芯片上的球状体、类器官和肾脏Spheroids,organoids and kidneys‑on‑chips - how complex human

AbstractKidneydiseaseisoneoftheleadingcausesofmorbidityworldwide,emphasizingtheimportanceforphysiologicallyaccu-ratediseasemodels.WithmostoftheapprovedrenaldrugsfailingtoperformaswellinhumanclinicaltrialsastheydidinOneoptionistousepatientderivedcelllines,growninbothtwo-dimensional(2D)andthree-dimensional(3D)structures,knownasspheroidsandorganoids.Despitetheircontributionstothefeld,thelackofphysiologicalaccuracy,includingtheabsenceoffuidfow,andmechanisticefectsinthese2Dand3Dmodelsmeansthereisstillroomforimprovement.Organ-on-a-chip(OOAC)technologyofersitselfasapotentialcandidatemodeltoovercometheselimitations.OverrecentyearsOOACtechnologyhasgrowninpopularity,withmultipleorgansystems,includinglung,liver,andkidneydescribedintheliterature.Inthisreview,traditionalhumancellularbasedmodels,includingmonolayer,spheroidandorganoidmodelswillbediscussed.Humankidney-on-a-chipmodelswillalsobediscussed,whileexploringtheadvantagesandpotentiallimita-tionsofthisrapidlyemergingfeldforthestudyofhumankidneydiseaseanddrugtesting.1IntroductionThekidneysareapairofhighlyspecializedinganestimated1millionnephrons,thefunctionalunitsofthekidney(Bertrametal.2011).EachindividualnephroncontainsabundleofcapillariesknownastheglomerulihousedintheBowman’scapsulewhereglomerularfltra-tionoccurs(Fig.1).Theprimaryfltratepassesoutintotherenaltubule,alongtheproximalconvolutedtubule,loopofHenleanddistalconvolutedtubuleandtothecortical1TranslationalandClinicalResearchInstitute,FacultyofMedicalSciences,NewcastleUniversity,InternationalCentreforLife,CentralParkway,NewcastleUponTyneNE2RenalServices,TheNewcastleUponTynFoundationsTrust,New3NIHRNewcastleBiomedicalReandmedullarycollectingducts(Fig.1).Alongthetubulereabsorptionofwater,electrolytesandnutrientsoccursinanephronsegmentspecifcmanner.Thefnalurineexitsthekidneysfromthecollectingductsintotherenalpelvis,downtheureter,intothebladderandisvoidedviatheure-thra.Besidestheproductionofurine,kidneysareinvolvedinthemaintenanceofbloodpressureviareninproducingcellsinthemaculadense(SuzukiandSaruta2004).Theintricatearchitectureofthekidneys,includingthehighlyheterogeneouscellularpopulation(Fig.1)providesmanyonbothacellularandmolecularlevel.Highbloodpressureandcontinuoushyperglycemiaindiabeticsandgeneticpre-kidneydisease(CKD)(Websteretal.2017).With1.2millionpeoplethoughttohavediedfromsomeformofCKDin2017(GBDChronicKidneyDiseaseCol-mechanisms,aswellaspotentialtreatmentsareextensivelyciallyavailableimmortalizedrenalcelllines,andanimalmodelsincludingmice(Rossetal.2006;Kurbegovicanda灬cortex,medulla,calyx,andrenalcapsule.Containedwithinkidneyaremillionsofnephplecomponents.Specifcrenalcelltypesarecolourcodedwiththerespectivenephronregionoforigin.Celproximaltubuleingreemultipleoriginsinyellow.Simplifedexamplesofhowrenalcellsareusedincellularmodelsareshown.Createdourfgureonline)Trudel2013;Ramsbottometal.2020),zebrafsh(Huangetal.2014),andprimates(Tsukiyamaetal.2019),havebeenusedtogaininsightsintothemechanismsofreeasesincludingrenalciliopathies.Thisgroupofdiseasesarecausedbydysfunctions,ortheabsenceofprimaryciliamicrotubulebasedcellularprotrusionsfoundonthesurfaceofalmosteverymammaliancell(SatirandChristensen2007)(Fig.2).Todate,therearenow35primaryrenalcilopathydiseases,with187establishedcausativegenesand241candidategenesforotherprimaryciliopathies(ReiterandLeroux2017).ExamplesofprimaryciliopathieswhichmayincludearenalphenotypeincludeJoubertSyndrome(JBTS),Oral-facial-digitalSyndrome(OFD),autosomaldominantorrecessivepolycystickidneydiseases(ADPKDandARPKDrespectively),nephronophthisis(NPHP),andBardet-Biedlsyndrome(BBS)(Fig.3).However,limitationsincludinginterspeciesdiferencesandtranslationaldifcultiesduringclinicaltrials(deCaes-teckeretal.2015)havedriventheshifttopatient-specifcstudies.Toachieveahuman-specifcapproach,renalcellsderivedfrombothtissuebiopsies(Murrayetal.2021)andurine(Molinarietal.2018;Molinarietal.2020;Srivastavaetal.2017a,b;Bondueetal.2021)havebeenukidneydiseases.Toprogressknowledgefurther,patientderivedcellshavealsobeenusedtocreatemorecomplex,self-organizedthree-dimensional(3D)structures.currentcellularmodelsusedtostudyhumanrenaldiseases.Wewillalsoexploretheadvancesmadeinrenalmodels,particularlytheinvolvementofmianddiscussthepossiblelimitations(Fig.4).2IsolationandselectionofcelltypesforuseinrenalstudiesHistorically,thegoldstandardtostudykidneymorphogen-esis,patterninganddiseasehasbeentheimplementationofanimalstudies,predominantlymouseandzebrafsh.Recentstitutionofanimalmodels,forthosegeneratedusingpri-maryrenalcelllinesfrommultipleorigins(Table1).ontheabilitytostudyhumanrenaldiseaseswithouttheneedforanimalmodels.Oneexampleofhowthishasbeenachievedhasbeentheuseofhumanpluripotentstemcellsa灬associatedgenesandtheirlocuswithinthecell.Humanprimaryciliaarecomposedofninemicrotubuciliumarehighlighted.CreatedwithPSCscanbeisolatedfrommultiplestartingmaterials,includingliver,kidney,andmostfrequentlydermalfbro-blasts.In2019,athree-stagediferentiationprogrammedwasdescribed,capableofproducingfvedistincttermi-nalandtwoprecursorrenalcelllinesfromhPSCs(Hari-haranetal.2019).Inbrief,theprocedureinvolved4daysofmetanephricinduction,4daysmesenchymalepithelialtransitionofnephronprogenitors,concludingwith6daysofnephronalcellspecifcation(Hariharanetal.2019).CelltypesgeneratedfromhPSCsusingthismethodincludedmesangial,proximaltubular,distaltubular,collectingductepithelialcellsandpodocyteprecursorsasearlyas14d(Hariharanetal.2019).Primaryrenalcellscanbeisolatedfromtissuebiopsies.However,toobtainhealthykidneycellsviabiopsyisdif-fcult,asthisinvasiveprocedureisnotapprovedunlessdis-easeissuspected.Proximaltubule,podocyteandglomeruli(Maetal.2015;Sanchez-Romeroetal.2019;Meneandkidneysarefrstdecapsulatedbyremovingtherenalcor-tex,beforetissuesamplesarecutinto1mm3samplesaregentlywashed,beforeundergoingenzymaticdigestionviacollagenase.Thedigestedmaterialisthensieved,andcelltypesselectedfor,beforeplatingintheappropriatemedia.Thisprotocolwasusedtogeneratetheimmortalizedhumankidneycell2(HK-2,Table1)(Detrisacetal.1984),acelllinewhichhasbeenusedinnumerousrenalstudiessince.Fibroblastsderivedfromskinbiopsieshaveoftenbeenusedtostudyciliopathiesasprimaryciliacanbeinducedfollowingashortperiodofserumstarvation(Pollaraetal.2022;Shamseldinetal.2020).However,inconsistentpro-liferation,viability(Genovaetal.2019)andorgan-specifcdiferencesareassociatedlimitationswhenusingfbroblastsmilkteethfromchildrenhavealsoproventobeasourceofciliatedcelllines(Ajzenbergetal.2015).However,viabil-ity,andaccessibilityarelimitingfactorsforthewidespreaduseofteethasasourceofciliatedcells,astoothlossisagelimited.Inresponsetothis,urinehasbeenusedasanalternativesourceofrenalcellsforover50years(SutherlandandBain1972;Linder1976;Felsourceofprimaryrenalcellsisfreshurinesamples,oftenreferredtoasliquidbiopsies.Briefytrifugedsothatanycellspresentintheurinesamplecana灬ciliopathieswhichpresenarenalphenotype.Othercom-monphenotypesassociatedeachprimaryciliopathyarebeseparatedfromtheurine.Fromhere,thecellsarethenwashed,beforeresuspensionforplatingforexpansiontostudybothrenaldiseases,andprimaryciliopathies.includingcancer(Elliottetal.1976),andprimaryciliopa-thies(Molinarietal.2018,2020;Srivastavaetal.2017a,b;urine-derivedrenalepithelialcells(hURECs)in2Dculturehasledtothediscoveryofdiseasespecifcciliarypheno-typesincludingtheelongationofprimaryciliainJBTShURECswithCEP290mutations(Srivastavaetal.2017a,b);elongatedandswollenprimaryciliainARPKDduetoPKHD1mutations(Molinarietal.2020);ationofIFT88atthetipofprimaryciliainhURECsderivedfromapatientwithnoveletal.2018).Rathersurprisingly,theseurinederivedcellshavealsobeenusedtostudyaspectsofcardiac(Steinle2019),vascular(Liuetal.2018),liver(Huetal.2020),andneurologicaldiseases(Guanetal.2014;Staoetal.2019).Selectionofcell-lineorigincanalsoproveinfuentialonitedself-renewalpotential,withincreasedvariationfoundbetweendonortypes,resultingindifcultywithreproduci-whenconsideringdiseasedriversonapatient-specifcbaInfact,theuseofprimarycelllineshasallowedfortheidentifcationofnovelmutations,andthecharacterizationofdisease-specifcphenotypes(Molinarietal.2018;Devaneetal.2022).Incomparison,immortalizedcelllinescanofernumerousadvantagestoprimarycelllinesincluding:unlimitedsupply(astheyarearecostefective,andtendtobeclearofinfectiousorhaz-ardouspathogens(KaurandDufour2012).3Two‑dimensional(2D)cellmodelstostudykidneydiseasesThe2Dcultureofhumancelllinesprovidesdatathatiseasilytranslated,usessimplemethods,islowincost,andhasthepotentialtobeupscaledtohighthroughputwiththepossibilitytostudymultiplediseases,mechanisms,andtreatmentsinshorttimeframes.Furthermore,mostcellu-larbasedassaysavailableonthemarkethavebeendevel-opedspecifcallyfor2Dbasedmodels,reducingtimespentoptimizingprotocols.Numerousadvancesindiseaseswitha灬rentcell-basedmodelsustostudyrenaldisecanbederivedfromindpatients’viaeitherareestablishedintraditithenbeseededon/intoMatrigscafolds,orspecialisedchipstogenerate3DcellmodeandAPRKD(Wilsonetal.1986),andnephronophthisiscellsgrownunder2Dconditions.Relatingtothestudyofciliopathies,bothdermalderivedfbroblasts,andhURECs(Table1)havebeenutilizedinthegenerationofnumerous2Dmodels.However,despitethemajorcontributiontothefeldofrenaldiseases,theseurine-derived2Dmodelsfacelimita-tions.Examplesofsuchincludethefactthatcellulardifer-entiationisheavilyreliantuponnotonlycell–cellinterac-tions,butalsoacellsiknownastheextra-cellularmatrix(ECM)(Majoetal.2020).TherenalECMiscriticalforphenotypicfeatures,cellgrowth,diferentiation,andgeneexpression(Makinoabsentintraditional2D-basedmodels.Toaddressesthisissue,SendaiVirusreprogrammed(ECM)coatingsincludingECMharvestedfromdecellular-izedhumanforeskinfbroblasts,ECMatrix™-511,recombi-nanthumanlaminin-521andtruncatedrecombinanthumanvitronectin(VTN-N),allunder2Dconditions(Murphyetal.2022).Followinggrowthonthesecoatings,pluripo-grownonforeskinfbroblastcoatingswerefoundtohaveincreasedlevelsofthedermalfbroblastmarkerHSP47,whilethoseculturedoncommerciallyavailableECMsshowedincreasedlevelsofpluripotentmarkersHoechst,NanogandPOU5F1(Murphyetal.2022).FollowingtheinitialECMcharacterizationtests,hPSCswerethendifer-entiatedintopodocyte-likecellsandstainedwithspecifcmarkers(synatopodinandWT1)andfollowingprincipialcomponentanalysis(PCA)showedgoodclusteringofallGeneexpressionanalysisalsosupportedthis,withhighlevelsofpodocyte-specifcmarkersPALLDandCOL4A3(Murphyetal.2022).Thisstudyhighlightsthebeneftsofusing2Dmodelstostudythehumankidney;byaddinganECMcoatingtoassistincelldiferentiation,itispossibletogeneratedesiredrenalcelltypes.Althoughthemainaimofthisstudywastoidentifynon-animalalternativesourcesofECMcoatingsforhPSCdiferentiationintopodocytes,themethodsdescribedcouldbefurtheradaptedandoptimizedforarangeofapplicationsincludingunderstandingkidneydiseasedrivers,andpotentiallyinvestigatingdrugtoxicityinpodocytesin2Dcultures.Theefectsoftheadditionoffuidfowstress(FFSS)andtensilestresshavebeenreportedin2Dpodocyteculturestostudycongenitalanomaliesofthekidneyandurinarytract(CAKUT)(Srivastavaetal.2017a,b).Hyperfltration-mediatedinjuryisoneofthemaincausea灬a灬a灬Table1CommonlyusedrenalceDisease/mechanismmoHumanPluripotentStemCHumanurine-derivedRenalEpitlungfbroblastsderivedfromurinarycellsRenalcorticaltissuecollectedfImmortalizedcelllines(e.g.,ImmortalisedhealthyhumanadultFollowingisolationofurinarycellsviacentrifugation,hPSCreprogrammedusingtheSen-daivirusbasednon-integrationinstructionsKidneysaredecapsulatintoEBSSonice.Corticaltissueisconsistencybeforesieflters,Glomeruliarethenwashed,priortodigestionviacollawithantibiotics.Cellularpeareresuspendedinprimarymedia,withcellsplated,andcultuundernormalconditionsbbeingplacedontothesurfaceofaT25faskcoatedwithcollagen.Flaskswereplacedinapostionfor30min,roturebeforeinversiontoposition.ExplantswereculF12basalmediasupplementwithinsulin,transferrin,seleniuhydrocortisone,triiodothyroandepidermalgrowthfGenerationofmultiplereGenerationof3DbasedJoubertSyndromePrimaryciliopathiesDrugtransportermodelsetal.2016b;Hariharan,(Molinarietal.2018Disease/mechanismmoPodocytes/glomeruliCelHumankidneyepithelialRenalcorticaltissuecollectedfCationicColloidalSilica-FerromagneticNanoparticles.Fopieces,sampleswerecollagenase.Sampleswestrained,beforecelmagneticfeldAsdescribedabovefortubuleepithelialcells,freglomeruliwerecollectedontopwashsesglomeruliwereForpodocytes,glomerularsuspen-forviaanti-podocaboundtoDynabeads,andmagnKidneytissuewasdcollagenase,withcellsisusinganti-humanCD34mono-clonalantibodiesconjugiron-containingmicrobeads.CwerethenculturedinEPsubstitutedwithbFGF,Idiopathicnephroticsyaa灬this,itisvitaltoreplicatestressconditionsonpodocytes.Tensile(orcompressive)stresswasachievedbyculturingpodocytesonfexiblemembranesofspeciallyconstructedculturedishes(tomimiccapillarywalls),wherenegativepressure(viavacuum)wasappliedtostretchpodocytes(Srivastavaetal.2017a,b).Itwasfoundthattensilestresswasresponsiblefortheformationofactin-richcentersandvatesp38-MAPKandERK1/2pathways(Srivastavaetal.2017a,b).IncomparisonFFSSisapplieddirectlytothesurfaceofcells,inthisinstancespecifcallytotheslitdia-phragmsofpodocytes.InthisstudyFFSSwasappliedfol-lowingthedevelopmentofafowchamberwhereof0.2dynes/cm2wasapplied(Srivastavaetal.2017a,b).ouslybeenreportedwithpodocytesinculture(Huangetal.inducedbyFFSSincludeddisruptionofactinstressfberswiththeformationofacorticalactinring,upregulationofCOX-2andEP2,increasedPGE2levels,andtheactivationofAkt-GSk3β-β-cateninandc-src/PLD1/mTORpathways(Srivastavaetal.2017a,b).DespitetheadditionofECMcoatingsstressestogenerate2Drenalmodels,thereremainslimita-tionwhenconsideringotheraspectsofrenalstudies.Forexample,inrelationtodrugtoxicityrelatedstudies,ithaspreviouslybeenshownthatdrugsshowntohavehighef-cacyin2Dinvitromodels,whentranslatedintopatientshavelowefcacy(Shoemaker2006).Thisisalimitationassociatedwiththelackofcomplex3Dstructure,difusionetal.2015).4Three‑dimensional(3D)cellmodelstostudykidneydiseasestotheuseof3Dmodelsincludingspheroidsandorganoids.Throughouttheliterature,thetermspheroidandorganoidhasbeenusedinterchangeably,ofusionwhendiscussingmodels(SimianandBissell2017).Ingeneral,spheroidshailfromsingle-celllineagesandaredescribedasfree-foatingself-organizedsphericalcellularaggregates,withalowlevelofcomplexity(LancasterandKnoblich2014).Incomparison,organoidsareconsideredmorecomplexthanspheroids,notonlyintheirphysicalstructurebutalsotheirdefnition,withnumerousuniquecellculturetechniquesdescriliterature(SimianandBissell2017).Organoidstendtoasformstructuralunitswhicharemoaspecifcorganstructureaswellasfunction(LancasterandKnoblich2014).Spheroidsandorganoidshaveboththeirownuniqueandsomeoverlapintheirpurposes,protocols,andorigins.Itisforthesereasons,thatcautionisadvisedthereplicationofcellularorganization,signalingnetworks,diferentiationanddrugscreeningsuccess,spheroidsandorganoidsaregrowingincreasingpopularityintherenalfeld.5RenalspheroidmodelsforstudyingdiseaseIn2011,humankidneyepithelialcells(hKEpCs,Table1)weredescribedtoaggregateinto3Dspheroidstructuresundernon-adherentconditions(Buzhoretal.2011).ThesehKEpCscouldmaintainaproportionofrenaldevelopmen-talandstem-celllikemarkerswhencomparedtothosecellsgrownin2D(Buzhoretal.2011).Furthermore,renalspheroidshavesuccessfullybeenestablishedfromdissoci-atedandundiferentiatedhPSCsseededbetweenlayersofMatrigel(Freedmanetal.2015).ThesehPSCderivedrenalspheroidsexpressedoctamer-bindingtranscriptionfactor4(OCT4),sex-determiningregionYbox-2(SOX2)NANOGOncediferentiatedintoatubularidentity,spheroidswerebuslectin(LTL),Lin11-Is11-Mec3(LIM)homeobox1manetal.2015).Together,thedescribedfndingsdemon-stratetheimportanceforconsiderationofmodelselectionwhenwishingtoinvestigatespecifcrenalmarkersfordis-easepurposes.RNAsequencing(RNA-seq)of3Dnephrospherescul-turedonplatespre-coatedwithpoly(2-hydroxyethylmeth-acrylate)ECM,derivedfrom2DhKEpCs,foundsignifcantgeneontology(GO)enrichdiferentiationoftheepithelialsegmentsofadulthumankidney(Harari-Steinbergetal.2020).Nephrosphereswerealsofoundtohaveenrichmentforvariousnephronspe-cifcmarkers,anddiferentiationtowardsarenalepithelialphenotypeafter1weekinculture(Harari-Steinbergetal.fcellsurvival,proliferation,organization,anddiferentiationprocesseswereallobserved,implyingatubulogenicandtherapeuticpotentialfornephrospheres(Harari-Steinbergetal.2020).Forapatient-specifcmodel,humanurine-derivedrenalepithelialcells(hURECs)canwithapicobasalpolarity,ciliaformation,andcompletea灬inMatrigelfullyformedspheroidswerevisible.Thestudyformciliainboth2Dand3Dconditions,whilsthURECsderivedfromJBTSpatientscouldonlyformciliain2Dconditions(Ajzenbergetal.2015).Thesignifcanceofthisfndinghighlightstheadvantageof3Dcellsystemsforthestudyofrenalciliopathiesviatheuseofurinederivedcells.usedtogeneratespheroidsfrpartialrescueofciliarydefectsfoltreatment(Hynesetal.2014).Morerecently,auniquemethodofrenalspheroidfor-mationinvolvingtheincorporationofimmortalizedrenalproximaltubuleepithelialcells(RPTEC),andfbrinogenasabioinkwasreported(Tröndleetal.2021).ThisbioinkwasdepositedontoahydrogellayerofMatrigel,CollagenIandFibrinbeforesealingwithasecondlayerofhydrogelepithelialcellself-assemblyintospheroids,withindividualdropletsleadingtotheformationofindividuatersremovingtheneedforcomplex3Dhandlingtechniques(Tröndleetal.2021).Dependingupontheconcentrationofbioinkused,thegroupobservedtheformationoftubulesasearlyasday1,withlumentypicallyformingaroundday4(Tröndleetal.2021).Furthermore,followingRNA-sequencing,31outof34kidney-specifcgeneswerefoundcomparedtothosecellsgrownin2Dconditions(Tröndleetal.2021).Ofthoseup-regulatedgenes,GOenrichmentanalysisshowedenrichmentinfbroblastgrowthfactorandorganicaniontransport,twotermswhicharethoughttobeassociatedwithtubularkidneystructureformationinvivo(Tröndleetal.2021).Collectively,thesefndingshighlightthebeneftofusingcomplexcellularmodelsforthestudrenalconditions,astheycanretainrenalspecifcsignatureswhichareoftenlostinsimpler2Dmodels.6RenalorganoidmodelstostudydiseaseEarlyrenalorganoidmodelsarepresentintheliteraturefromaround2015(Takasatoetal.2015)andarebecomingmorecommon.These3DcellularstrucinthestudyofNPHP(Forbesetal.2018),WilmsTumorsKuraokaetal.2020).Renalorganoids,pairedwithsingle-cellRNAsequencing,haveallowedforthofkeydiseasespecifcinteractionsindicatingfuturegeneticdisease-specifcresearchfocus(Lowetal.2019;Wuetal.2018).Kidneymicro-organoidscontainingaroundsixtotennephrons,surroundedbyendothelialandstromalpopula-tionshavebeendescribedinsuspensioncultures(Kumaretal.2019).Thisstudyfoundthatfollowingextendedtimeinculture(from7daysonwards),cystformation,andmesenchymalexpansionwaspresentwithvariationdependentuponcelllineoforigin(Kumaretal.2019).Thisfndingwasconsistentwithothersuspensiongrownmicro-organoids(Cruzetal.2017;Czernickietal.2018;Przepiorskietal.2018).Theuseofthesestructurescanassistinfurtheringtheunderstandingofthedrivingfactorsofrenalcystformation,andpotentiallybeusedtoidentifytherapeuticsforthetreatmentandpotentialpreventionofcysts,withouttheneedforanimalmodels.Cystformationwithinorganoidstructureshasbeenusedtomodelpolycystickidneydiseases(PKD),includingADPKDwhichhaspreviouslybeenattributedtohomozy-gousmutationsinPKD1(Cruzetal.2017;Kuraokaetal.2020).UpontheintroductionoftruncatingPKD1andPKD2genesintokidneyorganoidsderivedfromhPSCsspontaneouscystformationwasobserved(Freedmanetal.2015).Thepresenceoffuidaccumulation,andprolifera-tiveepithelialcellsliningthecysticregionaccuratelyrep-licatedtheADPKDcellularphenotypeinorganoidmod-els(Cruzetal.2017).FollowingforskolintreatmentoforganoidsderivedfromtheiPSCsofanADPKDandgeneeditedPKD1mutantcells,cystformationwasobservedandusedtomodeltheearlystagesofcystogenesisinADPKD(Kuraokaetal.2020).Incomparison,ARPKDisthoughttoarisebecauseofmutationsinthePKHD1gene(Sharpetal.2005).ARPKDrenalorganoidsgener-atedfrompatientiPSCshavealsobeenusedtostudythedilationofcysts(Lowetal.2019)andtheuretericepi-theliumcystformationofARPKD(Howdenetal.2021).TreatmentofbothADPKDandARPKDpatient-derivedrenalorganoidswiththecysticfbrosistransmembraneconductanceregulator(CFTR)wasshowntoblockcystformation(Lowetal.2019;Shimizuetal.2020).Again,thesestudiesshowpotentialfortheuseofrenalorganoidsasdiseasescreeningtoolsforthestudyofcystformation.Podocytopathieshavealsobenefitedfromtheuseofgenitalnephroticsyndrome(Romero-Guevaraetal.2020).FollowingCRISPR/cas9geneeditinginhiPSC-derivedorganoidswasfoundtorestorepodcytetranscriptionalpro-fleswhilstalsorescuingNPHS1-associateddiseasepheno-types(Haleetal.2018).Podocytedevelopmentalpathwaysingcellswithanegativesurfacecharge(Freedmanetal.2015;Kimetal.2017).Epitheliallumenformation,andpodocytetightjunctionformation,aswellasmicrovillifor-mation,areallheavilyreliantonthepresenceofnegativea灬chargesonpodocytecellsurfaces(Freedmanetal.2015;Kimetal.2017).AnotherexampleofagenetickidneydiseasestudiedviaorganoidmodelsincludeMucin1kidneydisease(MKD)(Dvela-Levittetal.(MUC1-fs),MKDischaracterizedbytheinheritanceoftubulo-interstitialkidneydisease,withpatientsrequiringeitherdialysisorkidneytransplantationduringtheirlife-time(Kirbyetal.2013).AftertreatmentviaBRD4780,itfromintracellularcompartmentsofiPSC-derivedpatientorganoids(Dvela-Levittetal.2019).WildtypeiPSCshavepreviouslybeendiferentiatedintomesodermcellsunder2Dconditions(Moraisetal.2022).Thesemesodermcellswerethendiferentiatedintokidneyorganoids,positiveforglomerularandtubularstructuresafter18daysinorganoidculture(Moraisetalowingimmunofuorescence,basementmembranesequenceofformationwasconfrmedintheseorganoidsassuitableforinves