Experimental Dermatology. 2019;28:355–366. wileyonlinelibrary.com/journal/exd  | 355© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Received:12October2018  |  Revised:2January2019  |  Accepted:8January2019DOI: 10.1111/exd.13891

O R I G I N A L A R T I C L E

Self- organizing hair peg- like structures from dissociated skin progenitor cells: New insights for human hair follicle organoid engineering and Turing patterning in an asymmetric morphogenetic field

Erin L. Weber1,2 | Thomas E. Woolley3 | Chao-Yuan Yeh1 | Kuang-Ling Ou1,4,5  |  Philip K. Maini6 | Cheng-Ming Chuong1,7

1DepartmentofPathology,KeckSchoolofMedicineoftheUniversityofSouthernCalifornia,LosAngeles,California2DivisionofPlasticandReconstructiveSurgery,KeckSchoolofMedicineoftheUniversityofSouthernCalifornia,LosAngeles,California3CardiffSchoolofMathematics,CardiffUniversity,Cardiff,UK4OstrowSchoolofDentistryoftheUniversityofSouthernCalifornia,LosAngeles,California5DivisionofPlasticandReconstructiveSurgery,DepartmentofSurgery,Tri-ServiceGeneralHospital,NationalDefenseMedicalCenter,Taipei,Taiwan6WolfsonCentreforMathematicalBiology,MathematicalInstitute,Oxford,UK7IntegrativeStemCellCenter,ChinaMedicalUniversity,Taichung,Taiwan

CorrespondenceCheng-MingChuong,KeckSchoolofMedicineoftheUniversityofSouthernCalifornia,LosAngeles,CA.Email:cmchuong@usc.edu

Funding informationNationalInstituteofArthritisandMusculoskeletalandSkinDiseases,Grant/AwardNumber:AR47364andAR60306;NationalScienceFoundation,Grant/AwardNumber:DMS1440386;CaliforniaInstituteforRegenerativeMedicine;AmericanCollegeofSurgeons;L.K.WhittierFoundation;A.P.GianniniFoundation

AbstractHumanskinprogenitorcellswillformnewhairfollicles,althoughatalowefficiency,wheninjectedintonudemouseskin.Tobetterstudyandimproveuponthisregen-erativeprocess,wedevelopedaninvitrosystemtoanalysethemorphogeneticcellbehaviourindetailandmodulatephysical-chemicalparameterstomoreeffectivelygeneratehairprimordia.Inthisthree-dimensionalculture,dissociatedhumanneona-tal foreskin keratinocytes self-assembled into a planar epidermal layerwhile fetalscalpdermalcellscoalescedintostripes,thenlargeclusters,andfinallysmallclustersresemblingdermalcondensations.Atsitesofdermalclustering,subjacentepidermalcells protruded to form hair peg-like structures,molecularly resembling hair pegswithinthesequenceoffolliculardevelopment.Thehairpeg-likestructuresemergedinacoordinated,formativewave,movingfromperipherytocentre,suggestingthatthedropletcultureconstitutesamicrocosmwithanasymmetricmorphogeneticfield.Invivo,hairfolliclepopulationsalsoforminaprogressivewave,implyingthesumma-tionoflocalperiodicpatterningeventswithanasymmetricglobalinfluence.Tofur-ther understand this global patterning process, we developed a mathematicalsimulation using Turing activator-inhibitor principles in an asymmetricmorphoge-neticfield.Together,ourculturesystemprovidesasuitableplatformto(a)analysetheself-assemblybehaviourofhairprogenitorcells intoperiodicallyarrangedhairprimordiaand(b)identifyparametersthatimpacttheformationofhairprimordiainanasymmetricmorphogeneticfield.Thisunderstandingwillenhanceourfutureabil-itytosuccessfullyengineerhumanhairfollicleorganoids.

K E Y W O R D S

hairfollicle,organogenesis,periodicpatternformation,skinreconstitution,tissueengineering

356  |     WEBER Et al.

1  | INTRODUC TION

Thebasic tenetofplastic surgery is the restorationof formandfunction.However,replacingskinandfunctionalappendagesre-mainschallenging.Thehairfollicleisamini-organ,which,inasso-ciationwiththeattachedsebaceousgland,playsacrucialrole inskinmoisture, thermal regulation, protective sensation and aes-thetic appearance. For burn patients, the loss of pilosebaceousunitsleadstodry,brittleskinwhichismoresusceptibletoinjury.Whiletransplantationiscurrentlythebestoptionforhairfolliclereplacement, theprocess requiresa largenumberofdonor folli-cles,whichburnpatientstypicallylack,andtargetsonlythescalp.Theabilitytotissueengineeranunlimitedsourceofpilosebaceousunitsfortransplantation,eithersinglyorappropriatelypatternedwithinbioengineeredskin,wouldprovideamuch-neededsolutionformanypatients.

Multiple different approaches have attempted to produce re-constituted skinwith hair inmouse and humans.[1] In themouse,wedemonstratedthatdissociatedepidermalanddermalcellsfromnewbornmouseskinself-assembleinvitrointomulti-layeredskinor-ganoidscontainingplacodesanddermalcondensates,thetwostemcell populations necessary for hair follicle development.[2,3]Whengraftedontoa full thicknessdermalwoundonanudemouse, theculturedorganoidsformedmature,cyclinghairfollicleswithinapla-narskinconfiguration.Transcriptomicanalysisofthemurineskinor-ganoidshasidentifiedfactorsthatcanrescuethehairformingabilityof adultmouse cells.[4]However, similar successwith human cellshasbeenmoredifficult.Adulthumanscalpcellswillproducenewfolliclesininvivomousemodels,albeitatlowrates.[5,6]Theuseoffetal,ratherthanadult,scalpenhancestheefficiencyofhumanhairfollicleregenerationbutapersistentlagtimeof3monthstofollicleformation indicatesthatmoremustbeunderstoodaboutfollicularmorphogenesis.[7,8]Despiteseveraldifferentapproaches,efficient,large-scale, therapeutic tissue engineering and transplantation ofreconstitutedhumanskinwithpilosebaceousunitsremainsachal-lengetothefield.

There are two different strategies to produce hair folliclesfromdissociated cells.One is touse3Dprinted tissue scaffoldsandplacecellsatkeypositionsforfurthermorphogenesis;[9] theother is to rely on the self-organizing ability of skin progenitorcells.[4]Differentprogenitorcellstatescanbeutilizedfortheself-organizingstrategy,suchasinducedpluripotentcells(iPS).[10] On someoccasions, cells need “help” to interactwith other cells orrequireparticularmolecularsignals tomoveforwardtothenextstage.Currently,intheemergingfieldofsyntheticbiology,meth-odsareunderdevelopment toprovide cellswith “help” in topo-logicalarrangement[11,12]ormolecularsignallingattherighttimeandplace.[13]

But, to effectively adopt the synthetic biology approach, wemust learn more about organoid cultures made of cells from dif-ferentages,locationsorspecies,sowecanapplykeymoleculestorestorehair formingability.[4]To thisend,wesought todevelopathree-dimensional, culturesystem inwhichdifferent typesof skin

progenitors,suchasepidermal-ordermal-likesomaticcells,embry-onicstemcellsoriPScells,canbeguidedtoformectodermalorgansin a planar configuration (Figure S1).[14]Wehope that this culturemodelmayserveasaplatformtoidentifythecriticalfactorsneeded,stepbystep,forthedevelopmentofindividualectodermalorgans.Here,wepresentourprogresstowardstheformationofhumanhairfollicleorganoids.Withinthisinvitromodel,weobservedtwodis-tinctandnovelphenomena.First,hairpeg-likestructuresemergedafter only 4days in culture and possessed molecular and cellularcharacteristics similar to authentic human hair pegs. Second, theformative process of periodic patterningwas quite apparent: dis-sociateddermalcellsassembledintostripes,clusters,thendistinctdermal condensations, followedby epidermal “stalks”withdermalpapilla-like “caps.” The process reproducibly began at the dropletboundaryandemanatedasacircumferentialwavetowardsthecen-treoftheculture.

Invivo,periodichairandfeatherplacodesforminaprogressivewave,propagatingindifferentdirectionsdependingonbodysite(e.gscalpand trunk).This implies that theprocess isacombinationoflocalperiodicpatterningeventsandanasymmetricglobalinfluencethatmake themorphogenetic fieldasymmetric.The localperiodicpatterningeventmayinvolvechemicalandmechanicalfeedbackbe-tweencellsandtheirenvironment.[15,16]Severalmodelshavebeenproposed, ranging from chemical-based reaction-diffusionmodelsto ones where the “reactants” are cells themselves to mechano-chemicalmodelswhich couple cell interactionswith chemical sig-nals.[17–19] The self-organizingpatternsobservedexperimentally inourculturesystemresemblepatternsmostsimplyillustratedbytheTuring activator-inhibitormodel.[20,21] The global behaviour of thesystemcanbedescribedbytheoccurrenceofaTuringinstabilityonanasymmetricmorphogeneticfield.Suchasymmetryisspeculatedtobecausedbymechanicalorchemicalforcesorunevencellprolif-erationordeath.[22,23]

The droplet culture system described here provides a uniqueopportunitytostudybothperiodicpatterningandglobaleventsinhumanhairfollicleformation.Theformationofhairpeg-likestruc-turesoccursmore rapidly thanother currentmethodsand, yet, isslowenoughtopermittheanalysisandoptimizationofthesequenceofcellularevents.Mathematicalmodellingoftheformationwaveinthehairpegpopulationallowsustoanalysetheself-assemblypro-cessandpredictconditionsthatmayenhanceorganoidformation.Translationally, this culture systemprovidesproofof concept thatstructures resembling human hair follicle precursors can be engi-neeredinvitroinatime-efficientmannerandservesasaplatformtoidentifytheoptimalconditionswithwhichtoefficientlyengineerhumanhairfolliclesfortransplantation.

2  | METHODS

2.1 | In vitro hair follicle reconstitution assay

Epidermal and dermal cells were enzymatically and mechanicallyseparated fromneonatal foreskinandsecond-trimester fetal scalp

     |  357WEBER Et al.

(estimatedgestationalage(EGA)17-19weeks),respectively.2×106 cultured neonatal foreskin keratinocytes and 3×106 fresh fetalscalpdermalcellswere resuspended in140ulofF12:DMEM(1:1)mediumwith5%FBSandP/S/Aandplatedasadropletonasix-wellcellcultureinsert.Thedropletswereincubatedat37°Cand5%CO2 for 4-7days. Growth factors were added daily. See supplementalmethodsfordetails.

2.2 | Patch assay

2×106neonatalforeskinkeratinocytesand3×106fetalscalpder-malcellswereinjectedsubcutaneouslyintothedeepdermisof6-to12-week-oldhairlessnudemice.Subcutaneousnoduleswithformedhairfollicleswereharvested8weekslater.

2.3 | Immunostaining, lentiviral vectors and live cell imaging

Seesupplementalmethod,TablesS1andS2.

2.4 | Mathematical modelling

Areaction-diffusionmodelwasdevelopedtosimulatethe interac-tion of two, as of yet, experimentally unidentified, differentmor-phogen populations. Details, equations and parameter definitionsareincludedinsupplementalmethods.

3  | RESULTS

3.1 | Human fetal scalp dermal cells induce the self- organization of hair peg- like structures in droplet culture

Dissociated neonatal human foreskin keratinocytes and 17- to19-week EGA human fetal scalp dermal cells were mixed andco-cultured in three-dimensional droplets (Figure1A). Within24hours, the epidermal and dermal cells segregated into twolayers, with epidermal cells adhering to the cell culture insertmembrane at thebaseof thedroplet anddermal cells overlyingthekeratinocytes inamoresuperficial layer (Figure1B).Around48hours, dermal cells began to organize, forming a trabecularmeshpattern,whichthenevolved intopunctatecellclusters.By72hours, keratinocytes abutting the dermal clusters rearrangedinto a concentric pattern and, within 96hours, keratinocyte“stalks”protruded,againstgravity,intothedropletspace,inasso-ciationwithadermalcell“cap”(Figure1B,MovieS1A-C).Incom-parisonwith17-weekEGAfetalscalpsections,thenewlyformedstructures resemble early hair pegs, a stage in follicle develop-mentinwhichtheinvaginatingkeratinocytesprotrudedownwardintothedermalplane,guidedbythedermalpapilla(Figure1C).Ofnote,while therewasaclearandearlysegregationofepidermalanddermalcells,wefrequentlyencounteredscattered, large, in-tenselykeratin-positivecellsinterspersedwithinthedermallayer,

whichexhibitedcharacteristicsconsistentwith terminallydiffer-entiated,anucleatedkeratinocytes.These“cells”donotappeartoparticipateinthemorphologicalevents.

Dermalfibroblastsareknowntoself-aggregateinnon-adherentculture.Todemonstrate that thehairpeg-likestructureswerenotanartifactoftheculturesystemorsimplyaresultofdermalfibro-blastself-aggregation,humanfetalscalpdermalcells,intheabsence

F IGURE  1 Humanneonatalforeskinkeratinocytesandfetalscalpdermalcellsself-organizedtoformhairpeg-likestructuresinvitro.A,Schematicoftheinvitrohairfolliclereconstitutionassay.Follicularorganoids,composedofepidermal(green)anddermalcells(red),protrudefromamulti-layeredkeratinocytesheet(green).hNFKs=humanneonatalforeskinkeratinocytes,hFSDs=humanfetalscalpdermalcells.B,Serialbrightfieldandconfocalimagesoftheculturedroplettakenevery24hdemonstratedtheformationofperiodicallyarrangedthree-dimensionalconfigurationsby96h,correspondingtohairpeg-likestructurescomposedofanepidermalstalkanddermalcap.Wholemountconfocalimagesinthesecondandthirdrows,withpancytokeratindenotingepidermalcellsingreenandnucleiinred,weretakenfromtheperipheryofthedroplet,asrepresentedbythewhitedottedboxinthebrightfieldimageinthefirstrow.Inthesecondrow,thewhitedottedlinedemarcatestheperipheryofthedroplet.Thefourthrowofimagesistriple-stainedsections,withpancytokeratin(panCK)markingepidermalcells(green),vimentinmarkingdermalcells(red)andnuclei(blue)stainedwithTO-PRO-3iodide.Thescalebaristhesameforallimagesperrow.Thelargegreenlobulesinthe48hsamplearedeadcellartifactswhichhavetrappedthefluorescentantibody(n=25).C,Invitrostructuresat96h(leftpanel)resembledhairpegsfoundin19-wkhumanfetalscalp(rightpanel).p63isamarkerofepidermalprogenitorcells(n=25).D,Humanfetalscalpdermalcellsaloneandadultscalpdermalcellsmixedwithneonatalforeskinkeratinocytesdidnotproduceanyhairpeg-likestructuresafter96hinculture.Theimagesaretakenfromtheperipheryoftheculturedroplet,asexemplifiedbytheblackdottedboxinB.hASDs=humanadultscalpdermalcells.E,Wheninjectedsubcutaneouslyintoanudemouse,humanneonatalforeskinkeratinocytesandfetalscalpdermalcellsproducedmaturehairfolliclescomposedofcellsofhumanorigin.Pancytokeratin(green)andvimentin(cyan)antibodiesarehuman-specific.Sectionsofmouseskinwereincludedtoconfirmspeciesspecificityoftheantibodies(bottompanels)(n=3)

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offoreskinkeratinocytes,wereculturedunderidenticalconditions.Fetal scalp dermal cells also formed a trabecular pattern but didnot form any three-dimensional structures (Figure1D). Similarly,adultdermalcells,fromhair-bearingadultscalp,wereculturedwithneonatal foreskin keratinocytes. Adult scalp dermal cells formedthick, dense sheets. Neither combination produced hair peg-likestructures.

3.2 | Hair peg- like structures in vitro displayed cytoarchitecture and molecular markers similar to those observed in vivo

Underdefined conditions, epidermal anddermal cells rapidly self-assembledandtransitionedthroughstagesreminiscentoffolliculardevelopmenttoformhairpeg-likestructuresbutfailedtoprogressfurtherinvitro.Toverifythatthehumanneonatalforeskinkeratino-cytesandhumanfetalscalpdermalcellspossessedfullregenerativepotential,thesameratioofepidermalanddermalcellswasinjectedsubcutaneouslyintonudemiceinatraditionalpatchassay.[2]Eightweekslater,completehairfollicles,includinghairshafts,wereclearlyvisible in the subcutaneous tissue encircling a central keratinizedmass (Figure1E). Immunostaining with human-specific antibodiesconfirmedthatcellsoftheepidermalouterrootsheathsanddermalpapillaewereofhumanorigin(Figure1E).

Akintohairpegsindevelopingfetalskin,thereconstitutedhairpeg-likestructureswerekeratin-14positiveandkeratin-10negative(Figure2A).Keratin-10and involucrin,markersof suprabasal cells,wereexpressedinallcellsoftheepidermalsheetexceptthebasallayer, consistent with normal patterns of epidermal stratification(Figure2A).Whileepidermalcellsoriginallystratifiedwithbasementmembranefacingtheinsert,thepolarityofstratificationwasalteredonceepidermaldowngrowthbegan,withepidermal“stalks”andas-sociateddermal“caps”projectingupwardsintotheculturethroughmoredifferentiated layersofepidermis.Wesuspect this isdue tophysicallimitationsofthedropletculturesystem.KeratinocytesoftheepidermalstalkexpressedK17,K18andE-cadherin,allknowntobeexpressedinthe innerorouterrootsheath layersofmaturefollicles,thoughatthehairpegstage,distinctepidermalsheathlay-ershavenotyetformedandlessisknownabouttheexpectedloca-tionsforexpressionoftheseproteins(Figure2A).Someofthelargerhairpeg-like structuresdisplayed longer, curvingepidermal stalks,whichwhenviewedattherightangle,appearedtopossessacentralkeratin-positivecoresurroundedbyconcentricallyorientedepider-mal cells, possibly indicating progression in development towardsthe bulbous peg stage (Figure2A). These advanced hair peg-likestructuresoccurred infrequently,however,making further charac-terizationdifficult.

p63,amarkerofepidermalstemcells,wasinitiallypresentinallkeratinocytesat24hours.Asisseeninnormalhairfollicledevelop-ment, p63 expression became limited to the basal layer followingepidermal stratification and p63-positive cells were reproduciblynotedattheleadingedgeoftheepidermalstalk,adjacenttotheder-malcap(Figure2B).PCNAimmunostainingdemonstratedactivecell

division inboth theepidermalbasal layer and the leadingedgeofthestalk,whiletheremainingepidermalcellswithinthestalkwerequiescent (Figure2B).Thepresenceof focal, replicatingepidermalprogenitorcellsattheleadingedgeofthestalksuggeststhatlocal-izedproliferationmaycontributetodowngrowthandwehypothe-sizethattheseproliferatingcellsmaybeputativehairmatrixcells.However,we cannot ruleout thepossible contributionof cellmi-grationfromtheadjacentstratifiedepidermisinhairpegformationandthemechanismbywhichepidermaldowngrowthoccurs isnotyetknown.

Consistentwith a dermal lineage, dermal cap cells synthesizedcollagens IandIII (Figure2C).Basementmembraneproteins,colla-gen IVand laminin, typically locatedat the interfacebetweenepi-dermalandmesenchymalcellswithinthehairfollicle,werepresentat the junction of epidermal stalk and the dermal cap (Figure2C,MovieS2A).Furthermore,thedermalcapcellsassociatedwiththehair peg-like structures displayedmarkers also present in the der-malcondensateanddermalpapilla.Thedermalcapwascomposedofaheterogeneousmixtureofdermalcellswithacentralcompart-mentalized area positive for alpha-smooth muscle actin (α-SMA,Figure2C,MovieS2B).Whilealkalinephosphataseisaclassicmarkerofthemurinedermalpapillaandisexpressedinthedermalpapillaeof17-weekhuman fetal scalp, there is limitedandconflictingdataregardingtheexpressionofalkalinephosphatasevsα-SMAinhumandermalpapillacellsinculture.Somepublicationsshowpersistental-kalinephosphataseexpressioninculturedhumandermalpapillacellsbutothersdemonstraterapidlossofalkalinephosphataseexpressionandupregulationofα-SMAexpression.[24–29] In reality, theexpres-sionof dermal papillamarker genes is easily influencedby cultureconditions. Inoursystem,α-SMAexpressionwaspresentwhileal-kalinephosphataseexpressionwasnot.Versican,anothercommonlyusedmarkerforthedermalcondensateandpapilla,wasstronglyex-pressed in thedermal cap (Figure2B,C).While thesedermal capsrepresentthedevelopmentalprogressionofdermalstripestoclus-ters to condensations and dermal papillae-like aggregates, whichcanfunctionallyinducehairpeg-likestructures,webelievetheyareincompleteorimmaturedermalpapillaebecausetheyexpresssome,butnotall,dermalpapillamolecularmarkersandinducetheforma-tionofhairpeg-likestructuresinsteadofcompletehairfollicles.

3.3 | The formation of hair peg- like structures in vitro mimics the sequential stages of development in vivo

Foreskin keratinocytes and fetal scalp dermal cells progressedthrough stages similar to native hair follicle development.[30] Between 48 and 72hours in culture, epidermal cells underlyingfocal dermal cell collections formed a concentric pattern, distinctfrom the cobblestonepatternof the surrounding epidermal sheet(Figure2D,Movie S3A,B).[31] β-catenin, known to be expressed inthe epidermal placode and required for hair follicle morphogen-esis,wasfocallyenriched inepidermalcellsabuttingtheclustereddermalcells,butabsentfromtheadjacentepidermisatthetimeof

     |  359WEBER Et al.

epidermal downgrowth (Figure2E).[32–34] Cells within the dermalclustersexpressedCD34,amarkerofthehumandermalcondensateandearlydermalpapilla(Figure2E).[35]Theformationofhairgerm-likestructuresand,then,hairpeg-likestructuresensuedbetween72and96hoursinculture.

Livecellconfocalimagingofthedropletculturewasdevelopedto visualize the cell-cell interactions and collective cell move-ments during hair peg formation. Visual discrimination betweenepidermalanddermalcellswasachievedusingepidermal-specificpromoters.Lentiviraltransductiontoexpresslineage-specificflu-orescentmarkers did not perturb hair peg development in vitro(FigureS2).Aviewfromthetopofatwo-colour, livecellculturedropletdemonstrateddistinctsphericaldermalcapsareshownintheSupplementMaterial (FigureS2,MovieS4).Nucleiofcellsattheperipheryof thedermalcapexhibitedacurvedmorphology,and cells near the centre of the dermal cap displayed increasedlocalcellmotionwhilecellsattheperipheryweremorestationary,suggesting a heterogeneity of dermal cell function. In contrast,cellswithintheepidermalsheetremainedstatic.Three-colourliveimagingdistinguishedK14+epidermalcells(yellow),p63+epider-mal precursor cells (magenta) and dermal cells (cyan)within thehairpegandadjacentepidermalsheet(FigureS2,MovieS5A,B).As seen in static confocal images,p63-positivecellswerenotedwithin theepidermal sheet aswell as theepidermal stalkof thehairpeg.Several stronglypositivep63cellswerepresentat theleadingedgeof theepidermal stalk, abutting thedermalcluster,and1-2cellswereconsistentlynotedattheoppositepoleofthedermalcluster,auniquepositionwhichcouldsuggestaninstruc-tiveroleindirectionalepidermaldowngrowth.

3.4 | Hair peg- like structure formation in the organoid droplet culture displays spatiotemporal patterning

Large-scaledermal cellpatternswithin thedroplet culturedem-onstrated a spatiotemporal progression, which initiated at the

droplet periphery and advanced towards the centre (Figure3A).At 24hours postplating, dissociated dermal cells remained dis-tributed in a homogeneous layerwithout a distinctmacroscopicpattern.Overthenext12hours,dermalcellscoalescedintolongundulating stripes of higher dermal cell density. By 48hours inculture,longstripeshadsubdividedintoshorterstripesand,over

F IGURE  2 Hairpeg-likestructuresformedinvitroexpressedappropriateepidermalanddermalmarkersandprogressedthroughreproduciblestagesreminiscentofearlyhairfollicledevelopment.A,Stainingofsectionsofhairpeg-likestructureswithkeratin-14(green)demonstratedclearseparationbetweenepidermalanddermalcells(topleft).Consistentwithpatternsofkeratinexpressioninhumanfetalscalp,epidermalcellswithintheinvitrohairpeg-likestructuresdidnotexpresskeratin-10(red).Involucrin(green),amarkerofkeratinocyteterminaldifferentiation,washighlyexpressedinonlyaportionoftheepidermalsheet,which,alongwithkeratin-10expression,suggestsstratification(bottomleft).Involucrinalsostronglymarkedcellsbelievedtobeterminallydifferentiated,anuclearcorneocytesthatbecameinappropriatelytrappedwithinthedermalcellcap.Theepidermalstalkswerekeratin-17(green),keratin-18(magenta)andE-cadherin(green)positive(wholemount,topright).Severalofthelargerhairpeg-likestructuresappearedtohaveepidermalstalkswithcentrallumensandconcentricallyorganizedkeratinocytes,markedbykeratin14(green)(wholemount,bottomright).B,At24h,allkeratinocytesexpressedp63(green),amarkerofepidermalstemness(sections,toppanel).By72and96h,p63-positivecellswerelocalizedtothebasallayeroftheepidermalsheetandtheleadingedgeoftheepidermalstalkabuttingthedermalcap(middlepanel).96-himagesarewholemountspecimens.PCNA-positive(green),activelyproliferatingcellswerepresentwithinthebasallayeroftheepidermalsheet,theepidermalstalkattheinterfacewiththedermalcapandtheperipheryofthedermalcap,similarto17-wksecond-trimesterhumanfetalscalp(sections,bottompanel).C,ThecellsofthedermalcapexpressedcollagenI(green,sectionandwholemount)andcollagenIII(green,wholemount)(topleft).K10=keratin-10.CollagenIV(red)andlaminin-332(green),markersofthedermalpapillabasementmembrane,wereexpressedattheinterfacebetweenepidermalanddermalcellswithinthehairpeg-likestructuresinvitro(wholemount,bottomleft).PanCK=pancytokeratin,nuc=nuclei,vim=vimentin.α-SMA(green),amarkerofhumandermalpapillacellsinculture,wasexpressedwithinthecentreofthedermalcap(wholemount,topright).Humandermalpapillacellsinvivoexpressalkalinephosphatase(alkphos(green),leftimage),amarkerwhichistypicallylostduringinvitroculture.Thedermalcapwasalsopositiveorversican,acommonlyuseddermalcondensateordermalpapillamarker(wholemount,bottomright).D,Serialopticalsectionsofadermalaggregateat48himagedatincreasingdropletdepthsdemonstratedarounded,densedermalclusteratopanepidermalsheetwithconcentricallyarrangednuclei,reminiscentoftheepidermalplacode.E,β-cateninwasexpressedthroughouttheepidermalsheetat48hbutwasrestrictedtothoseepidermalcellsassociatedwiththedermalcapby72h(sections,toppanels).DermalcellswithinthedermalcapwerepositiveforCD34,amarkeroftheearlydermalpapillastemcell(bottompanels).The48-himageisawholemountspecimen.Allotherimagesaresections.Allstainingwasperformedonmultiplehairpeg-likestructuresfromatleastthreebiologicalreplicates(n=3)

Kera�n-14 nuclei17 wk fetal scalp 96 h

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time, short stripesbecame rounded,CD34-positivedermal clus-ters(Figure2F).Between72and96hours,hairpeg-likestructuresformed (Figure3B), first at the droplet periphery. In addition toformingearliest,elongatedstructuresapproximatingmorematurehair peg-like structures formed more densely at the periphery(Figure3C, D). Centrally, dermal aggregates were 60% larger indiameter,whichcorrelatedwiththeformationoflessmaturehairpeg-likestructuresand,inmanycases,abnormalaggregatespos-sessingmultipleepidermalstalks(Figure3D).Theformativewaveof“longstripe—shortstripe—roundedcluster—peg-likestructure”advanced from theperiphery towards thecentreof thedroplet,witheachnewchangeinmorphology,andthestripeandspotpat-ternsarereminiscentoftheperiodicpatternspredictedbyTuringactivator-inhibitorprinciples.[36,37]

3.5 | Mathematical modelling simulates the observed spatiotemporal patterns

Reaction-diffusionsystemsarecapableofspontaneouslyproducingsustainedspatialpatterns.Specifically,spots,stripesandlabyrinthinepatterns are all possiblewithin the framework of diffusion-driveninstability, knownasTuringpatterns.Once formed,generallyonlyoneofthesepatternsisselectedandremainsfixed.[38]Incontrast,inourdropletculture,multipledistinctpatternsoccursimultaneouslyandareformedinserialprogressionatdifferentlocationswithinthedroplet.Asmentioned, suchpatterning complexity canarise fromseveraldifferentsources.Wechosetouseareaction-diffusionde-scriptionbecause the transitionbetween spots and stripes iswellunderstood.[38,39]Critically, intwodimensions,Turingpatternscanproducespotsand/orstripes,buttypicallynotatthesametime.[21] Itissimplythecompetitionbetweenthequadraticandcubictermsoftheactivatorkineticsthatdeterminewhichpatternmodeisob-tained.[21]Thus,ifthecorrectpatternkineticsarechosentoproduceinphase,oroutofphase,concentrationpatterns, thenanyTuringsystemcanbeguidedtogiverisetospotsand/orstripes.Further,a parameter's influence is extremely local in Turing patterns.[22] Thus,allwerequiretoconvertasystemfromspotstostripesistouse a gradient that influences the competitionbetween the cubicandquadraticterm.Hence,thisisacompletelygeneralandrobustmechanismforproducingsuchdynamics.Basedonthis,weproposethatanasymmetricspatiotemporalgradientispresenttoexplainthemixedspectrumofpatternswithinthespace(organoiddroplet)andthetransitionofpatternsovertime.Theworkimpliesthatthedrop-letrepresentsanasymmetricmorphogeneticfield.Indeed,inembry-onicdevelopment,hairsandfeathersforminpropagativewavesindifferentbodydomains,ratherthansimultaneously.[40–42]ThisgaveusthemotivationtodevelopasimulationofTuringpatterningoccur-ringinanasymmetricmorphogeneticfield(Figure3E).

Tomakethesimulationmodelmorebroadlyapplicable,wepur-poselyassignedthemorphogensgenericactivatororinhibitorfunc-tions, rather than focusingonspecific signallingmolecules (pleaserefertothesupplementalmethodsforamoredetaileddescription).Critically, this work is not about specifying the exact underlying

F IGURE  3 Collectively,astripe-to-dotformativegradientformsfromthecentretoperipheryoftheculturedropletandsuggestsaTuringperiodicpatteringprocessonanasymmetricfield.A,Brightfieldimagesofasingleculturedroplettakenevery12hdemonstratedtheformationofaninitialtrabecularpattern,whichthengavewaytotheperiodicallyarrangedstripesandcellclusters(toprow).Linedrawingscreatedfrombrightfieldimages(middlerow)andaschematicwhereredrepresentsdermalcellsandgreenrepresentsepidermalcells(bottomrow)emphasizethetransitionsindistinctperiodicpatternsastheyrelatetodevelopmentalstages(n=4).B,High-powermagnificationdemonstratesthehairpeg-likearchitectureunderbrightfieldimaging(n=4).C,Thefieldwasdividedintofiveconcentriczones.Anatomichairpeg-likestructuresdevelopedatahigherdensityinzones4-5,towardstheperipheryofthedroplet.Inpairedttestcomparisons,theaveragedensityofhairpeg-likestructuresattheperipheryoftheculturedropletwasstatisticallydifferentfrommorecentralzones(*P<0.05).Errorbarsrepresentstandarderrorofthemean(n=11).D,Dermalclustersofasmallerdiameterweremorelikelytobeassociatedwithsinglestalkedhairpeg-likestructuresthanwerelargerdermalclustersfoundatthecentreofthedroplet.Averageclusterdiameterisplotted.Pairedttestcomparisonswereusedtoexaminestatisticallysignificantdifferencesbetweengroups.Errorbarsrepresentstandarderrorofthemean.*P<0.05,**P < 0.01 (n=3).E,Numericalsimulationsofreaction-diffusionequationsarepresentedinthemethodssection.Thetoprowillustrateshowthedensityofactivator(u)altersovertime,withdarkercolourspresentinghigh-densityregionsandlightercoloursrepresentinglow-densityregions.Thebottomrowillustratestheradiallysymmetricspatiotemporalgradientfieldthataltersthepropertiesofthereaction-diffusionequationsheterogeneouslyacrossthedomain.Astimeincreases,thevalueofthefieldattheboundaryincreasestoamaximumvalueandthegradientgetssteeper.Weseethatasthegradientsteepens,theactivatorpatterntransitionsfromspotsattheperipherytolabyrinthinepatternsinthecentre,whichrecapitulatestheinvitroperiodicpatterns.AdditionalparametervaluesaregiveninTableS3

*

Hair peg-likestructures

100 μm

96 h(B)

1mm

1 mm 1 mm

Hair peg-likestructures

1

2

3

4

5

(C) 6

5

4

3

2

1

0

Dens

ity o

f hai

r peg

s (pe

r mm

2 )1 2 3 4 5

zones

160

120

80

40

0

Diam

eter

of d

erm

al c

onde

nsat

e (μ

m)

Peg

Mul

�-st

alk

No

peg

24 h 36 h 48 h 60 h 72 h 84 h

1 mm

(A)

1 mm

**** *

*

(D)

(E)

     |  361WEBER Et al.

kinetics.Indeed,wedonothavesufficientlydetailedinformationtodeterminethesystemtothisaccuracy.Thespecificuseofourmodelis to highlight that the transition seen in the experiments can becaptured,quitegenerally,usingasimple radiallysymmetric, linear,time-dependentgradient.Theobservedresultcouldbeobtainedinaninfinitenumberofmorecomplicatedways.However,ourresultshaveputalowerboundlimitonthecomplexityrequiredtomakeamodelconsistentwiththeobservedresults.

Sinceweobservethathairpeg-likestructuresfirstformintheperiphery, theasymmetrysuggests that theactivatorbecomes in-creasinglysensitivetotheinhibitormorphogenν,attheperiphery,or, alternatively, the activator becomes decreasingly sensitive totheactivator's self-activation response.Suchagradient caneasilyariseastheexperimentaldropletisanisotropic,andcouldbeduetochemicalsignalling(e.ggrowthfactors)orphysicalforcesinnature,orboth.Thus,theprinciplesofTuringactivatorandinhibitorremainthesame,butindifferentregions,weanticipatethefieldcanbehet-erogeneouslypredisposedwithparametersthatfavourorsuppressperiodicpatterning.Astimeprogresses, thegradient increasesto-wardstheperiphery(bottomsimulationsoffigure“simulation”),andpatterns transit from labyrinthinestripes to spots (topsimulationsoffigure“simulation)(Figure3E).Thespatiotemporalheterogeneityismodelledasalinearspatialgradientthatincreasesatthedropletboundaryandfixesovertime(Figure3E,MovieS6).Thevisualiza-tionofthegradientexhibitsitselfasahairpegformativewavetrav-ellingfromtheperipherytowardsthecentreofthefield,matchingexperimentalresultsobservedinthedropletcultures.Critically,theproposedasymmetrycouldbewrappedupinsidetheequations,butthiswould obscure the essential requirement of a spatiotemporalgradientappearing.Thus,wechoosetobeexplicitwiththeadditionofsuchcomplexity.

3.6 | A platform to modulate hair peg morphogenesis in vitro

Toincreasethenumberofhairpeg-likestructuresandtostimulatedevelopmentbeyondthehairpegstage,wemodulatedmultiplepa-rameterswithin the droplet culture system. The greatest numberofhairpeg-like structuresperdroplet culturewasgeneratedwitha 150 μL volumedroplet, 5%FBS concentration and epidermal todermalcellratioof2:3(Figure4A).Anaverageof286hairpeg-likestructures(±138)percm2withaninterfolliculardistanceof350μm wasproducedunderoptimalconditions(Figure4B).Forcomparison,endogenoushairpegsfrom17-weekfetalscalparespaced,onaver-age,235μmapart.Theinvitrohairpeg-likestructuresweresimilarin overall shape to hair pegs of 17-week fetal scalp but exhibitedsignificantlydifferentstructuralproportions.Thereconstitutedhairpeg-likestructurespossessedshorter,narrowerkeratinocytestalksandwiderdermalcapswhiletheheightofthedermalcapremainedconsistentwithendogenousfetalhairpegs(Figure4B).Nativefetalscalpexhibitedhairpegsofvariousepidermalstalkheights.There-constitutedhairpeg-likestructureswere,onaverage,shorterthantheendogenoushairpegsbutmorecloselyresembledtheshorter,

earlyhairpegs innativeskin,suggestingthat,accordingtonormaldevelopmental patterns, the reconstitutedhair peg-like structurescould be expected to elongate further before transitioning to thebulbouspegstage.However,ourreconstitutedhairpeg-likestruc-turesfailedtoprogressfurtherwhentheyweremaintainedforthreeadditionaldaysinculture.Clearly,otherfactorsarerequired.

Althoughwe have not been able to achievemoremature hairfollicleformation,theself-organizationofperiodicallyarrangedhairpeg-like structures fromdissociated cells is a remarkable process.Detailedanalysisoftheprocessenablesthisdropletculturetoserveas a platform for large-scale screeningof experimental conditions

F IGURE  4 Modulationofhairpegmorphogenesisinvitro.A,Here,weexaminetheconditionsthatcaninfluencethenumber,sizeandprogressionofthehairpeg-likestructures.Hairpeg-likestructuresformedmorefrequentlywhenculturemediumcontained5%FBSandwhenanepidermaltodermalcellratioof2:3wasused.Theaveragenumberofhairpeg-likestructuresformedperconditionisplotted,witherrorbarsrepresentingstandarderrorofthemeanandstatisticalsignificanceassessedwithpairedttests.*P<0.05(n=6).B,Theinvitrohairpeg-likestructures(blackbars)showsimilararchitecturetohairpegsin17-wkhumanfetalscalp(greybars).However,theaveragestalkheight,stalkwidthandcapwidthoftheinvitrohairpeg-likestructuresweresignificantlysmaller.Whiletheinvitrohairpeg-likestructuresformedatregularintervals,therewasalargeraverageinterfolliculardistancethanisfoundinfetalscalptissue.AnasteriskdenotesaP-valueof<0.05,whencomparedtonativehumanfetalscalp,viapairedttests.Errorbarsrepresentstandarderrorofthemean(n=5).C,Theadditionofgrowthfactorstothedropletculturescausedsignificantchangesincertainaspectsoftheepidermalstalkanddermalcapdimensions,butdidnotinducefurtherdevelopmentintoabulbouspegstructure.Dimensionsmeasuredthefollowing:1)epidermalstalkwidth,2)epidermalstalklength,3)epidermalstalkareaatthestructuremidpoint,4)dermalcapwidth,5)dermalcapheight,6)epidermalstalk-dermalcapoverlapand7)dermalcapareaatthestructuremidpoint.Growthfactors:A)negativecontrol,B)Shh1μg/mL,C)Tgfβ2 0.5 μg/mL,D)RARantagonistER50891 1 μmol/L,E)FGF7+FGF101μg/mL,F)FGF21μg/mL,G)FGF2+Shh1μg/mL,H)FGF2+Wnt7a1μg/mL,I)PKCi660nmol/Lchelerythrinechlorideand10nmol/LbisindolylmaleimideIandJ)Noggin1μg/mL.Allmeasurementswerenormalizedtothenegativecontrol,andaveragedimensionsareshown.Errorbarsrepresentstandarderrorofthemean.Statisticalsignificancebetweentwogroupswascalculatedusingpairedttests.AnasteriskdenotesaP-value<0.05,comparedtothenegativecontrolwithoutaddedfactors(n≥3)

Cap width (4)

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Nor

mal

ized

leng

thor

are

a

Cap height (5) Cap-stalk overlap (6) Cap area (7)

Stalk width (1)

A B C D E F G H I J

Stalk area (3)Stalk height (2)

2.0

1.5

1.0

0.5

0

Nor

mal

ized

leng

th

or a

rea

1

4

2

5

6

3

7

*

* *

*

*

*

*

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A B C D E F G H I J A B C D E F G H I J

A B C D E F G H I J A B C D E F G H I J A B C D E F G H I J A B C D E F G H I J

*

**

*

*

*

vimen�n nuclei

400

300

200

100

Heig

ht o

r wid

th (μ

m)

Stal

k he

ight

Stal

k w

idth

Cap

heig

ht

Cap

wid

th

Inte

r-pe

gdi

stan

ce

**

*

*

17 week human fetal scalp

50 µm

0% 1% 5% 10%FBS concentra�on

300

(A) (B) (C)

250

200

150

100

50

0

HF p

recu

rsor

s per

dro

plet

2:1 2:3 1:5 1:9 1:12Epidermal:Dermal ra�o

HF p

recu

rsor

s per

dro

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300

250

200

150

100

50

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*

362  |     WEBER Et al.

to optimize in vitro follicle formation.We identified four possiblesignalsthatmightsupportdevelopmentalprogression:(a)increaseddermalsignallingforepidermaldowngrowth(Shh,Tgfβ2),(b)stron-ger dermal papilla inductivity (Wnt7a, FGF2), (c) inhibition of pre-maturekeratinocytedifferentiation(proteinkinaseC(PKC),Noggin,retinoicacidreceptors(RAR))and(d)stimulationofkeratinocytedif-ferentiationand/orstratification(FGF2,FGF7/10).[43–50]

Exogenous growth factorswere added to the culturemediumevery24hours.Arangeofconcentrationswastestedforeachpro-tein; results for the concentration which produced the greatesteffect are shown (Figure4C). Thus far, noneof the added factorshave resulted in progression to the next stage, the bulbous hairpeg.However,adetailedanalysisofdermalcapandepidermalstalkwidth,heightandareaidentifiedsignificantchangesmediatedbytheaddedgrowthfactors,which,withmoreinvestigation,mayholdthekey to stimulating true follicle formation in culture.Cap and stalksagittalareasmaintainedalinearrelationshipwiththetotalcapandstalkvolumes,emphasizingtheradialsymmetryofthesestructuresandallowingustosimplifyanalysisbymeasuringtheareaofeachstructure at themidpoint corresponding tomaximalwidth (FigureS3).Duringtheearlypegtobulbouspegtransition,thedermalcapbecomesmorecompactandisencapsulatedbythebaseoftheelon-gatingepidermalsheath.[30]Theadditionof1μmol/LShhstimulatedepidermaldowngrowth,resultinginlongerepidermalstalks,aswellasachange in thedermalcapshape,withan increasedwidthandcap-stalkoverlap,suggestingthatShhmaystimulatedermalcellmi-grationproximallyalongtheepidermalstalkor,conversely,epider-malstalkdisplacementofdermalcapcells (Figure4C).TheproteinkinaseC inhibitors,chelerythrinechlorideandbisindolylmaleimideI, produceda similar effect,with increasedepidermal stalk lengthandoverallstalkarea,aswellasincreaseddermalcapareaandcap-stalkoverlap.FGF2,incombinationwithShh,decreaseddermalcapheightandareaandtheretinoicacidreceptorantagonistER50891enhanced total stalk area. Though subtle changes were evidentwhenexogenousfactorswereadded,theywereinsufficienttoalterthegrossmorphologyofthehairpeg-likestructureandpushdevel-opmentintothebulbouspegstage.

4  | DISCUSSION

Theabilitytotissueengineerhumanhairfolliclesfortransplantationwould eliminate a treatment gap for numerouspatients.Over theyears, our group's research has focused on themorphogenesis ofskinappendages.Recently,weexaminedtheself-organizingbehav-iourofdissociatedepidermalanddermalnewbornmousecellsandtheirability to reconstitute functional follicles.[4]Similarstudiesofhumanfollicularmorphogenesishavebeendifficulttoachieve,duetothelowefficiencyoffollicleformationfromreadilyavailableadultcellsandthelongtimetofollicleformation.Here,wedemonstratedtheproductionofhumanhairpeg-likestructuresinvitrofromawell-definedmixtureofprogenitorcells.Inthisthree-dimensionalorga-noiddropletculture,dissociatedneonatalepidermalandfetaldermal

cellsprogressed,viaself-organization,throughthefollowingrepro-ducible and recognizable stages akin to early follicle developmenttoreachcellularconfigurationssimilartohairpegsinsitu:(a)mixeddissociatedcells, (b) cell sheets, (c)dermal stripesandclusters, (d)dermal clusterswith associated epidermal placode-like collectionsand (e) distinct hair peg-like structureswith spatial periodicity.[30] Thedevelopmentalprocessproceededrapidlywithin96hoursandwasdependentonepidermal:dermalcellratioandfactorconcentra-tion, suggesting theneed foranappropriatebalanceofepithelial-mesenchymalsignallingfactorsorcell-cellinteractions.Thisinvitroculturesystemdemonstratestheinitiationandrapidprogressionofearlystagesofhumanfollicle-likedevelopment. Italsoshowsthathumanandmousecellsutilizedifferentmorphogeneticpathsinthemorphospaceofepidermal-dermalmulticellularconfigurationsandmayexplainwhyithasbeendifficulttoachieverobusthumanhairreconstitution.Wehypothesizethatthedifferencesbetweenhumanandmousehairfolliclereconstitutionmaybeduetothreefactors:epidermal cell plasticity, the inducing ability of dermal cells and morpho-genetic field competence.

1. The plasticity of foreskin keratinocytes is known to wane withprolongedculture,resultinginreducedhairfollicleformation.[51] We hypothesized that a loss of epidermal plasticity inhibitedfurther follicle organoid development in vitro beyond the pegstage. Protein kinase C (PKC) and retinoic acid pathways playa role in epidermaldifferentiation and stratificationduring skindevelopment. Excessive retinoic acid causes cessation of hairfollicledevelopment at thegerm stage inmice,while inhibitionof PKCpromotes folliculogenesis from adultmouse cells.[4,47,49] TheadditionofPKCinhibitorsandanRARantagonistexhibitedpositive effects on the length and diameter of the epidermalstalk butwas insufficient to drive further folliculogenesis, sug-gesting that other factors are required for progressive devel-opment. We suspect that the less primitive epigenetic stateof the keratinocytes used may be the molecular basis forsuboptimal competence. In future studies, we will search forfactors that can “reprogram” these keratinocytes or use moreresponsive keratinocytes.

2. The inducing ability of dermal cells isasecondcriticalcomponentforfolliculogenesis.Thedermalpapillareleasesmultiplefactors,whichparticipateinepidermal-mesenchymalsignallingduringfol-liculogenesis. Shh and Tgfβ are necessary for epidermal down-growthandmicewhich lackShhsignallingpossesshair follicleswhicharestalledatthegerm/pegstage.[43,44]TheadditionofShhto the droplet cultures stimulated additional epidermal down-growthbutdidnotcausestructuralprogression to thebulbouspegstage.WealsoexaminedWnt7aandFGF2,whichhavebeenshowntomaintainproliferationandinductivityinculturedmurinedermalpapillacells.[45,46]Yet,wedidnotobservesignificantpro-gressinorganoiddevelopment.Thedermalpapilla-likecellsinourculturedonotappearfullyfunctionalastheycanonlysupporttheinduction of hair peg-like structures, not mature follicles.However, this system provides a promising platform for the

     |  363WEBER Et al.

continued search for factors or conditions which enhanceinductivity.

3. The morphogenetic field,comprisedofepidermalcells,dermalcellsandextracellularmatrixtogether,mustenteracompetentstageforperiodicpatterningtobegin.Thedevelopingembryoisahet-erogeneousmorphogeneticfieldwithanisotropicgrowthinwhichchemical factors,cell typesandmechanical forcesareunevenlydistributedinthreespatialdimensionsandonetemporaldimen-sion.Here,ourorganoidculturedemonstratesobviousasymme-trywithin the droplet, as patterns began at the periphery andmigratedcentrally.Labyrinthinestripesofdermalcellswerenotedinitially, which subsequently transformed into periodically ar-rangeddermalcellclusters.BothstripesanddermalclusterscanbeproducedbyasimpleTuringmodelandcanreflectaninterme-diatestageofthefinalperiodicpatternsifthereisanunevenmor-phogenetic field.[38] What can account for the difference inprogression through the periodic patterning process? While asimplegenericradialgradienteffectedbyonecomponentmaybesufficient foraTuringactivator-inhibitorsystemtoproducethepatternhere,itmaynotbesufficienttoproducethecomplexspa-tiotemporal patterning transitions we observed experimen-tally.[36,37]Here,wepurposelydesignedagenericmodel,tohavewiderconceptualapplication.Simulationwithmathematicalmod-elling suggests similar patterning sequences can be achievedthroughunevenchemicalsignallingactivities[52] (e.ghighercon-centrationof activatormorphogensat thedropletperipheryorhighersensitivityofcellsatthedropletperiphery)orunevenme-chanical forces[53] (e.g cellular tension or matrix rigidity favourperiodic formation at the droplet periphery). Themathematicalandexperimentalmodelspresentedherewillhelpusidentifythemolecularbasisof thesepatterningprocesses in theanalysabledropletinvitroandinthecomplexdevelopingembryoinvivointhefuture.

In vivo, chicken feather buds form exquisite hexagonal patternsprogressively from themidline to the flank.Earlierworkshave sug-gestedthisresultsfromalocalTuringeventandaglobalpropagatingevent.[54]However,thenatureoftheglobaleventwasunknown.Thisispartofthemotivationforthisstudy,tousetheorganoiddroplettoun-derstandmoreaboutthenatureofthesequentialappearanceofhairorfeatherprimordia.ItistimelythatapaperreportingaglobalEdawavespreadingfromthemidlinetotheflankisjustreported,whichsuggestsEdainducesFGF20,followedbydermalcellaggregateformation,thusfacilitatingTuringpatterningviamechano-chemicalcoupling.[55]Basedonthisandotherstudies,weproposeanewunderstandingonthepro-cessofpropagativeformationoffeatherarraysinvivo:alocalTuringperiodicpatterningoccursandworkstogetherwithaglobalpropaga-tionmechanism.[56] In thisnewstudy,progressiveexpressionofEdaandactivationofEdasignallingfromthemidlinetotheflankisshowntomediatetheglobalprocessbyloweringthethresholdofdermalcon-densation.[55]Yet,whatisupstreamtoEdaremainsunclear.Thus,theglobalmechanismcanbechemicalormechanicalinnature,aslongasitcantilttheTuringactivator/inhibitorsystemratio.[56]Theasymmetric

morphogenetic field in theorganoidculturestudiedherepresentsagoodmodeltofurtherinvestigatehowthisglobalasymmetrymech-anismworks.

Withtwocellularcomponents,theinitialepidermalanddermalcellratiowillinfluencethefinalstablepositioninthemorpho-spaceof two-component multi-cellular assemblies.[4] The initial condi-tions,determinedbytheprobabilityofcellcollisionandtherelativestrengthof cell adhesion, control the initialmulti-cellular configu-ration. In the human cell culture droplet, epidermal-matrix adhe-sionsappeartodominate,leadingtotheformationoftheepidermallayerfirst.Dermal-dermalinteractionsarestrongerthanepidermal-dermal adhesions, leading to the formation of dermal stripes anddermal clusters.However, duringmorphogenetic processes, therecan be “qualitative changes” of cellular collectives. Following theformation of dermal condensations, epidermal-dermal condensateadhesion increases and can induce the formation of hair peg-likestructures,uptotheextentthatepidermalbasementmembranepo-larity isreversed.Thus,thehigh-resolutionanalysisoftheprocessofhairpegformationprovidesanexcellentopportunitytofine-tunekeycellularevents.

Insummary,wehavedemonstratedthathumanfetalscalpder-malcells, inassociationwithcompetentepidermalcells,candirectthe rapid regeneration of human hair peg-like organoids in vitro.Theopportunitytostudytheasymmetricspatiotemporalsequenceofperiodicpatterningwithinthedropletprovidesinsightsintotheself-organizingbehaviourofskinprogenitorcells.Furthermore,thisinvitroculturesystemprovidesanopportunitytostudywaystore-storeandoptimizehairfollicleregenerationfromeasilyobtainableadultdermalcellsandmaysupporttheproductionofcompletehairfolliclesfortransplantationinthefuture.[57]

ACKNOWLEDG EMENTS

ThisworkwassupportedbyfundingfromtheAmericanCollegeofSurgeons(E.L.W.),theCaliforniaInstituteforRegenerativeMedicine(E.L.W.), theA.P.GianniniFoundation (E.L.W.), a graduate fellow-shipfromtheMinistryofNationalDefenseofTaiwan(K.L.O.),theL.K.WhittierFoundation (C.M.C.), theNational InstituteofArthritisandMusculoskeletalandSkinDiseasesoftheNationalInstitutesofHealth (AR47364,AR60306;andGM125322 toC.M.C.) and theNationalScienceFoundation (DMS1440386toT.E.W.andP.K.M.).WethankDr.ChinLinGuoforhelpfuldiscussionandacknowledgetheUSCResearchCenterforLiverDiseaseCellandTissueImagingCoreandtheUSCStemCellMicroscopyCoreFacilityfortheirassis-tanceandparticipation.WethankDr.R.B.Widelitzforhelpfulinput.T.E.W. and P.K.M. thank the Mathematical Biosciences Institute(MBI)atOhioStateUniversityforhelpingto initiatethisresearch.MBIreceives its fundingthroughtheNationalScienceFoundationgrantDMS1440386.

CONFLIC T OF INTERE S T

Theauthorshavedeclarednoconflictofinterest.

364  |     WEBER Et al.

AUTHOR CONTRIBUTIONS

E.L.W. and C.M.C. conceived the idea and experimental design.E.L.W.andK.L.O.conductedtheexperiments.C.Y.Y.providedimag-ingexpertise.T.E.W.andP.K.M.producedmathematicalmodelling.E.L.W.,T.E.W.andC.M.C.preparedthemanuscript.

ORCID

Kuang-Ling Ou https://orcid.org/0000-0001-9536-2677

R E FE R E N C E S

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

Figure S1. A generic working model for planar skin reconstitutionwithhairformation.Epidermalanddermalcellsaremixedandplatedon tissue culture insert in high cell density as a droplet. Differentepidermal and dermal cells can be used, those derive from new-born skin, adult skin, adult hair follicle, and ES or iPS derived cells Figure S2.Livecellimagingofhairpeg-likestructures.A,Overexpressionoffluorescentproteinsdidnotaffecttheabilitytoformhairpeg-likestructures.Inthisimage,epidermalcellnucleiweremarkedwithgreenfluorescentproteinanddermalcellnucleiwerepreferentiallymarkedwithorangefluorescentprotein.Thedottedlineoutlinestheepider-malstalk.Epi=epidermal,D=dermal,E=epidermal(n=7).B,Astillimagefromatwo-colorliveimagingvideo(FigureS5A),lookingdownfromthetopoftheculturedroplet,demonstratesadermalcap.p63-positivekeratinocytenucleiaremagenta,dermalcellnucleiarecyan.Epi SC=epidermal stemcell (n=5).C,A single lateral image takenfromathree-colorliveimagingvideo(FigureS5B)demonstratesahairpeg-like structure.Keratinocytenuclei areorange,p63-positiveke-ratinocytenucleiaremagenta,anddermalcellnucleiarecyan(n=5) Figure S3.Reconstitutedhairpeg-likestructuresdisplayedradialsym-metry.Cap and stalk sagittal areasmaintained a linear relationshipwiththetotalcapandstalkvolumes,emphasizingtheradialsymmetryofthesestructuresandallowingustosimplifyanalysisbymeasuringtheareaofeachstructureatthemidpointcorrespondingtomaximalwidth

Movie S1 Three-dimensional z-stack reconstructions of humanhairpeg-like structures formed inculture.A,Wholemountcon-focal z-stack images of multiple hair peg-like structures immu-nostainedwithkeratin-14 (green),vimentin (red), andTO-PRO-3iodide(nuclei,blue)demonstratetheperiodicpatterningandfor-mationofdistinct structureswithina425μm2 area.Cellswithinthekeratinized sheetweredifficult to stainwithpancytokeratindue to poor antibody penetration. B,Whole mount confocal z-stackimagesofhairpeg-likestructuresimmunostainedwithpan-cytokeratin(green)andpropidiumiodide(nuclei,red)demonstratethe spherical configuration of the dermal cap and the tubularstructureoftheepidermalstalk.Sandwichingofthewholemountculturebeneathacoverslip forconfocal imagingcausedthehairpegstoappearbentor flattenedagainst thekeratinocytesheet.C,Highermagnificationviewofasinglereconstitutedhumanhairpeg-likestructureimmunostainedwithpancytokeratin(green)andpropidiumiodide(nuclei,red).Noteepidermalcellsstarttowraparoundthedermalcap

Movie S2 Three-dimensional z-stack reconstructions of reconsti-tutedhumanhairpeg-likestructuresdemonstratemarkersofdermalpapillageneexpression.A,Wholemountconfocalz-stack imagingdemonstratesα-SMAstaininginacentrallocationwithinthedermal

cap of a hair peg-like structure. α-SMA (green), propidium iodide(red). B,Wholemount confocal z-stack imaging demonstrates thepresenceofextracellularcollagenIVattheepidermal-dermalinter-face.CD34-anearlydermalpapillamarker(green),collagenIV(red),TO-PRO-3iodide(blue).Thelargegreenlobulesareartifactsrepre-sentingdeadcellswhichhavetrappedthedye

Movie S3 Three-dimensional z-stack reconstructions of epidermalplacode-like structuresanddermal clusters at48hours.A,Wholemountconfocalz-stackimagingdemonstratesmultipledermalclus-tersatopakeratinocytesheetandalteredkeratinocytearrangementpatternaroundthedermalclusters.Pancytokeratin(green),propid-iumiodide(red).The largegreenlobulesareartifactsrepresentingdead cellswhichhave trapped thedye.B,Wholemount confocalz-stack imaging demonstrating vimentin-positive immunostainingofthedermalclustersat48hourspost-plating.Vimentin(red),TO-PRO-3iodide(blue)

Movie S4 Livecell imagingofa reconstitutedhumanhairpeg-likestructure. Time-lapse movie highlighting dermal cell shape andmovementwithinthedermalcapofareconstitutedhumanhairpeg-likestructure,asviewedfromthetopofaculturedroplet.Theepi-dermalstalkisnotvisibleinthisview.Az-stackimagewasrecordedevery10minutesfrom101-103hourspost-platingandisreplayedatarateof5framespersecond.Theentireculturedescendedalongthez-axisduringimaging,resultinginpartialmovementoutofthefocalplaneovertime.p63-positiveepidermalcellswerelabelledwithnu-cleareGFPfluorescentprotein(magenta).DermalcellswerelabelledwithnuclearmCerulean3fluorescentprotein(cyan).Notethevarieddermalcellmovementandnuclearshapewithinthedermalcap.Fewp63-positiveepidermalcellsarevisibleinthistop-downview,astheepidermalstalkisobscuredbythecellsofthedermalcap.However,reproducibly,1-3p63-positiveepidermalcellswerenotedwithinthedermalcap,frequentlyattheapex,asseenhere

Movie S5 Live cell imagingof a reconstitutedhumanhairpeg-likestructure.Time-lapsemovieofareconstitutedhumanhairpeg-likestructure, viewed from top-down (A) and lateral (B) orientations.Az-stack imagewas recordedevery10minutes from83-85hourspost-platingand is replayedata rateof10 framespersecond.Allepidermalnucleiwerepre-labelledwithmOrange2fluorescentpro-tein(yellow).p63-positiveepidermalnucleiwerelabelledwitheGFP(magenta).DermalnucleiwerelabelledwithmCerulean3fluorescentprotein(cyan).Notethattheentirespecimendriftsduringimaging.However, the epidermal cells within the epidermal sheet remainstatic,asevidencedbynochangeinpositionalrelationshipwithad-jacentepidermalcells.Thepositionofthedermalcapdoesmoveinspace,relativetotheepidermalsheet,becausetheepidermalstalkisflexibleandswayswithinthedropletculturemedium

Movie S6Mathematical simulation of human hair follicle periodicpatternformationinvitro.Thechangesinperiodicpatterningfromlongstripestoshortstripestopunctateclusters,correspondingto

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dermalclustersandthenhairpeg-likestructures,isrepresentedhereby a Turing-basedmathematical simulation. The periodic patternsformsequentiallyontheleft,astheradiallysymmetricspatiotempo-ralgradientincreasesinthemiddleandright-sideddiagrams.

Appendix S1SupplementalMethodsTable S1AntibodiesTable S2Primersforlentiviralvectorconstruction.gDNA = genomicDNATable S3Parametervaluesforequations(1)-(4)

How to cite this article:WeberEL,WoolleyTE,YehC-Y,OuK-L,MainiPK,ChuongC-M.Self-organizinghairpeg-likestructuresfromdissociatedskinprogenitorcells:NewinsightsforhumanhairfollicleorganoidengineeringandTuringpatterninginanasymmetricmorphogeneticfield.Exp Dermatol.2019;28:355-366.https://doi.org/10.1111/exd.13891