalleltothisworkinourlabcalled
SPRING,thatinourexperiencedoesbetterinanalyzingcontinuousprocessesinsinglecelldata.SPRING
hasthreecoresteps:first,itreducesdimensionalitytoa50dimPCAspace;second,itconstructsaknearest-neighbor(kNN)graphinthisspace;finally,itrendersaninteractive2DvisualizationofthiskNN
graphusingaforcedirectedlayout.InthisvisualizationedgesofthekNNgraphareliterallyspringsthat
pulltogethersimilarcells,whileeverycellhasamagneticrepulsionforcethatpushesitawayfromother
cellsandanintrinsicgravity.Thebalanceoftheseforcesilluminatesthetopologyofhowcellsare
positionedinhigh-dimensionwithrespecttooneanother.Inthisvisualizationnodescanbemoved
aroundtorotatetheprojectionandfindtheclearestrepresentation;ineachnewpositionthegraphrerelaxesaccordingtotheunderlyingphysicsoftheforcedirectedlayout.Thespecificstepswetookto
generateFig.3Aareasfollows.NotethatasmallmanualcorrectionwasmadetothepositionoftheESC
andLMNpopulationstoreducewhitespace,butdidnotdistortrelationshipsbetweenpopulations.
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1. LoadthecellsalongeachdifferentiationpathintoSPRINGseparately.
2. Removedoublets.Doubletswereidentifiedbythreecriteria.1)theyarerare,inlinewithour
experimentalexpectationforcelldoublets,2)theyformedlongrangeconnectionsbetween
largecellgroupsinthesametimepointandsample,3)theydonotpossessanyuniquemarker
genes;allgenestheyexpressarealinearcombinationoftwoothercellstates.Weidentified
approximately80doubletsinthestandardprotocol(<3%ofallcells),andapproximately40in
thedirectprogrammingapproach(<2%ofallcells).
3. LoadthefilteredcellsfrombothprotocolsintoSPRINGtogether.Tomakeplotsthecoordinates
fromtheSPRINGrepresentationwereexportedintoMATLAB;cellswereeithercoloredbycell
state(Fig.3A),orgeneexpression(Fig.3B).
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ComparisonofdifferentiationpathsII:Pairwisecosinesimilarityofcellstatecentroids
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Wealsoaskedhowthedirectprogrammingandstandardmotorneurondifferentiationpathswere
relatedtoeachotherbyperformingapairwisecomparisonofcellstatecentroids.Centroidsarethe
averageorcenterofmassingeneexpressionspaceforacollectionofcells.Centroidscanprovideavery
accurateestimateofglobalgeneexpressionthataveragesoverthenoiseintrinsictosingle-cellRNA
sequencingatthelevelofindividualcells.Bycomputingthecosinesimilaritybetweentwocellstate
centroidsweareaskinghowsimilarthesestatesareinaveragegeneexpression.Ifthepathsdoindeed
26
bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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splitandthenreconvergeourexpectationwastoseesimilaritybetweenearlyandlatestatesinboth
progressions,butnotbetweentheintermediatestates.WechosetoperformthesecomparisonsinaPVgenespaceconstructedfromcellsinbothprotocolstomakeourcomparisonsassensitiveaspossible.
Weobtainedsimilarbutlesssensitiveresultsusingallgenesaboveaminimumexpressionvalue(not
shown).Asummaryofthestepsofthisanalysisis:
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1. Combineallcellsfrombothprotocols,extractPV-genes(asdescribedabove),andz-score
normalizetheirexpression.
2. CalculatethecentroidofeveryclusterfromeachtrajectoryinthisPV-genespace.
3. Computethepairwisecosinesimilarityforalldirectprogrammingclustersversusallstandard
protocolclusters.
4. Visualize:wechosetouseaheatmap(Fig.3C).
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ComparisonofdifferentiationpathsIII:Maximumlikelihoodassignmentofsinglecellstocellstate
centroidsbetweentrajectories
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Finally,weperformedanindependentcomparisonofthedifferentiationpathsthatdidnotdependon
ourdefinitionofclusterboundariesinthedirectprogrammingtrajectory.Weaskedwhetherany
potentiallyrareindividualcellsorsubpopulationsinthedirectprogrammingtrajectoryresemblethe
intermediatestatesofthestandardtrajectory.Becausewewerenowdealingwithsinglecells,not
averagesofmanymeasurements,carewasnecessaryinthisanalysistoremainrobustrelativetothe
noiseinsinglecellmeasurements.WethereforetookaBayesianapproachandreasonedasfollows.In
ourdata,eachcellisavectorofcounts,withoneelementforeverygene.Thesecountscanbeviewed
asamultinomialsamplefromsomeunderlyingdistributionofgeneexpression.Sinceeachstateofthe
standarddifferentiationtrajectoryisdefinedbyaparticulargeneexpressiondistribution,wecanask:
whatistheprobabilityagivendirectprogrammingprotocolcellwassampledfromeachstandard
differentiationtrajectorycluster?Inthisusage,probabilityamountstoameasureofsimilarity,withhigh
probabilityindicatinghighsimilarity.WeobtainedsimilarresultsworkingineitheraPV-geneexpression
space,orconsideringallgenesexpressedaboveaminimumcountsthreshold.Theresultsofthisanalysis
usingPV-genesarepresentedinFigure3D.Wealsoobtainedsimilar(butmorenoisy)resultsusing
cosinesimilarityasanalternativedistancemetric(notshown).Thespecificstepsofourcomputationare
asfollows:
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1. Extractasetofgeneswithwhichtomakecomparisons(eitherPV-genesorallgenesabovea
minimumexpressionthreshold).
2. Foreachclusterofthestandardtrajectory,calculatetheprobabilityofobservingagivengene
(i.e.thefractionofcountsinthatclusterfromthegene).Forgenesthatarenotdetectedadd
1e-07totalcounts.
3. Foreachcellinthedirectprogrammingtrajectory,calculatethelog-likelihoodthatitwasdrawn
fromeachoftheseclusters.Thislog-likelihoodisfromthemultinomialdistributionfunction
usingtheprobabilitiesobtainedinstep2.
4. Identifyandtallythemaximumlikelihoodassignmentsofalldirectprogrammingcells.
Normalizerawassignmentssothattheysumto100(givingthepercentage).Plotthepercentage
ofdirectprogrammingcellsassignedtoeachstandardprotocolstate.
27
bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Cellcyclegeneexpressionanalysis
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Cell-cycleactivitycanbeestimatedfromacellcycleassociatedgeneexpressionsignature;populations
thatexpresshigheraveragelevelsofcellcyclegenesaremostlikelycyclingathigherfrequencythana
populationwithlowerlevelexpressionofthesegenes.InFig.4bandFig.S4weperformedananalysisin
thisspirittodeterminewhichpartsoftheDPandSPdifferentiationtrajectoriesappearedtobe
proliferative,andtoestimatewherecellsareexitingthecellcycle.Wecomputedaproliferationscore
thatwastheaggregateexpressionofapanelof21cellcyclerelatedgenes:Aurka,Top2a,Ccna2,Ccnd1,
Ccnd2,Ccnd3,Ccne1,Ccne2,Ccnb1,Cdk4,Cdk6,Cdk2,Cdk1,Cdkn2b,Cdkn2a,Cdkn2c,Cdkn2d,Cdkn1a,
Cdkn1b,Cdkn1c,Mcm6,Cdc20,Plk1,andPcna.Wealsocomputedacellcycleexitscoreonthebasisof
theaggregateexpressionofapanelof4tumorsuppressorgenesthatinhibitthecellcycle:Cdkn1c,
Cdkn1b,Cdkn1a,andCdkn2d.InFig.S4weshowtheexpressionofrepresentativeindividualgenesfrom
thisscore;ingeneralcellcyclegeneswerecorrelatedwitheachotherintheirexpressionovercells,as
werecellcycleexitgenes.
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Comparisonofmotorneuronsinvitrowithprimarymotorneuronsusingsingle-celldata
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Howdoesthetranscriptionalstateofmotorneuronsproducedbybothprotocolscomparetothatof
motorneuronsinvivo?Toanswerthisquestionweleveragedtheabilityofsingle-cellRNAsequencingto
comparecellstatesevenwithinpopulationsthatarenotpure(alsoseebelowforfunctional
comparisonsofthephenotypes).Weperformedthreeanalyses.First,wecomputedthecosinesimilarity
betweenthecentroidsofeachcellstateinbothprotocolsandprimarymotorneurons(Fig.5B).The
specificstepsofthisanalysiswereasfollows:
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1. Combineallcellstatesfrombothprotocols,andfromHB9+E13.5primarytissue,extractPVgenes(asdescribedabove),andz-scorenormalizetheirexpression.
2. Calculatethecentroidofeveryclusterfromeachtrajectory,andfromprimarymotorneurons,in
thisPV-genespace.
3. Computethecosinesimilarityforallinvitropopulationsversusprimarymotorneurons,and
visualizeasabargraph.
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Second,weperformedaco-clusteringanalysis(Supp.Fig.6).Ifcellsclustertogetherthisisanindication
ofsimilarity.Anadvantageofco-clusteringisthatitallowsonetomakestatementsatasinglecelllevel
aboutthefractionofcellswithinastatethatresembleanotherstate,forexample.Thespecificstepsof
ourcoclusteringanalysiswere:
1. Forstandardprotocol,directprogramming,andprimaryHB9+cells,filterforneurons:inthis
analysiswedefinedneuronsascellswith>2.5UMIsfromuniversalneuronalmarkerTubb3.
2. PerformtSNEandlocaldensitygradientclustering(asabove;Supp.Fig.6A).
3. Foreverycondition,countthefractionofcellsbelongingtoeachcluster.Similarstatesshould
belongtothesamecluster.
4. Visualize:wechosetouseapiechart.
Third,weperformeddifferentialgeneexpressionanalysis,comparingthemostmaturemotorneuron
statesfromeachprotocolwithprimaryHB9+motorneurons(Supp.Fig.7).Thisanalysiswasperformed
asdescribedabove.Wealsoperformeddifferentialexpressionanalysisofthesepopulationsusingbulk
microarraysasvalidation(seebelow).
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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qPCR
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RNAwasisolatedusingQiagenRneasyPlusKit.PurifiedRNAwasthenreversetranscribedusingBiorad'siSCRIPTcDNAsynthesiskit.QuantitativePCRwasperformedusingBio-rad'sSYBRgreenSupermix
onaCFX96Real-TimePCRsystem.
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Immunostaining
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Antibodiesusedforimmunostainingwereanti-Tubb3(CellSignalingD71G9;1:100),anti-Map2(Sigma
M9942;1:200),anti-VACht(SynapticSystems139103;1:200),anti-Isl1(Abcamab109517;1:1000),and
anti-Hb9(DSHB81.5C10;1:100).Differentiatedcellswerefixedin4%paraformaldehydefor20minutes
andthenpermeabilizedwith0.1%Triton-Xfor15minutes.Afterprimaryincubationfor1hour,samples
werelabeledwithasecondaryantibodyconjugatedtoAlexaFluor647.Sampleswereco-stainedwith
DAPIbeforeimagingonaNikonEclipseTE2000-Emicroscope.
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Electrophysiology
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Allrecordingswerecarriedoutatroomtemperaturewithin6daysofplatingtheneuronsin35mmdish.
Whole-cellvoltageclamprecordingsweremadewithaMulticlamp700Bamplifier(MolecularDevices)
andpatchpipetteswithresistancesof2˗3MΩ.Pipettesolutionwas135mMK-Gluconate,10mMKCl,1
mMMgCl2,5mMEGTA,10mMHEPES,pH7.2,adjustedwithNaOH.Theexternalsolutionwas140mM
NaCl,5mMKCl,2mMCaCl2,2mMMgCl2,10mMHEPES,and10mMD-glucose,pH7.4,adjustedwith
NaOH.WeusedgravityperfusionsystemconnectedwithPerfusionPencil®withMulti-BarrelManifold
Tip(AutoMateScientific)toexternallyapply0.5µMtetrodotoxin,100µMAMPA,kainate,GABA,or
glycinetothecells.CommandprotocolsweregeneratedanddatawasdigitizedwithaDigidata1440A
A/DinterfacewithpCLAMP10software.
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Co-culturemusclecontractionassays
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C2C12myoblastsweregrownin10%FBS+DMEMmediaandthendifferentiatedintomyotubesby
incubatingindifferentiationmedium(2%horseserum+DMEM).Aftermyotubeswereformed,the
neuronsweredissociatedbytrypsinizationandreseededontopofthedifferentiatedmuscletoallow
contractionstodevelop.VideoofcontractionsweretakenusingMetamorphsoftwareandmanually
countedover5secondintervals.Forstoppingassay,300µMTubocurarine(Sigma)wasaddedtothe
mediaasanacetylcholinecompetitor.Forlabelingofacetylcholinereceptors,bungarotoxin(Invitrogen)
wasusedaftercellswerefixedwithparaformaldehyde.SimilarlytoC2C12cells,EScellsover-expressing
MyoDwerealsodifferentiatedtomyotubesusingdifferentiationmediumandsubjectedtoco-culture
withneurons.
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MicroarrayAnalysis
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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ThemRNAofundifferentiatediNILEScellsandNIH3T3cells(growinDMEM+10%FBS)werecollected
andpurifiedbyRNAextractionusingRNeasyPlusExtractionKit(Qiagen).Neuronsdifferentiatedby
eitherprotocolwerefirstsortedbyflowcytometryonaBDFACSariaIImachine(BecktonDickinson,
USA)tocollectHb9::GFP+cells,andthenweresubjectedtoRNAextractioninasimilarfashion.
CollectedRNAwasthenamplifiedandhybridizedtoAffymetrixGeneChipMouseTranscriptomeArrays
(MTA1.0).ResultswereprocessedbytheChildren’sHospitalMicroarrayCoreFacility,andwere
analyzedusingAffrymetrix’sTranscriptomeAnalysisConsoleandExpressionConsolesoftware.
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bioRxiv preprint first posted online Apr. 5, 2017; doi: http://dx.doi.org/10.1101/124594. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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